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12.3: Virulence Factors in Infection - Biology

12.3: Virulence Factors in Infection - Biology


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Learning Objectives

  • Explain how virulence factors contribute to signs and symptoms of infectious disease
  • Differentiate between endotoxins and exotoxins
  • Describe and differentiate between various types of exotoxins
  • Describe virulence factors unique to fungi and parasites
  • Compare virulence factors of fungi and bacteria
  • Explain the difference between protozoan parasites and helminths
  • Describe how helminths evade the host immune system
  • Describe the mechanisms viruses use for adhesion and antigenic variation

In the previous section, we explained that some pathogens are more virulent than others. This is due to the unique virulence factors produced by individual pathogens, which determine the extent and severity of disease they may cause. A pathogen’s virulence factors are encoded by genes that can be identified. When genes encoding virulence factors are inactivated, virulence in the pathogen is diminished. In this section, we examine various types and specific examples of virulence factors and how they contribute to each step of pathogenesis.

Virulence Factors for Adhesion

As discussed in the previous section, the first two steps in pathogenesis are exposure and adhesion. Recall that an adhesin is a structure, such as a protein or glycoprotein, found on the surface of a pathogen that attaches to receptors on the host cell. Adhesins are found on bacterial, viral, fungal, and protozoan pathogens. One example of a bacterial adhesin is type 1 fimbrial adhesin, a molecule found on the tips of fimbriae of enterotoxigenic E. coli (ETEC). Recall that fimbriae are hairlike protein bristles on the cell surface. Type 1 fimbrial adhesin allows the fimbriae of ETEC cells to attach to the mannose glycans expressed on intestinal epithelial cells. Table (PageIndex{1}) lists common adhesins found in some of the pathogens we have discussed or will be seeing later in this chapter.

Table (PageIndex{1}): Some Bacterial Adhesins and Their Host Attachment Sites
PathogenDiseaseAdhesinAttachment Site
Streptococcus pyogenesStrep throatProtein FRespiratory epithelial cells
Streptococcus mutansDental cariesAdhesin P1Teeth
Neisseria gonorrhoeaeGonorrheaType IV piliUrethral epithelial cells
Enterotoxigenic E. coli (ETEC)Traveler’s diarrheaType 1 fimbriaeIntestinal epithelial cells
Vibrio choleraeCholeraN-methylphenylalanine piliIntestinal epithelial cells

Clinical Focus: part 3

The presence of bacteria in Michael’s blood is a sign of infection, since blood is normally sterile. There is no indication that the bacteria entered the blood through an injury. Instead, it appears the portal of entry was the gastrointestinal route. Based on Michael’s symptoms, the results of his blood test, and the fact that Michael was the only one in the family to partake of the hot dogs, the physician suspects that Michael is suffering from a case of listeriosis.

Listeria monocytogenes, the facultative intracellular pathogen that causes listeriosis, is a common contaminant in ready-to-eat foods such as lunch meats and dairy products. Once ingested, these bacteria invade intestinal epithelial cells and translocate to the liver, where they grow inside hepatic cells. Listeriosis is fatal in about one in five normal healthy people, and mortality rates are slightly higher in patients with pre-existing conditions that weaken the immune response. A cluster of virulence genes encoded on a pathogenicity island is responsible for the pathogenicity of L. monocytogenes. These genes are regulated by a transcriptional factor known as peptide chain release factor 1 (PrfA). One of the genes regulated by PrfA is hyl, which encodes a toxin known as listeriolysin O (LLO), which allows the bacterium to escape vacuoles upon entry into a host cell. A second gene regulated by PrfA is actA, which encodes for a surface protein known as actin assembly-inducing protein (ActA). ActA is expressed on the surface of Listeria and polymerizes host actin. This enables the bacterium to produce actin tails, move around the cell’s cytoplasm, and spread from cell to cell without exiting into the extracellular compartment.

Michael’s condition has begun to worsen. He is now experiencing a stiff neck and hemiparesis (weakness of one side of the body). Concerned that the infection is spreading, the physician decides to conduct additional tests to determine what is causing these new symptoms.

Exercise (PageIndex{1})

  1. What kind of pathogen causes listeriosis, and what virulence factors contribute to the signs and symptoms Michael is experiencing?
  2. Is it likely that the infection will spread from Michael’s blood? If so, how might this explain his new symptoms?

Bacterial Exoenzymes and Toxins as Virulence Factors

After exposure and adhesion, the next step in pathogenesis is invasion, which can involve enzymes and toxins. Many pathogens achieve invasion by entering the bloodstream, an effective means of dissemination because blood vessels pass close to every cell in the body. The downside of this mechanism of dispersal is that the blood also includes numerous elements of the immune system. Various terms ending in –emia are used to describe the presence of pathogens in the bloodstream. The presence of bacteria in blood is called bacteremia. Bacteremia involving pyogens (pus-forming bacteria) is called pyemia. When viruses are found in the blood, it is called viremia. The term toxemia describes the condition when toxins are found in the blood. If bacteria are both present and multiplying in the blood, this condition is called septicemia.

Patients with septicemia are described as septic, which can lead to shock, a life-threatening decrease in blood pressure (systolic pressure <90 mm Hg) that prevents cells and organs from receiving enough oxygen and nutrients. Some bacteria can cause shock through the release of toxins (virulence factors that can cause tissue damage) and lead to low blood pressure. Gram-negative bacteria are engulfed by immune system phagocytes, which then release tumor necrosis factor, a molecule involved in inflammation and fever. Tumor necrosis factor binds to blood capillaries to increase their permeability, allowing fluids to pass out of blood vessels and into tissues, causing swelling, or edema(Figure (PageIndex{1})). With high concentrations of tumor necrosis factor, the inflammatory reaction is severe and enough fluid is lost from the circulatory system that blood pressure decreases to dangerously low levels. This can have dire consequences because the heart, lungs, and kidneys rely on normal blood pressure for proper function; thus, multi-organ failure, shock, and death can occur.

Exoenzymes

Some pathogens produce extracellular enzymes, or exoenzymes, that enable them to invade host cells and deeper tissues. Exoenzymes have a wide variety of targets. Some general classes of exoenzymes and associated pathogens are listed in Table (PageIndex{2}). Each of these exoenzymes functions in the context of a particular tissue structure to facilitate invasion or support its own growth and defend against the immune system. For example, hyaluronidase S, an enzyme produced by pathogens like Staphylococcus aureus, Streptococcus pyogenes, and Clostridium perfringens, degrades the glycoside hylauronan (hyaluronic acid), which acts as an intercellular cement between adjacent cells in connective tissue (Figure (PageIndex{2})). This allows the pathogen to pass through the tissue layers at the portal of entry and disseminate elsewhere in the body (Figure (PageIndex{2})).

Table (PageIndex{2}): Some Classes of Exoenzymes and Their Targets
ClassExampleFunction
GlycohydrolasesHyaluronidase S in Staphylococcus aureusDegrades hyaluronic acid that cements cells together to promote spreading through tissues
NucleasesDNAse produced by S. aureusDegrades DNA released by dying cells (bacteria and host cells) that can trap the bacteria, thus promoting spread
PhospholipasesPhospholipase C of Bacillus anthracisDegrades phospholipid bilayer of host cells, causing cellular lysis, and degrade membrane of phagosomes to enable escape into the cytoplasm
ProteasesCollagenase in Clostridium perfringensDegrades collagen in connective tissue to promote spread

Pathogen-produced nucleases, such as DNAse produced by S. aureus, degrade extracellular DNA as a means of escape and spreading through tissue. As bacterial and host cells die at the site of infection, they lyse and release their intracellular contents. The DNA chromosome is the largest of the intracellular molecules, and masses of extracellular DNA can trap bacteria and prevent their spread. S. aureus produces a DNAse to degrade the mesh of extracellular DNA so it can escape and spread to adjacent tissues. This strategy is also used by S. aureus and other pathogens to degrade and escape webs of extracellular DNA produced by immune system phagocytes to trap the bacteria.

Enzymes that degrade the phospholipids of cell membranes are called phospholipases. Their actions are specific in regard to the type of phospholipids they act upon and where they enzymatically cleave the molecules. The pathogen responsible for anthrax, B. anthracis, produces phospholipase C. When B. anthracis is ingested by phagocytic cells of the immune system, phospholipase C degrades the membrane of the phagosome before it can fuse with the lysosome, allowing the pathogen to escape into the cytoplasm and multiply. Phospholipases can also target the membrane that encloses the phagosome within phagocytic cells. As described earlier in this chapter, this is the mechanism used by intracellular pathogens such as L. monocytogenes and Rickettsia to escape the phagosome and multiply within the cytoplasm of phagocytic cells. The role of phospholipases in bacterial virulence is not restricted to phagosomal escape. Many pathogens produce phospholipases that act to degrade cell membranes and cause lysis of target cells. These phospholipases are involved in lysis of red blood cells, white blood cells, and tissue cells.

Bacterial pathogens also produce various protein-digesting enzymes, or proteases. Proteases can be classified according to their substrate target (e.g., serine proteases target proteins with the amino acid serine) or if they contain metals in their active site (e.g., zinc metalloproteases contain a zinc ion, which is necessary for enzymatic activity).

One example of a protease that contains a metal ion is the exoenzyme collagenase. Collagenase digests collagen, the dominant protein in connective tissue. Collagen can be found in the extracellular matrix, especially near mucosal membranes, blood vessels, nerves, and in the layers of the skin. Similar to hyaluronidase, collagenase allows the pathogen to penetrate and spread through the host tissue by digesting this connective tissue protein. The collagenase produced by the gram-positive bacterium Clostridium perfringens, for example, allows the bacterium to make its way through the tissue layers and subsequently enter and multiply in the blood (septicemia). C. perfringens then uses toxins and a phospholipase to cause cellular lysis and necrosis. Once the host cells have died, the bacterium produces gas by fermenting the muscle carbohydrates. The widespread necrosis of tissue and accompanying gas are characteristic of the condition known as gas gangrene (Figure (PageIndex{3})).

Toxins

In addition to exoenzymes, certain pathogens are able to produce toxins, biological poisons that assist in their ability to invade and cause damage to tissues. The ability of a pathogen to produce toxins to cause damage to host cells is called toxigenicity.

Toxins can be categorized as endotoxins or exotoxins. The lipopolysaccharide (LPS) found on the outer membrane of gram-negative bacteria is called endotoxin (Figure (PageIndex{4})). During infection and disease, gram-negative bacterial pathogens release endotoxin either when the cell dies, resulting in the disintegration of the membrane, or when the bacterium undergoes binary fission. The lipid component of endotoxin, lipid A, is responsible for the toxic properties of the LPS molecule. Lipid A is relatively conserved across different genera of gram-negative bacteria; therefore, the toxic properties of lipid A are similar regardless of the gram-negative pathogen. In a manner similar to that of tumor necrosis factor, lipid A triggers the immune system’s inflammatory response. If the concentration of endotoxin in the body is low, the inflammatory response may provide the host an effective defense against infection; on the other hand, high concentrations of endotoxin in the blood can cause an excessive inflammatory response, leading to a severe drop in blood pressure, multi-organ failure, and death.

A classic method of detecting endotoxin is by using the Limulus amebocyte lysate (LAL) test. In this procedure, the blood cells (amebocytes) of the horseshoe crab (Limulus polyphemus) is mixed with a patient’s serum. The amebocytes will react to the presence of any endotoxin. This reaction can be observed either chromogenically (color) or by looking for coagulation (clotting reaction) to occur within the serum. An alternative method that has been used is an enzyme-linked immunosorbent assay (ELISA) that uses antibodies to detect the presence of endotoxin.

Unlike the toxic lipid A of endotoxin, exotoxins are protein molecules that are produced by a wide variety of living pathogenic bacteria. Although some gram-negative pathogens produce exotoxins, the majority are produced by gram-positive pathogens. Exotoxins differ from endotoxin in several other key characteristics, summarized in Table (PageIndex{3}). In contrast to endotoxin, which stimulates a general systemic inflammatory response when released, exotoxins are much more specific in their action and the cells they interact with. Each exotoxin targets specific receptors on specific cells and damages those cells through unique molecular mechanisms. Endotoxin remains stable at high temperatures, and requires heating at 121 °C (250 °F) for 45 minutes to inactivate. By contrast, most exotoxins are heat labile because of their protein structure, and many are denatured (inactivated) at temperatures above 41 °C (106 °F). As discussed earlier, endotoxin can stimulate a lethal inflammatory response at very high concentrations and has a measured LD50 of 0.24 mg/kg. By contrast, very small concentrations of exotoxins can be lethal. For example, botulinum toxin, which causes botulism, has an LD50 of 0.000001 mg/kg (240,000 times more lethal than endotoxin).

Table (PageIndex{3}): Comparison of Endotoxin and Exotoxins Produced by Bacteria
CharacteristicEndotoxinExotoxin
SourceGram-negative bacteriaGram-positive (primarily) and gram-negative bacteria
CompositionLipid A component of lipopolysaccharideProtein
Effect on hostGeneral systemic symptoms of inflammation and feverSpecific damage to cells dependent upon receptor-mediated targeting of cells and specific mechanisms of action
Heat stabilityHeat stableMost are heat labile, but some are heat stable
LD50HighLow

The exotoxins can be grouped into three categories based on their target: intracellular targeting, membrane disrupting, and superantigens. Table (PageIndex{4}) provides examples of well-characterized toxins within each of these three categories.

Table (PageIndex{4}): Some Common Exotoxins and Associated Bacterial Pathogens
CategoryExamplePathogenMechanism and Disease
Intracellular-targeting toxinsCholera toxinVibrio choleraeActivation of adenylate cyclase in intestinal cells, causing increased levels of cyclic adenosine monophosphate (cAMP) and secretion of fluids and electrolytes out of cell, causing diarrhea
Tetanus toxinClostridium tetaniInhibits the release of inhibitory neurotransmitters in the central nervous system, causing spastic paralysis
Botulinum toxinClostridium botulinumInhibits release of the neurotransmitter acetylcholine from neurons, resulting in flaccid paralysis
Diphtheria toxinCorynebacterium diphtheriaeInhibition of protein synthesis, causing cellular death
Membrane-disrupting toxinsStreptolysinStreptococcus pyogenesProteins that assemble into pores in cell membranes, disrupting their function and killing the cell
PneumolysinStreptococcus pneumoniae
Alpha-toxinStaphylococcus aureus
Alpha-toxinClostridium perfringensPhospholipases that degrade cell membrane phospholipids, disrupting membrane function and killing the cell
Phospholipase CPseudomonas aeruginosa
Beta-toxinStaphylococcus aureus
SuperantigensToxic shock syndrome toxinStaphylococcus aureusStimulates excessive activation of immune system cells and release of cytokines (chemical mediators) from immune system cells. Life-threatening fever, inflammation, and shock are the result.
Streptococcal mitogenic exotoxinStreptococcus pyogenes
Streptococcal pyrogenic toxinsStreptococcus pyogenes

The intracellular targeting toxins comprise two components: A for activity and B for binding. Thus, these types of toxins are known as A-B exotoxins (Figure (PageIndex{5})). The B component is responsible for the cellular specificity of the toxin and mediates the initial attachment of the toxin to specific cell surface receptors. Once the A-B toxin binds to the host cell, it is brought into the cell by endocytosis and entrapped in a vacuole. The A and B subunits separate as the vacuole acidifies. The A subunit then enters the cell cytoplasm and interferes with the specific internal cellular function that it targets.

Four unique examples of A-B toxins are the diphtheria, cholera, botulinum, and tetanus toxins. The diphtheria toxin is produced by the gram-positive bacterium Corynebacterium diphtheriae, the causative agent of nasopharyngeal and cutaneous diphtheria. After the A subunit of the diphtheria toxin separates and gains access to the cytoplasm, it facilitates the transfer of adenosine diphosphate (ADP)-ribose onto an elongation-factor protein (EF-2) that is needed for protein synthesis. Hence, diphtheria toxin inhibits protein synthesis in the host cell, ultimately killing the cell (Figure (PageIndex{6})).

Cholera toxin is an enterotoxin produced by the gram-negative bacterium Vibrio cholerae and is composed of one A subunit and five B subunits. The mechanism of action of the cholera toxin is complex. The B subunits bind to receptors on the intestinal epithelial cell of the small intestine. After gaining entry into the cytoplasm of the epithelial cell, the A subunit activates an intracellular G protein. The activated G protein, in turn, leads to the activation of the enzyme adenyl cyclase, which begins to produce an increase in the concentration of cyclic AMP (a secondary messenger molecule). The increased cAMP disrupts the normal physiology of the intestinal epithelial cells and causes them to secrete excessive amounts of fluid and electrolytes into the lumen of the intestinal tract, resulting in severe “rice-water stool” diarrhea characteristic of cholera.

Botulinum toxin (also known as botox) is a neurotoxin produced by the gram-positive bacterium Clostridium botulinum. It is the most acutely toxic substance known to date. The toxin is composed of a light A subunit and heavy protein chain B subunit. The B subunit binds to neurons to allow botulinum toxin to enter the neurons at the neuromuscular junction. The A subunit acts as a protease, cleaving proteins involved in the neuron’s release of acetylcholine, a neurotransmitter molecule. Normally, neurons release acetylcholine to induce muscle fiber contractions. The toxin’s ability to block acetylcholine release results in the inhibition of muscle contractions, leading to muscle relaxation. This has the potential to stop breathing and cause death. Because of its action, low concentrations of botox are used for cosmetic and medical procedures, including the removal of wrinkles and treatment of overactive bladder.

Another neurotoxin is tetanus toxin, which is produced by the gram-positive bacterium Clostridium tetani. This toxin also has a light A subunit and heavy protein chain B subunit. Unlike botulinum toxin, tetanus toxin binds to inhibitory interneurons, which are responsible for release of the inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA). Normally, these neurotransmitters bind to neurons at the neuromuscular junction, resulting in the inhibition of acetylcholine release. Tetanus toxin inhibits the release of glycine and GABA from the interneuron, resulting in permanent muscle contraction. The first symptom is typically stiffness of the jaw (lockjaw). Violent muscle spasms in other parts of the body follow, typically culminating with respiratory failure and death. Figure (PageIndex{7}) shows the actions of both botulinum and tetanus toxins.

Membrane-disrupting toxins affect cell membrane function either by forming pores or by disrupting the phospholipid bilayer in host cell membranes. Two types of membrane-disrupting exotoxins are hemolysins and leukocidins, which form pores in cell membranes, causing leakage of the cytoplasmic contents and cell lysis. These toxins were originally thought to target red blood cells (erythrocytes) and white blood cells (leukocytes), respectively, but we now know they can affect other cells as well. The gram-positive bacterium Streptococcus pyogenes produces streptolysins, water-soluble hemolysins that bind to the cholesterol moieties in the host cell membrane to form a pore. The two types of streptolysins, O and S, are categorized by their ability to cause hemolysis in erythrocytes in the absence or presence of oxygen. Streptolysin O is not active in the presence of oxygen, whereas streptolysin S is active in the presence of oxygen. Other important pore-forming membrane-disrupting toxins include alpha toxin of Staphylococcus aureus and pneumolysin of Streptococcus pneumoniae.

Bacterial phospholipases are membrane-disrupting toxins that degrade the phospholipid bilayer of cell membranes rather than forming pores. We have already discussed the phospholipases associated with B. anthracis, L. pneumophila, and Rickettsia species that enable these bacteria to effect the lysis of phagosomes. These same phospholipases are also hemolysins. Other phospholipases that function as hemolysins include the alpha toxin of Clostridium perfringens, phospholipase C of P. aeruginosa, and beta toxin of Staphylococcus aureus.

Some strains of S. aureus also produce a leukocidin called Panton-Valentine leukocidin (PVL). PVL consists of two subunits, S and F. The S component acts like the B subunit of an A-B exotoxin in that it binds to glycolipids on the outer plasma membrane of animal cells. The F-component acts like the A subunit of an A-B exotoxin and carries the enzymatic activity. The toxin inserts and assembles into a pore in the membrane. Genes that encode PVL are more frequently present in S. aureus strains that cause skin infections and pneumonia.1 PVL promotes skin infections by causing edema, erythema (reddening of the skin due to blood vessel dilation), and skin necrosis. PVL has also been shown to cause necrotizing pneumonia. PVL promotes pro-inflammatory and cytotoxic effects on alveolar leukocytes. This results in the release of enzymes from the leukocytes, which, in turn, cause damage to lung tissue.

The third class of exotoxins is the superantigens. These are exotoxins that trigger an excessive, nonspecific stimulation of immune cells to secrete cytokines (chemical messengers). The excessive production of cytokines, often called a cytokine storm, elicits a strong immune and inflammatory response that can cause life-threatening high fevers, low blood pressure, multi-organ failure, shock, and death. The prototype superantigen is the toxic shock syndrome toxin of S. aureus. Most toxic shock syndrome cases are associated with vaginal colonization by toxin-producing S. aureus in menstruating women; however, colonization of other body sites can also occur. Some strains of Streptococcus pyogenes also produce superantigens; they are referred to as the streptococcal mitogenic exotoxins and the streptococcal pyrogenic toxins.

Exercise (PageIndex{2})

  1. Describe how exoenzymes contribute to bacterial invasion.
  2. Explain the difference between exotoxins and endotoxin.
  3. Name the three classes of exotoxins.

Virulence Factors for Survival in the Host and Immune Evasion

Evading the immune system is also important to invasiveness. Bacteria use a variety of virulence factors to evade phagocytosis by cells of the immune system. For example, many bacteria produce capsules, which are used in adhesion but also aid in immune evasion by preventing ingestion by phagocytes. The composition of the capsule prevents immune cells from being able to adhere and then phagocytose the cell. In addition, the capsule makes the bacterial cell much larger, making it harder for immune cells to engulf the pathogen (Figure (PageIndex{8})). A notable capsule-producing bacterium is the gram-positive pathogen Streptococcus pneumoniae, which causes pneumococcal pneumonia, meningitis, septicemia, and other respiratory tract infections. Encapsulated strains of S. pneumoniae are more virulent than nonencapsulated strains and are more likely to invade the bloodstream and cause septicemia and meningitis.

Some pathogens can also produce proteases to protect themselves against phagocytosis. As will be discussed later, the human immune system produces antibodies that bind to surface molecules found on specific bacteria (e.g., capsules, fimbriae, flagella, LPS). This binding initiates phagocytosis and other mechanisms of antibacterial killing and clearance. Proteases combat antibody-mediated killing and clearance by attacking and digesting the antibody molecules (Figure (PageIndex{8})).

In addition to capsules and proteases, some bacterial pathogens produce other virulence factors that allow them to evade the immune system. The fimbriae of certain species of Streptococcus contain M protein, which alters the surface of Streptococcus and inhibits phagocytosis by blocking the binding of the complement molecules that assist phagocytes in ingesting bacterial pathogens. The acid-fast bacterium Mycobacterium tuberculosis (the causative agent of tuberculosis) produces a waxy substance known as mycolic acid in its cell envelope. When it is engulfed by phagocytes in the lung, the protective mycolic acid coat enables the bacterium to resist some of the killing mechanisms within the phagolysosome.

Some bacteria produce virulence factors that promote infection by exploiting molecules naturally produced by the host. For example, most strains of Staphylococcus aureus produce the exoenzyme coagulase, which exploits the natural mechanism of blood clotting to evade the immune system. Normally, blood clotting is triggered in response to blood vessel damage; platelets begin to plug the clot, and a cascade of reactions occurs in which fibrinogen, a soluble protein made by the liver, is cleaved into fibrin. Fibrin is an insoluble, thread-like protein that binds to blood platelets, cross-links, and contracts to form a mesh of clumped platelets and red blood cells. The resulting clot prevents further loss of blood from the damaged blood vessels. However, if bacteria release coagulase into the bloodstream, the fibrinogen-to-fibrin cascade is triggered in the absence of blood vessel damage. The resulting clot coats the bacteria in fibrin, protecting the bacteria from exposure to phagocytic immune cells circulating in the bloodstream.

Whereas coagulase causes blood to clot, kinases have the opposite effect by triggering the conversion of plasminogen to plasmin, which is involved in the digestion of fibrin clots. By digesting a clot, kinases allow pathogens trapped in the clot to escape and spread, similar to the way that collagenase, hyaluronidase, and DNAse facilitate the spread of infection. Examples of kinases include staphylokinases and streptokinases, produced by Staphylococcus aureusand Streptococcus pyogenes, respectively. It is intriguing that S. aureus can produce both coagulase to promote clotting and staphylokinase to stimulate the digestion of clots. The action of the coagulase provides an important protective barrier from the immune system, but when nutrient supplies are diminished or other conditions signal a need for the pathogen to escape and spread, the production of staphylokinase can initiate this process.

A final mechanism that pathogens can use to protect themselves against the immune system is called antigenic variation, which is the alteration of surface proteins so that a pathogen is no longer recognized by the host’s immune system. For example, the bacterium Borrelia burgdorferi, the causative agent of Lyme disease, contains a surface lipoprotein known as VlsE. Because of genetic recombination during DNA replication and repair, this bacterial protein undergoes antigenic variation. Each time fever occurs, the VlsE protein in B. burgdorferi can differ so much that antibodies against previous VlsE sequences are not effective. It is believed that this variation in the VlsE contributes to the ability B. burgdorferi to cause chronic disease. Another important human bacterial pathogen that uses antigenic variation to avoid the immune system is Neisseria gonorrhoeae, which causes the sexually transmitted disease gonorrhea. This bacterium is well known for its ability to undergo antigenic variation of its type IV pili to avoid immune defenses.

Exercise (PageIndex{3})

  1. Name at least two ways that a capsule provides protection from the immune system.
  2. Besides capsules, name two other virulence factors used by bacteria to evade the immune system.

Eukaryote Virulence

Although fungi and parasites are important pathogens causing infectious diseases, their pathogenic mechanisms and virulence factors are not as well characterized as those of bacteria. Despite the relative lack of detailed mechanisms, the stages of pathogenesis and general mechanisms of virulence involved in disease production by these pathogens are similar to those of bacteria.

Fungal Virulence

Pathogenic fungi can produce virulence factors that are similar to the bacterial virulence factors that have been discussed earlier in this chapter. In this section, we will look at the virulence factors associated with species of Candida, Cryptococcus, Claviceps, and Aspergillus.

Candida albicans is an opportunistic fungal pathogen and causative agent of oral thrush, vaginal yeast infections, and cutaneous candidiasis. Candida produces adhesins (surface glycoproteins) that bind to the phospholipids of epithelial and endothelial cells. To assist in spread and tissue invasion, Candida produces proteases and phospholipases (i.e., exoenzymes). One of these proteases degrades keratin, a structural protein found on epithelial cells, enhancing the ability of the fungus to invade host tissue. In animal studies, it has been shown that the addition of a protease inhibitor led to attenuation of Candida infection.2 Similarly, the phospholipases can affect the integrity of host cell membranes to facilitate invasion.

The main virulence factor for Cryptococcus, a fungus that causes pneumonia and meningitis, is capsule production. The polysaccharide glucuronoxylomannan is the principal constituent of the Cryptococcus capsule. Similar to encapsulated bacterial cells, encapsulated Cryptococcus cells are more resistant to phagocytosis than nonencapsulated Cryptococcus, which are effectively phagocytosed and, therefore, less virulent.

Like some bacteria, many fungi produce exotoxins. Fungal toxins are called mycotoxins. Claviceps purpurea, a fungus that grows on rye and related grains, produces a mycotoxin called ergot toxin, an alkaloid responsible for the disease known as ergotism. There are two forms of ergotism: gangrenous and convulsive. In gangrenous ergotism, the ergot toxin causes vasoconstriction, resulting in improper blood flow to the extremities, eventually leading to gangrene. A famous outbreak of gangrenous ergotism occurred in Eastern Europe during the 5th century AD due to the consumption of rye contaminated with C. purpurea. In convulsive ergotism, the toxin targets the central nervous system, causing mania and hallucinations.

The mycotoxin aflatoxin is a virulence factor produced by the fungus Aspergillus, an opportunistic pathogen that can enter the body via contaminated food or by inhalation. Inhalation of the fungus can lead to the chronic pulmonary disease aspergillosis, characterized by fever, bloody sputum, and/or asthma. Aflatoxin acts in the host as both a mutagen (a substance that causes mutations in DNA) and a carcinogen (a substance involved in causing cancer), and has been associated with the development of liver cancer. Aflatoxin has also been shown to cross the blood-placental barrier.3 A second mycotoxin produced by Aspergillus is gliotoxin. This toxin promotes virulence by inducing host cells to self-destruct and by evading the host’s immune response by inhibiting the function of phagocytic cells as well as the pro-inflammatory response. Like Candida, Aspergillus also produces several proteases. One is elastase, which breaks down the protein elastin found in the connective tissue of the lung, leading to the development of lung disease. Another is catalase, an enzyme that protects the fungus from hydrogen peroxide produced by the immune system to destroy pathogens.

Exercise (PageIndex{4})

  1. List virulence factors common to bacteria and fungi.
  2. What functions do mycotoxins perform to help fungi survive in the host?

Protozoan Virulence

Protozoan pathogens are unicellular eukaryotic parasites that have virulence factors and pathogenic mechanisms analogous to prokaryotic and viral pathogens, including adhesins, toxins, antigenic variation, and the ability to survive inside phagocytic vesicles.

Protozoans often have unique features for attaching to host cells. The protozoan Giardia lamblia, which causes the intestinal disease giardiasis, uses a large adhesive disc composed of microtubules to attach to the intestinal mucosa. During adhesion, the flagella of G. lamblia move in a manner that draws fluid out from under the disc, resulting in an area of lower pressure that facilitates adhesion to epithelial cells. Giardia does not invade the intestinal cells but rather causes inflammation (possibly through the release of cytopathic substances that cause damage to the cells) and shortens the intestinal villi, inhibiting absorption of nutrients.

Some protozoans are capable of antigenic variation. The obligate intracellular pathogen Plasmodium falciparum (one of the causative agents of malaria) resides inside red blood cells, where it produces an adhesin membrane protein known as PfEMP1. This protein is expressed on the surface of the infected erythrocytes, causing blood cells to stick to each other and to the walls of blood vessels. This process impedes blood flow, sometimes leading to organ failure, anemia, jaundice (yellowing of skin and sclera of the eyes due to buildup of bilirubin from lysed red blood cells), and, subsequently, death. Although PfEMP1 can be recognized by the host’s immune system, antigenic variations in the structure of the protein over time prevent it from being easily recognized and eliminated. This allows malaria to persist as a chronic infection in many individuals.

The virulence factors of Trypanosoma brucei, the causative agent of African sleeping sickness, include the abilities to form capsules and undergo antigenic variation. T. brucei evades phagocytosis by producing a dense glycoprotein coat that resembles a bacterial capsule. Over time, host antibodies are produced that recognize this coat, but T. brucei is able to alter the structure of the glycoprotein to evade recognition.

Exercise (PageIndex{5})

Explain how antigenic variation by protozoan pathogens helps them survive in the host.

Helminth Virulence

Helminths, or parasitic worms (in the animal kingdom), are multicellular eukaryotic parasites that depend heavily on virulence factors that allow them to gain entry to host tissues. For example, the aquatic larval form of Schistosoma mansoni, which causes schistosomiasis, penetrates intact skin with the aid of proteases that degrade skin proteins, including elastin.

To survive within the host long enough to perpetuate their often-complex life cycles, helminths need to evade the immune system. Some helminths are so large that the immune system is ineffective against them. Others, such as adult roundworms (which cause trichinosis, ascariasis, and other diseases), are protected by a tough outer cuticle.

Over the course of their life cycles, the surface characteristics of the parasites vary, which may help prevent an effective immune response. Some helminths express polysaccharides called glycans on their external surface; because these glycans resemble molecules produced by host cells, the immune system fails to recognize and attack the helminth as a foreign body. This “glycan gimmickry,” as it has been called, serves as a protective cloak that allows the helminth to escape detection by the immune system.4

In addition to evading host defenses, helminths can actively suppress the immune system. mansoni, for example, degrades host antibodies with proteases. Helminths produce many other substances that suppress elements of both innate nonspecific and adaptive specific host defenses. They also release large amounts of material into the host that may locally overwhelm the immune system or cause it to respond inappropriately.

Exercise (PageIndex{6})

Describe how helminths avoid being destroyed by the host immune system.

Viral Virulence

Although viral pathogens are not similar to bacterial pathogens in terms of structure, some of the properties that contribute to their virulence are similar. Viruses use adhesins to facilitate adhesion to host cells, and certain enveloped viruses rely on antigenic variation to avoid the host immune defenses.

Viral Adhesins

One of the first steps in any viral infection is adhesion of the virus to specific receptors on the surface of cells. This process is mediated by adhesins that are part of the viral capsid or membrane envelope. The interaction of viral adhesins with specific cell receptors defines the tropism (preferential targeting) of viruses for specific cells, tissues, and organs in the body. The spike protein hemagglutinin found on Influenzavirus is an example of a viral adhesin; it allows the virus to bind to the sialic acid on the membrane of host respiratory and intestinal cells. Another viral adhesin is the glycoprotein gp20, found on HIV. For HIV to infect cells of the immune system, it must interact with two receptors on the surface of cells. The first interaction involves binding between gp120 and the CD4 cellular marker that is found on some essential immune system cells. However, before viral entry into the cell can occur, a second interaction between gp120 and one of two chemokine receptors (CCR5 and CXCR4) must occur. Table (PageIndex{5}) lists the adhesins for some common viral pathogens and the specific sites to which these adhesins allow viruses to attach.

Table (PageIndex{5}): Some Viral Adhesins and Their Host Attachment Sites
PathogenDiseaseAdhesinAttachment Site
InfluenzavirusInfluenzaHemagglutininSialic acid of respiratory and intestinal cells
Herpes simplex virus I or IIOral herpes, genital herpesGlycoproteins gB, gC, gDHeparan sulfate on mucosal surfaces of the mouth and genitals
Human immunodeficiency virusHIV/AIDSGlycoprotein gp120CD4 and CCR5 or CXCR4 of immune system cells

Antigenic Variation in Viruses

Antigenic variation also occurs in certain types of enveloped viruses, including influenza viruses, which exhibit two forms of antigenic variation: antigenic drift and antigenic shift (Figure (PageIndex{9})). Antigenic drift is the result of point mutations causing slight changes in the spike proteins hemagglutinin (H) and neuraminidase (N). On the other hand, antigenic shift is a major change in spike proteins due to gene reassortment. This reassortment for antigenic shift occurs typically when two different influenza viruses infect the same host.

The rate of antigenic variation in influenza viruses is very high, making it difficult for the immune system to recognize the many different strains of Influenzavirus. Although the body may develop immunity to one strain through natural exposure or vaccination, antigenic variation results in the continual emergence of new strains that the immune system will not recognize. This is the main reason that vaccines against Influenzavirus must be given annually. Each year’s influenza vaccine provides protection against the most prevalent strains for that year, but new or different strains may be more prevalent the following year.

For another explanation of how antigenic shift and drift occur, watch this video.

Exercise (PageIndex{7})

  1. Describe the role of adhesins in viral tropism.
  2. Explain the difference between antigenic drift and antigenic shift.

Key Concepts and Summary

  • Virulence factors contribute to a pathogen’s ability to cause disease.
  • Exoenzymes and toxins allow pathogens to invade host tissue and cause tissue damage. Exoenzymes are classified according to the macromolecule they target and exotoxins are classified based on their mechanism of action. Bacterial toxins include endotoxin and exotoxins.
  • Endotoxin is the lipid A component of the LPS of the gram-negative cell envelope. Exotoxins are proteins secreted mainly by gram-positive bacteria, but also are secreted by gram-negative bacteria.
  • Bacterial pathogens may evade the host immune response by producing capsules to avoid phagocytosis, surviving the intracellular environment of phagocytes, degrading antibodies, or through antigenic variation.
  • Fungal and parasitic pathogens use pathogenic mechanisms and virulence factors that are similar to those of bacterial pathogens
  • Fungi initiate infections through the interaction of adhesins with receptors on host cells. Some fungi produce toxins and exoenzymes involved in disease production and capsules that provide protection of phagocytosis.
  • Protozoa adhere to target cells through complex mechanisms and can cause cellular damage through release of cytopathic substances. Some protozoa avoid the immune system through antigenic variation and production of capsules.
  • Helminthic worms are able to avoid the immune system by coating their exteriors with glycan molecules that make them look like host cells or by suppressing the immune system.
  • Viral pathogens use adhesins for initiating infections and antigenic variation to avoid immune defenses. Influenza viruses use both antigenic drift and antigenic shift to avoid being recognized by the immune system.

Footnotes

  1. V. Meka. “Panton-Valentine Leukocidin.” http://www.antimicrobe.org/h04c.file...L-S-aureus.asp
  2. K. Fallon et al. “Role of Aspartic Proteases in Disseminated Candida albicans Infection in Mice.” Infection and Immunity 65 no. 2 (1997):551–556.
  3. C.P. Wild et al. “In-utero exposure to aflatoxin in west Africa.” Lancet 337 no. 8757 (1991):1602.
  4. I. van Die, R.D. Cummings. “Glycan Gimmickry by Parasitic Helminths: A Strategy for Modulating the Host Immune Response?” Glycobiology 20 no. 1 (2010):2–12.

Contributor

  • Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at https://openstax.org/books/microbiology/pages/1-introduction)


12.3 Modes of Disease Transmission

Understanding how infectious pathogens spread is critical to preventing infectious disease. Many pathogens require a living host to survive, while others may be able to persist in a dormant state outside of a living host. But having infected one host, all pathogens must also have a mechanism of transfer from one host to another or they will die when their host dies. Pathogens often have elaborate adaptations to exploit host biology, behavior, and ecology to live in and move between hosts. Hosts have evolved defenses against pathogens, but because their rates of evolution are typically slower than their pathogens (because their generation times are longer), hosts are usually at an evolutionary disadvantage. This section will explore where pathogens survive—both inside and outside hosts—and some of the many ways they move from one host to another.


15.3 Virulence Factors of Bacterial and Viral Pathogens

In the previous section, we explained that some pathogens are more virulent than others. This is due to the unique virulence factor s produced by individual pathogens, which determine the extent and severity of disease they may cause. A pathogen’s virulence factors are encoded by genes that can be identified using molecular Koch’s postulates. When genes encoding virulence factors are inactivated, virulence in the pathogen is diminished. In this section, we examine various types and specific examples of virulence factors and how they contribute to each step of pathogenesis.

Virulence Factors for Adhesion

As discussed in the previous section, the first two steps in pathogenesis are exposure and adhesion. Recall that an adhesin is a protein or glycoprotein found on the surface of a pathogen that attaches to receptors on the host cell. Adhesins are found on bacterial, viral, fungal, and protozoan pathogens. One example of a bacterial adhesin is type 1 fimbrial adhesin , a molecule found on the tips of fimbriae of enterotoxigenic E. coli ( ETEC ). Recall that fimbriae are hairlike protein bristles on the cell surface. Type 1 fimbrial adhesin allows the fimbriae of ETEC cells to attach to the mannose glycans expressed on intestinal epithelial cells. Table 15.7 lists common adhesins found in some of the pathogens we have discussed or will be seeing later in this chapter.

Some Bacterial Adhesins and Their Host Attachment Sites
Pathogen Disease Adhesin Attachment Site
Streptococcus pyogenes Strep throat Protein F Respiratory epithelial cells
Streptococcus mutans Dental caries Adhesin P1 Teeth
Neisseria gonorrhoeae Gonorrhea Type IV pili Urethral epithelial cells
Enterotoxigenic E. coli (ETEC) Traveler’s diarrhea Type 1 fimbriae Intestinal epithelial cells
Vibrio cholerae Cholera N-methylphenylalanine pili Intestinal epithelial cells

Clinical Focus

Part 3

The presence of bacteria in Michael’s blood is a sign of infection, since blood is normally sterile. There is no indication that the bacteria entered the blood through an injury. Instead, it appears the portal of entry was the gastrointestinal route. Based on Michael’s symptoms, the results of his blood test, and the fact that Michael was the only one in the family to partake of the hot dogs, the physician suspects that Michael is suffering from a case of listeriosis.

Listeria monocytogenes , the facultative intracellular pathogen that causes listeriosis, is a common contaminant in ready-to-eat foods such as lunch meats and dairy products. Once ingested, these bacteria invade intestinal epithelial cells and translocate to the liver, where they grow inside hepatic cells. Listeriosis is fatal in about one in five normal healthy people, and mortality rates are slightly higher in patients with pre-existing conditions that weaken the immune response. A cluster of virulence genes encoded on a pathogenicity island is responsible for the pathogenicity of L. monocytogenes. These genes are regulated by a transcriptional factor known as peptide chain release factor 1 (PrfA). One of the genes regulated by PrfA is hyl, which encodes a toxin known as listeriolysin O (LLO), which allows the bacterium to escape vacuoles upon entry into a host cell. A second gene regulated by PrfA is actA, which encodes for a surface protein known as actin assembly-inducing protein (ActA). ActA is expressed on the surface of Listeria and polymerizes host actin. This enables the bacterium to produce actin tails , move around the cell’s cytoplasm, and spread from cell to cell without exiting into the extracellular compartment.

Michael’s condition has begun to worsen. He is now experiencing a stiff neck and hemiparesis (weakness of one side of the body). Concerned that the infection is spreading, the physician decides to conduct additional tests to determine what is causing these new symptoms.

  • What kind of pathogen causes listeriosis, and what virulence factors contribute to the signs and symptoms Michael is experiencing?
  • Is it likely that the infection will spread from Michael’s blood? If so, how might this explain his new symptoms?

Jump to the next Clinical Focus box. Go back to the previous Clinical Focus box.

Bacterial Exoenzymes and Toxins as Virulence Factors

After exposure and adhesion, the next step in pathogenesis is invasion , which can involve enzymes and toxins. Many pathogens achieve invasion by entering the bloodstream, an effective means of dissemination because blood vessels pass close to every cell in the body. The downside of this mechanism of dispersal is that the blood also includes numerous elements of the immune system. Various terms ending in –emia are used to describe the presence of pathogens in the bloodstream. The presence of bacteria in blood is called bacteremia . Bacteremia involving pyogens (pus-forming bacteria) is called pyemia . When viruses are found in the blood, it is called viremia . The term toxemia describes the condition when toxins are found in the blood. If bacteria are both present and multiplying in the blood, this condition is called septicemia .

Patients with septicemia are described as septic , which can lead to shock , a life-threatening decrease in blood pressure (systolic pressure <90 mm Hg) that prevents cells and organs from receiving enough oxygen and nutrients. Some bacteria can cause shock through the release of toxins (virulence factors that can cause tissue damage) and lead to low blood pressure. Gram-negative bacteria are engulfed by immune system phagocytes, which then release tumor necrosis factor , a molecule involved in inflammation and fever. Tumor necrosis factor binds to blood capillaries to increase their permeability, allowing fluids to pass out of blood vessels and into tissues, causing swelling, or edema (Figure 15.10). With high concentrations of tumor necrosis factor, the inflammatory reaction is severe and enough fluid is lost from the circulatory system that blood pressure decreases to dangerously low levels. This can have dire consequences because the heart, lungs, and kidneys rely on normal blood pressure for proper function thus, multi-organ failure, shock, and death can occur.

Exoenzymes

Some pathogens produce extracellular enzymes, or exoenzyme s, that enable them to invade host cells and deeper tissues. Exoenzymes have a wide variety of targets. Some general classes of exoenzymes and associated pathogens are listed in Table 15.8. Each of these exoenzymes functions in the context of a particular tissue structure to facilitate invasion or support its own growth and defend against the immune system. For example, hyaluronidase S, an enzyme produced by pathogens like Staphylococcus aureus , Streptococcus pyogenes , and Clostridium perfringens , degrades the glycoside hyaluronan (hyaluronic acid), which acts as an intercellular cement between adjacent cells in connective tissue (Figure 15.11). This allows the pathogen to pass through the tissue layers at the portal of entry and disseminate elsewhere in the body (Figure 15.11).

Some Classes of Exoenzymes and Their Targets
Class Example Function
Glycohydrolases Hyaluronidase S in Staphylococcus aureus Degrades hyaluronic acid that cements cells together to promote spreading through tissues
Nucleases DNAse produced by S. aureus Degrades DNA released by dying cells (bacteria and host cells) that can trap the bacteria, thus promoting spread
Phospholipases Phospholipase C of Bacillus anthracis Degrades phospholipid bilayer of host cells, causing cellular lysis, and degrade membrane of phagosomes to enable escape into the cytoplasm
Proteases Collagenase in Clostridium perfringens Degrades collagen in connective tissue to promote spread

Pathogen-produced nucleases, such as DNAse produced by S. aureus, degrade extracellular DNA as a means of escape and spreading through tissue. As bacterial and host cells die at the site of infection, they lyse and release their intracellular contents. The DNA chromosome is the largest of the intracellular molecules, and masses of extracellular DNA can trap bacteria and prevent their spread. S. aureus produces a DNAse to degrade the mesh of extracellular DNA so it can escape and spread to adjacent tissues. This strategy is also used by S. aureus and other pathogens to degrade and escape webs of extracellular DNA produced by immune system phagocytes to trap the bacteria.

Enzymes that degrade the phospholipids of cell membranes are called phospholipases . Their actions are specific in regard to the type of phospholipids they act upon and where they enzymatically cleave the molecules. The pathogen responsible for anthrax , B. anthracis, produces phospholipase C. When B. anthracis is ingested by phagocytic cells of the immune system, phospholipase C degrades the membrane of the phagosome before it can fuse with the lysosome, allowing the pathogen to escape into the cytoplasm and multiply. Phospholipases can also target the membrane that encloses the phagosome within phagocytic cells. As described earlier in this chapter, this is the mechanism used by intracellular pathogens such as L. monocytogenes and Rickettsia to escape the phagosome and multiply within the cytoplasm of phagocytic cells. The role of phospholipases in bacterial virulence is not restricted to phagosomal escape. Many pathogens produce phospholipases that act to degrade cell membranes and cause lysis of target cells. These phospholipases are involved in lysis of red blood cells, white blood cells, and tissue cells.

Bacterial pathogens also produce various protein-digesting enzymes, or proteases. Proteases can be classified according to their substrate target (e.g., serine proteases target proteins with the amino acid serine) or if they contain metals in their active site (e.g., zinc metalloproteases contain a zinc ion, which is necessary for enzymatic activity).

One example of a protease that contains a metal ion is the exoenzyme collagenase . Collagenase digests collagen, the dominant protein in connective tissue. Collagen can be found in the extracellular matrix, especially near mucosal membranes, blood vessels, nerves, and in the layers of the skin. Similar to hyaluronidase, collagenase allows the pathogen to penetrate and spread through the host tissue by digesting this connective tissue protein. The collagenase produced by the gram-positive bacterium Clostridium perfringens , for example, allows the bacterium to make its way through the tissue layers and subsequently enter and multiply in the blood (septicemia). C. perfringens then uses toxins and a phospholipase to cause cellular lysis and necrosis. Once the host cells have died, the bacterium produces gas by fermenting the muscle carbohydrates. The widespread necrosis of tissue and accompanying gas are characteristic of the condition known as gas gangrene (Figure 15.12).

Link to Learning

Two types of cell death are apoptosis and necrosis. Visit this website to learn more about the differences between these mechanisms of cell death and their causes.

Toxins

In addition to exoenzymes, certain pathogens are able to produce toxin s, biological poisons that assist in their ability to invade and cause damage to tissues. The ability of a pathogen to produce toxins to cause damage to host cells is called toxigenicity .

Toxins can be categorized as endotoxins or exotoxins. The lipopolysaccharide (LPS) found on the outer membrane of gram-negative bacteria is called endotoxin (Figure 15.13). During infection and disease, gram-negative bacterial pathogens release endotoxin either when the cell dies, resulting in the disintegration of the membrane, or when the bacterium undergoes binary fission. The lipid component of endotoxin, lipid A , is responsible for the toxic properties of the LPS molecule. Lipid A is relatively conserved across different genera of gram-negative bacteria therefore, the toxic properties of lipid A are similar regardless of the gram-negative pathogen. In a manner similar to that of tumor necrosis factor, lipid A triggers the immune system’s inflammatory response (see Inflammation and Fever). If the concentration of endotoxin in the body is low, the inflammatory response may provide the host an effective defense against infection on the other hand, high concentrations of endotoxin in the blood can cause an excessive inflammatory response, leading to a severe drop in blood pressure, multi-organ failure, and death.

A classic method of detecting endotoxin is by using the Limulus amebocyte lysate (LAL) test . In this procedure, the blood cells (amebocytes) of the horseshoe crab (Limulus polyphemus) is mixed with a patient’s serum. The amebocytes will react to the presence of any endotoxin. This reaction can be observed either chromogenically (color) or by looking for coagulation (clotting reaction) to occur within the serum. An alternative method that has been used is an enzyme-linked immunosorbent assay ( ELISA ) that uses antibodies to detect the presence of endotoxin.

Unlike the toxic lipid A of endotoxin, exotoxin s are protein molecules that are produced by a wide variety of living pathogenic bacteria. Although some gram-negative pathogens produce exotoxins, the majority are produced by gram-positive pathogens. Exotoxins differ from endotoxin in several other key characteristics, summarized in Table 15.9. In contrast to endotoxin, which stimulates a general systemic inflammatory response when released, exotoxins are much more specific in their action and the cells they interact with. Each exotoxin targets specific receptors on specific cells and damages those cells through unique molecular mechanisms. Endotoxin remains stable at high temperatures, and requires heating at 121 °C (250 °F) for 45 minutes to inactivate. By contrast, most exotoxins are heat labile because of their protein structure, and many are denatured (inactivated) at temperatures above 41 °C (106 °F). As discussed earlier, endotoxin can stimulate a lethal inflammatory response at very high concentrations and has a measured LD50 of 0.24 mg/kg. By contrast, very small concentrations of exotoxins can be lethal. For example, botulinum toxin , which causes botulism , has an LD50 of 0.000001 mg/kg (240,000 times more lethal than endotoxin).

Comparison of Endotoxin and Exotoxins Produced by Bacteria
Characteristic Endotoxin Exotoxin
Source Gram-negative bacteria Gram-positive (primarily) and gram-negative bacteria
Composition Lipid A component of lipopolysaccharide Protein
Effect on host General systemic symptoms of inflammation and fever Specific damage to cells dependent upon receptor-mediated targeting of cells and specific mechanisms of action
Heat stability Heat stable Most are heat labile, but some are heat stable
LD50 High Low

The exotoxins can be grouped into three categories based on their target: intracellular targeting, membrane disrupting, and superantigens. Table 15.10 provides examples of well-characterized toxins within each of these three categories.

Some Common Exotoxins and Associated Bacterial Pathogens
Category Example Pathogen Mechanism and Disease
Intracellular-targeting toxins Cholera toxin Vibrio cholerae Activation of adenylate cyclase in intestinal cells, causing increased levels of cyclic adenosine monophosphate (cAMP) and secretion of fluids and electrolytes out of cell, causing diarrhea
Tetanus toxin Clostridium tetani Inhibits the release of inhibitory neurotransmitters in the central nervous system, causing spastic paralysis
Botulinum toxin Clostridium botulinum Inhibits release of the neurotransmitter acetylcholine from neurons, resulting in flaccid paralysis
Diphtheria toxin Corynebacterium diphtheriae Inhibition of protein synthesis, causing cellular death
Membrane-disrupting toxins Streptolysin Streptococcus pyogenes Proteins that assemble into pores in cell membranes, disrupting their function and killing the cell
Pneumolysin Streptococcus pneumoniae
Alpha-toxin Staphylococcus aureus
Alpha-toxin Clostridium perfringens Phospholipases that degrade cell membrane phospholipids, disrupting membrane function and killing the cell
Phospholipase C Pseudomonas aeruginosa
Beta-toxin Staphylococcus aureus
Superantigens Toxic shock syndrome toxin Staphylococcus aureus Stimulates excessive activation of immune system cells and release of cytokines (chemical mediators) from immune system cells. Life-threatening fever, inflammation, and shock are the result.
Streptococcal mitogenic exotoxin Streptococcus pyogenes
Streptococcal pyrogenic toxins Streptococcus pyogenes

The intracellular targeting toxin s comprise two components: A for activity and B for binding. Thus, these types of toxins are known as A-B exotoxins (Figure 15.14). The B component is responsible for the cellular specificity of the toxin and mediates the initial attachment of the toxin to specific cell surface receptors. Once the A-B toxin binds to the host cell, it is brought into the cell by endocytosis and entrapped in a vacuole. The A and B subunits separate as the vacuole acidifies. The A subunit then enters the cell cytoplasm and interferes with the specific internal cellular function that it targets.

Four unique examples of A-B toxins are the diphtheria, cholera, botulinum, and tetanus toxins. The diphtheria toxin is produced by the gram-positive bacterium Corynebacterium diphtheriae , the causative agent of nasopharyngeal and cutaneous diphtheria. After the A subunit of the diphtheria toxin separates and gains access to the cytoplasm, it facilitates the transfer of adenosine diphosphate (ADP)-ribose onto an elongation-factor protein (EF-2) that is needed for protein synthesis. Hence, diphtheria toxin inhibits protein synthesis in the host cell, ultimately killing the cell (Figure 15.15).

Cholera toxin is an enterotoxin produced by the gram-negative bacterium Vibrio cholerae and is composed of one A subunit and five B subunits. The mechanism of action of the cholera toxin is complex. The B subunits bind to receptors on the intestinal epithelial cell of the small intestine. After gaining entry into the cytoplasm of the epithelial cell, the A subunit activates an intracellular G protein. The activated G protein, in turn, leads to the activation of the enzyme adenyl cyclase, which begins to produce an increase in the concentration of cyclic AMP (a secondary messenger molecule). The increased cAMP disrupts the normal physiology of the intestinal epithelial cells and causes them to secrete excessive amounts of fluid and electrolytes into the lumen of the intestinal tract, resulting in severe “rice-water stool” diarrhea characteristic of cholera.

Botulinum toxin (also known as botox ) is a neurotoxin produced by the gram-positive bacterium Clostridium botulinum . It is the most acutely toxic substance known to date. The toxin is composed of a light A subunit and heavy protein chain B subunit. The B subunit binds to neurons to allow botulinum toxin to enter the neurons at the neuromuscular junction. The A subunit acts as a protease, cleaving proteins involved in the neuron’s release of acetylcholine , a neurotransmitter molecule. Normally, neurons release acetylcholine to induce muscle fiber contractions. The toxin’s ability to block acetylcholine release results in the inhibition of muscle contractions, leading to muscle relaxation. This has the potential to stop breathing and cause death. Because of its action, low concentrations of botox are used for cosmetic and medical procedures, including the removal of wrinkles and treatment of overactive bladder.

Link to Learning

Click this link to see an animation of how the cholera toxin functions.

Click this link to see an animation of how the botulinum toxin functions.

Another neurotoxin is tetanus toxin , which is produced by the gram-positive bacterium Clostridium tetani . This toxin also has a light A subunit and heavy protein chain B subunit. Unlike botulinum toxin, tetanus toxin binds to inhibitory interneurons, which are responsible for release of the inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA). Normally, these neurotransmitters bind to neurons at the neuromuscular junction, resulting in the inhibition of acetylcholine release. Tetanus toxin inhibits the release of glycine and GABA from the interneuron, resulting in permanent muscle contraction. The first symptom is typically stiffness of the jaw (lockjaw). Violent muscle spasms in other parts of the body follow, typically culminating with respiratory failure and death. Figure 15.16 shows the actions of both botulinum and tetanus toxins.

Membrane-disrupting toxins affect cell membrane function either by forming pores or by disrupting the phospholipid bilayer in host cell membranes. Two types of membrane-disrupting exotoxins are hemolysin s and leukocidins , which form pores in cell membranes, causing leakage of the cytoplasmic contents and cell lysis. These toxins were originally thought to target red blood cells (erythrocytes) and white blood cells (leukocytes), respectively, but we now know they can affect other cells as well. The gram-positive bacterium Streptococcus pyogenes produces streptolysins , water-soluble hemolysins that bind to the cholesterol moieties in the host cell membrane to form a pore. The two types of streptolysins, O and S, are categorized by their ability to cause hemolysis in erythrocytes in the absence or presence of oxygen. Streptolysin O is not active in the presence of oxygen, whereas streptolysin S is active in the presence of oxygen. Other important pore-forming membrane-disrupting toxins include alpha toxin of Staphylococcus aureus and pneumolysin of Streptococcus pneumoniae .

Bacterial phospholipases are membrane-disrupting toxin s that degrade the phospholipid bilayer of cell membranes rather than forming pores. We have already discussed the phospholipases associated with B. anthracis, L. pneumophila, and Rickettsia species that enable these bacteria to effect the lysis of phagosomes. These same phospholipases are also hemolysins. Other phospholipases that function as hemolysins include the alpha toxin of Clostridium perfringens , phospholipase C of P. aeruginosa, and beta toxin of Staphylococcus aureus.

Some strains of S. aureus also produce a leukocidin called Panton-Valentine leukocidin (PVL) . PVL consists of two subunits, S and F. The S component acts like the B subunit of an A-B exotoxin in that it binds to glycolipids on the outer plasma membrane of animal cells. The F-component acts like the A subunit of an A-B exotoxin and carries the enzymatic activity. The toxin inserts and assembles into a pore in the membrane. Genes that encode PVL are more frequently present in S. aureus strains that cause skin infections and pneumonia. 8 PVL promotes skin infections by causing edema, erythema (reddening of the skin due to blood vessel dilation), and skin necrosis. PVL has also been shown to cause necrotizing pneumonia. PVL promotes pro-inflammatory and cytotoxic effects on alveolar leukocytes. This results in the release of enzymes from the leukocytes, which, in turn, cause damage to lung tissue.

The third class of exotoxins is the superantigen s. These are exotoxins that trigger an excessive, nonspecific stimulation of immune cells to secrete cytokines (chemical messengers). The excessive production of cytokines, often called a cytokine storm , elicits a strong immune and inflammatory response that can cause life-threatening high fevers, low blood pressure, multi-organ failure, shock, and death. The prototype superantigen is the toxic shock syndrome toxin of S. aureus. Most toxic shock syndrome cases are associated with vaginal colonization by toxin-producing S. aureus in menstruating women however, colonization of other body sites can also occur. Some strains of Streptococcus pyogenes also produce superantigens they are referred to as the streptococcal mitogenic exotoxins and the streptococcal pyrogenic toxins .

Check Your Understanding

  • Describe how exoenzymes contribute to bacterial invasion.
  • Explain the difference between exotoxins and endotoxin.
  • Name the three classes of exotoxins.

Virulence Factors for Survival in the Host and Immune Evasion

Evading the immune system is also important to invasiveness. Bacteria use a variety of virulence factors to evade phagocytosis by cells of the immune system. For example, many bacteria produce capsules , which are used in adhesion but also aid in immune evasion by preventing ingestion by phagocytes. The composition of the capsule prevents immune cells from being able to adhere and then phagocytose the cell. In addition, the capsule makes the bacterial cell much larger, making it harder for immune cells to engulf the pathogen (Figure 15.17). A notable capsule-producing bacterium is the gram-positive pathogen Streptococcus pneumoniae , which causes pneumococcal pneumonia, meningitis, septicemia, and other respiratory tract infections. Encapsulated strains of S. pneumoniae are more virulent than nonencapsulated strains and are more likely to invade the bloodstream and cause septicemia and meningitis.

Some pathogens can also produce proteases to protect themselves against phagocytosis. As described in Adaptive Specific Host Defenses, the human immune system produces antibodies that bind to surface molecules found on specific bacteria (e.g., capsules , fimbriae , flagella , LPS ). This binding initiates phagocytosis and other mechanisms of antibacterial killing and clearance. Proteases combat antibody-mediated killing and clearance by attacking and digesting the antibody molecules (Figure 15.17).

In addition to capsules and proteases, some bacterial pathogens produce other virulence factors that allow them to evade the immune system. The fimbriae of certain species of Streptococcus contain M protein , which alters the surface of Streptococcus and inhibits phagocytosis by blocking the binding of the complement molecules that assist phagocytes in ingesting bacterial pathogens. The acid-fast bacterium Mycobacterium tuberculosis (the causative agent of tuberculosis ) produces a waxy substance known as mycolic acid in its cell envelope. When it is engulfed by phagocytes in the lung, the protective mycolic acid coat enables the bacterium to resist some of the killing mechanisms within the phagolysosome.

Some bacteria produce virulence factors that promote infection by exploiting molecules naturally produced by the host. For example, most strains of Staphylococcus aureus produce the exoenzyme coagulase , which exploits the natural mechanism of blood clotting to evade the immune system. Normally, blood clotting is triggered in response to blood vessel damage platelets begin to plug the clot, and a cascade of reactions occurs in which fibrinogen, a soluble protein made by the liver, is cleaved into fibrin . Fibrin is an insoluble, thread-like protein that binds to blood platelets, cross-links, and contracts to form a mesh of clumped platelets and red blood cells. The resulting clot prevents further loss of blood from the damaged blood vessels. However, if bacteria release coagulase into the bloodstream, the fibrinogen-to-fibrin cascade is triggered in the absence of blood vessel damage. The resulting clot coats the bacteria in fibrin, protecting the bacteria from exposure to phagocytic immune cells circulating in the bloodstream.

Whereas coagulase causes blood to clot, kinases have the opposite effect by triggering the conversion of plasminogen to plasmin, which is involved in the digestion of fibrin clots. By digesting a clot, kinases allow pathogens trapped in the clot to escape and spread, similar to the way that collagenase, hyaluronidase, and DNAse facilitate the spread of infection. Examples of kinases include staphylokinases and streptokinases , produced by Staphylococcus aureus and Streptococcus pyogenes , respectively. It is intriguing that S. aureus can produce both coagulase to promote clotting and staphylokinase to stimulate the digestion of clots. The action of the coagulase provides an important protective barrier from the immune system, but when nutrient supplies are diminished or other conditions signal a need for the pathogen to escape and spread, the production of staphylokinase can initiate this process.

A final mechanism that pathogens can use to protect themselves against the immune system is called antigenic variation , which is the alteration of surface proteins so that a pathogen is no longer recognized by the host’s immune system. For example, the bacterium Borrelia burgdorferi , the causative agent of Lyme disease , contains a surface lipoprotein known as VlsE . Because of genetic recombination during DNA replication and repair, this bacterial protein undergoes antigenic variation. Each time fever occurs, the VlsE protein in B. burgdorferi can differ so much that antibodies against previous VlsE sequences are not effective. It is believed that this variation in the VlsE contributes to the ability B. burgdorferi to cause chronic disease. Another important human bacterial pathogen that uses antigenic variation to avoid the immune system is Neisseria gonorrhoeae , which causes the sexually transmitted disease gonorrhea . This bacterium is well known for its ability to undergo antigenic variation of its type IV pili to avoid immune defenses.

Check Your Understanding

  • Name at least two ways that a capsule provides protection from the immune system.
  • Besides capsules, name two other virulence factors used by bacteria to evade the immune system.

Clinical Focus

Resolution

Based on Michael’s reported symptoms of stiff neck and hemiparesis, the physician suspects that the infection may have spread to his nervous system. The physician decides to order a spinal tap to look for any bacteria that may have invaded the meninges and cerebrospinal fluid (CSF), which would normally be sterile. To perform the spinal tap, Michael’s lower back is swabbed with an iodine antiseptic and then covered with a sterile sheet. The needle is aseptically removed from the manufacturer’s sealed plastic packaging by the clinician’s gloved hands. The needle is inserted and a small volume of fluid is drawn into an attached sample tube. The tube is removed, capped and a prepared label with Michael’s data is affixed to it. This STAT (urgent or immediate analysis required) specimen is divided into three separate sterile tubes, each with 1 mL of CSF. These tubes are immediately taken to the hospital’s lab, where they are analyzed in the clinical chemistry, hematology, and microbiology departments. The preliminary results from all three departments indicate there is a cerebrospinal infection occurring, with the microbiology department reporting the presence of a gram-positive rod in Michael’s CSF.

These results confirm what his physician had suspected: Michael’s new symptoms are the result of meningitis , acute inflammation of the membranes that protect the brain and spinal cord. Because meningitis can be life threatening and because the first antibiotic therapy was not effective in preventing the spread of infection, Michael is prescribed an aggressive course of two antibiotics, ampicillin and gentamicin, to be delivered intravenously. Michael remains in the hospital for several days for supportive care and for observation. After a week, he is allowed to return home for bed rest and oral antibiotics. After 3 weeks of this treatment, he makes a full recovery.

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Viral Virulence

Although viral pathogens are not similar to bacterial pathogens in terms of structure, some of the properties that contribute to their virulence are similar. Viruses use adhesins to facilitate adhesion to host cells, and certain enveloped viruses rely on antigenic variation to avoid the host immune defenses. These virulence factors are discussed in more detail in the following sections.

Viral Adhesins

One of the first steps in any viral infection is adhesion of the virus to specific receptors on the surface of cells. This process is mediated by adhesins that are part of the viral capsid or membrane envelope . The interaction of viral adhesins with specific cell receptors defines the tropism (preferential targeting) of viruses for specific cells, tissues, and organs in the body. The spike protein hemagglutinin found on Influenzavirus is an example of a viral adhesin it allows the virus to bind to the sialic acid on the membrane of host respiratory and intestinal cells. Another viral adhesin is the glycoprotein gp20, found on HIV . For HIV to infect cells of the immune system, it must interact with two receptors on the surface of cells. The first interaction involves binding between gp120 and the CD4 cellular marker that is found on some essential immune system cells. However, before viral entry into the cell can occur, a second interaction between gp120 and one of two chemokine receptors (CCR5 and CXCR4) must occur. Table 15.11 lists the adhesins for some common viral pathogens and the specific sites to which these adhesins allow viruses to attach.

Some Viral Adhesins and Their Host Attachment Sites
Pathogen Disease Adhesin Attachment Site
Influenzavirus Influenza Hemagglutinin Sialic acid of respiratory and intestinal cells
Herpes simplex virus I or II Oral herpes, genital herpes Glycoproteins gB, gC, gD Heparan sulfate on mucosal surfaces of the mouth and genitals
Human immunodeficiency virus HIV/AIDS Glycoprotein gp120 CD4 and CCR5 or CXCR4 of immune system cells

Antigenic Variation in Viruses

Antigenic variation also occurs in certain types of enveloped viruses, including influenza viruses, which exhibit two forms of antigenic variation: antigenic drift and antigenic shift (Figure 15.18). Antigenic drift is the result of point mutations causing slight changes in the spike proteins hemagglutinin (H) and neuraminidase (N). On the other hand, antigenic shift is a major change in spike proteins due to gene reassortment. This reassortment for antigenic shift occurs typically when two different influenza viruses infect the same host.

The rate of antigenic variation in influenza viruses is very high, making it difficult for the immune system to recognize the many different strains of Influenzavirus. Although the body may develop immunity to one strain through natural exposure or vaccination, antigenic variation results in the continual emergence of new strains that the immune system will not recognize. This is the main reason that vaccines against Influenzavirus must be given annually. Each year’s influenza vaccine provides protection against the most prevalent strains for that year, but new or different strains may be more prevalent the following year.

Link to Learning

For another explanation of how antigenic shift and drift occur, watch this video.


References

Lowy, F.D. Staphylococcus aureus infections. N. Engl. J. Med. 339, 520–532 (1998).

DeLeo, F.R. & Chambers, H.F. Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era. J. Clin. Invest. 119, 2464–2474 (2009).

Klein, E., Smith, D.L. & Laxminarayan, R. Hospitalizations and deaths caused by methicillin-resistant Staphylococcus aureus, United States, 1999-2005. Emerg. Infect. Dis. 13, 1840–1846 (2007).

Arias, C.A. & Murray, B.E. Antibiotic-resistant bugs in the 21st century—a clinical super-challenge. N. Engl. J. Med. 360, 439–443 (2009).

Kloos, W.E. & Jorgensen, J.H. Staphylococci. in Manual of Clinical Microbiology edn. 4 (eds. Lennette, E.H., Balows, A., Hausler Jr., W.J. & Shadomy, H.J.) 143–153 (American Society for Microbiology, Washington, DC, 1985).

Liu, C.I. et al. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence. Science 319, 1391–1394 (2008).

Clauditz, A., Resch, A., Wieland, K.P., Peschel, A. & Götz, F. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infect. Immun. 74, 4950–4953 (2006).

Liu, G.Y. et al. Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. J. Exp. Med. 202, 209–215 (2005).

Pelz, A. et al. Structure and biosynthesis of staphyloxanthin from Staphylococcus aureus. J. Biol. Chem. 280, 32493–32498 (2005).

Kim, S.H. & Lee, P.C. Functional expression and extension of staphylococcal staphyloxanthin biosynthetic pathway in Escherichia coli. J. Biol. Chem. 287, 21575–21583 (2012).

Bischoff, M. & Berger-Bächi, B. Teicoplanin stress-selected mutations increasing sigma(B) activity in Staphylococcus aureus. Antimicrob. Agents Chemother. 45, 1714–1720 (2001).

Palma, M. & Cheung, A.L. sigma(B) activity in Staphylococcus aureus is controlled by RsbU and an additional factor(s) during bacterial growth. Infect. Immun. 69, 7858–7865 (2001).

Lan, L., Cheng, A., Dunman, P.M., Missiakas, D. & He, C. Golden pigment production and virulence gene expression are affected by metabolisms in Staphylococcus aureus. J. Bacteriol. 192, 3068–3077 (2010).

Fey, P.D. et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio 4, e00537–12 (2013).

Ding, Y. et al. Metabolic sensor governing bacterial virulence in Staphylococcus aureus. Proc. Natl. Acad. Sci. USA 111, E4981–E4990 (2014).

Liu, Y. et al. SsrA (tmRNA) acts as an antisense RNA to regulate Staphylococcus aureus pigment synthesis by base pairing with crtMN mRNA. FEBS Lett. 584, 4325–4329 (2010).

Duthie, E.S. & Lorenz, L.L. Staphylococcal coagulase mode of action and antigenicity. J. Gen. Microbiol. 6, 95–107 (1952).

Porretta, G.C. et al. Antifungal agents, Part 11. Biphenyl analogues of naftifine: synthesis and antifungal activities. Arch. Pharm. (Weinheim) 328, 667–672 (1995).

Gupta, A.K., Ryder, J.E. & Cooper, E.A. Naftifine: a review. J. Cutan. Med. Surg. 12, 51–58 (2008).

Wieland, B. et al. Genetic and biochemical analyses of the biosynthesis of the yellow carotenoid 4,4′-diaponeurosporene of Staphylococcus aureus. J. Bacteriol. 176, 7719–7726 (1994).

Russell, R.G. Ibandronate: pharmacology and preclinical studies. Bone 38 (suppl. 1), S7–S12 (2006).

Leejae, S., Hasap, L. & Voravuthikunchai, S.P. Inhibition of staphyloxanthin biosynthesis in Staphylococcus aureus by rhodomyrtone, a novel antibiotic candidate. J. Med. Microbiol. 62, 421–428 (2013).

Pantoliano, M.W. et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J. Biomol. Screen. 6, 429–440 (2001).

Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).

Raisig, A. & Sandmann, G. 4,4′-diapophytoene desaturase: catalytic properties of an enzyme from the C30 carotenoid pathway of Staphylococcus aureus. J. Bacteriol. 181, 6184–6187 (1999).

Furubayashi, M., Li, L., Katabami, A., Saito, K. & Umeno, D. Construction of carotenoid biosynthetic pathways using squalene synthase. FEBS Lett. 588, 436–442 (2014).

David, M.Z. & Daum, R.S. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin. Microbiol. Rev. 23, 616–687 (2010).

Kuroda, M. et al. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 357, 1225–1240 (2001).

El-Gohary, M. et al. Topical antifungal treatments for tinea cruris and tinea corporis. Cochrane Database Syst. Rev. 8, CD009992 (2014).

Belter, A. et al. Squalene monooxygenase—a target for hypercholesterolemic therapy. Biol. Chem. 392, 1053–1075 (2011).

Volkman, J.K. Sterols in microorganisms. Appl. Microbiol. Biotechnol. 60, 495–506 (2003).

Alsterholm, M., Karami, N. & Faergemann, J. Antimicrobial activity of topical skin pharmaceuticals—an in vitro study. Acta Derm. Venereol. 90, 239–245 (2010).

Nolting, S. & Bräutigam, M. Clinical relevance of the antibacterial activity of terbinafine: a contralateral comparison between 1% terbinafine cream and 0.1% gentamicin sulphate cream in pyoderma. Br. J. Dermatol. 126 (suppl. 39), 56–60 (1992).

Ciftci, E., Guriz, H. & Aysev, A.D. Mupirocin vs terbinafine in impetigo. Indian J. Pediatr. 69, 679–682 (2002).

Koning, S. et al. Interventions for impetigo. Cochrane Database Syst. Rev. 1, CD003261 (2012).

Hammond, R.K. & White, D.C. Inhibition of vitamin K2 and carotenoid synthesis in Staphylococcus aureus by diphenylamine. J. Bacteriol. 103, 611–615 (1970).

Raisig, A. & Sandmann, G. Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways. Biochim. Biophys. Acta 1533, 164–170 (2001).

Bae, T. et al. Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing. Proc. Natl. Acad. Sci. USA 101, 12312–12317 (2004).

Schaub, P. et al. On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase. PLoS One 7, e39550 (2012).

Lineweaver, H. & Burk, D. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658–666 (1934).


General Overviews

One could argue that virulence evolution is a field in which there almost are more reviews than research articles. The place to start is probably Ewald 1994, which summarizes the author’s view on adaptiveness for virulence. However, Zimmer 2003 is accessible to a wider audience. Some reviews offer a focus on specific aspects Bull 1994, for example, discusses virulence from the point of view of a molecular biologist, whereas Ebert and Herre 1996 focuses on transmission model and genetic constraints. Frank 1996 stands out as one of the first reviews of mathematical models for virulence evolution. Frank also discusses kin selection issues, a topic also raised in Chao, et al. 2000, but from a more experimental side. Mackinnon and Read 2004 is interesting because it analyses virulence evolution through works performed on a single parasite, Plasmodium. Finally, Schmid-Hempel 2011 is very detailed and contains a rich bibliography on the topic. In fact, many of the topics dealt with in this review are also discussed by Schmid-Hempel.

Bull, J. J. 1994. Virulence. Evolution 48.5: 1423–1437.

This general review detail the various explanations as to why pathogens harm their host, with a special emphasis on levels of selection. Available online for purchase or by subscription.

Chao, L., K. A. Hanley, C. L. Burch, C. Dahlberg, and P. E. Turner. 2000. Kin selection and parasite evolution: Higher and lower virulence with hard and soft selection. Quarterly Review of Biology 75.3: 261–275.

A review that focuses more specifically on how kin selection can help us understand virulence evolution, particularly the context of genetically diverse infections, where the relatedness among coinfecting parasites can vary. Available online for purchase or by subscription.

Ebert, D., and E. A. Herre. 1996. The evolution of parasitic diseases. Parasitology Today 12.3: 96–101.

A general review on virulence evolution, with a particular emphasis on genetic constraints and vertical transmission. Available online for purchase or by subscription.

Ewald, P. W. 1994. Evolution of infectious disease. Oxford: Oxford Univ. Press.

This book is one of the most accessible publications on the topic. Paul Ewald advocates his idea that virulence evolution can be understood and directed. The fact that this is a single-author book makes it easier to read. Note that a shorter version of his argument was published in 1993 in Scientific American.

Frank, S. A. 1996. Models of parasite virulence. Quarterly Review of Biology 71.1: 37–78.

This long review puts a special emphasis on mathematical approaches to virulence evolution, especially that based on kin selection theory. It also provides the reader with a detailed overview of the theory, and remains one of the most informative reviews. Available online for purchase or by subscription.

Mackinnon, M. J., and A. F. Read. 2004. Virulence in malaria: An evolutionary viewpoint. Philosophical Transactions of the Royal Society B: Biological Sciences 359.1446: 965–986.

A review focused on the case of malaria, which is one of the most studied organisms in this context. It illustrates how these general questions on virulence evolution can be applied to the case of a specific parasite.

Schmid-Hempel, P. 2011. Evolutionary parasitology: The integrated study of infections, immunology, ecology, and genetics. Oxford: Oxford Univ. Press.

A recent book focused on parasite evolution. Virulence evolution has a central place in it, and the chapter is very pedagogical, with helpful boxes. This is probably the first place to look for a detailed, recent, and clear overview of the topic. It is also the best place to find an extensive bibliography on the topic.

Zimmer, C. 2003. Taming pathogens: An elegant idea, but does it work? Science 300.5624: 1362–1364.

An article by science writer Carl Zimmer about the feasibility of controlling virulence evolution. An entertaining reading for a wide audience. Available online for purchase or by subscription.

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References

  1. ↑Assessment Technologies Institute of Nursing Education. “Wound Care: The anatomy and physiology of wound healing.”
  2. ↑Dowd, Scot E., Randall D. Wolcott, Yan Sun, Trevor McKeehan, Ethan Smith, and Daniel Rhoads. “Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP).” 2008. PLoS ONE 3(10): e3326. DOI: 10.1371/journal.pone.0003326.
  3. ↑Paddock, Catharine. “Bacteria living on skin may affect how wounds heal.” May 2, 2014. Medical News Today.
  4. ↑ 4.04.1Watters, Chase, Katrina DeLeon, Urvish Trivedi, John A. Griswold, Mark Lyte, Ken J. Hampel, Matthew J. Wargo, and Kendra P. Rumbaugh. “Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice.” 2013. Medical Microbiology and Immunology 202: 131-141. DOI: 10.1007/s00430-012-0277-7.
  5. ↑“IDF Diabetes Atlas Executive Summary.” 7th edition. 2015. International Diabetes Federation.
  6. ↑ 6.06.1Attinger, Christopher, and Randy Wolcott. “Clinically Addressing Biofilm in Chronic Wounds.” 2011. Advances in Wound Care, 1(3): 127-132. DOI: 10.1089/wound.2011.0333.
  7. ↑ 7.07.17.2Watters, Chase, Jake A. Everett, Cecily Haley, Allie Clinton, and Kendra P. Rumbaugh. “Insulin Treatment Modulates the Host Immune System To Enhance Pseudomonas aeruginosa Wound Biofilms.” 2014. Infection and Immunity 82(1): 92-100. DOI: 10.1128/IAI.00651-13.
  8. ↑ 8.08.1 Slonczewski, Joan L., and John W. Foster. Microbiology: An Evolving Science Chapter 4 Bacterial Culture, Growth, and Development. 2013.
  9. ↑ 9.09.1Joo, Hwang-Soo and Michael Otto. “Molecular basis of In Vivo Biofilm Formation by Bacterial Pathogens.” 2012. Chemistry & Biology 19: 1503-1513. DOI: 10.1016/j.chembiol/2012.10.022.
  10. ↑Ventolini, Gary. “Vaginal Lactobacillus: biofilm formation in vivo – clinical implications.” 2015. International Journal of Women’s Health 7: 243-247.
  11. ↑Donlan, Rodney M. “Biofilm Elimination on Intravascular Catheters: Important Considerations for the Infectious Disease Practitioner.” 2011. Clinical Infectious Diseases 52(8): 1038-1045. DOI: 10.1093/cid/cir077.
  12. ↑ 12.012.112.212.312.4Tuttle, Marie S., Eliot Mostow, Pranab Mukherjee, Fen Z. Hu, Rachael melton-Kreft, Garth D. Ehrlich, Scot E. Dowd, and Mohmoud A. Ghannoum. “Characterization of Bacterial Communities in Venous Insufficiency Wounds by Use of Conventional Culture and Molecular Diagnostic Methods.” 2011. Journal of Clinical Microbiology 49(11): 3812-3819. DOI: 10.1128/JCM.00847-11.
  13. ↑Percival, Steven L., and Philip G. Bowler. “Biofilms and Their Potential Role in Wound Healing.” 2004. Wounds 16(7).
  14. ↑Hill, Katja E., Sladjana Malic, Ruth McKee, Tracy Rennison, Keith G. Harding, David W. Williams, and David W. Thomas. “An in vitro model of chronic wound biofilms to test wound dressings and assess antimicrobial susceptibilities.” 2010. Journal of Antimicrobial Chemotherapy: 1-12. DOI: 10.1093/jac/dkq105.
  15. ↑ 15.015.1Bertesteanu, Serban, Stefanos Triaridis, Milan Stankovic, Veronica Lazar, Mariana Carmen Chifiriuc, Mihaela Vlad, and Raluca Grigore. “Polymicrobial wound infections: Pathophysiology and current therapeutic approaches.” 2014. International Journal of Pharmaceutics 463: 119-126. DOI: 10.1016/j.ijpharm.2013.12.012.
  16. ↑Dowd, Scot E., Randall D. Wolcott, Yan Sun, Trevor McKeehan, Ethan Smith, and Daniel Rhoads. “Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP).” 2008. PLoS ONE 3(10): e3326. DOI: 10.1371/journal.pone.0003326.
  17. ↑“Skin Bacteria Could Aid Wound Healing.” Advanced Tissue, May 21, 2014.
  18. ↑Jefferson, Kimberly K. “What drives bacteria to produce a biofilm?” 2004. FEMS Microbiology Letters 236(2): 163-173. DOI: 10.1111/j.1574-6968.2004.tb09643.x
  19. ↑Ito, Akinobu, Asami Taniuchi, Thithiwat May, Koji Kawata and Satoshi Okabe. “Increased Antibiotic Resistance of Escherichia coli in Mature Biofilms.” 2009. Applied and Environmental Microbiology 75(12): 4093-4100. DOI: 10.1128/AEM.02949-08.
  20. ↑Ryall, Ben, Marta Carrara, James E. A. Zlosnik, Volker Behrends, Xiaoyun Lee, Zhen Wong, Kathryn E. Lougheed, and Huw D. Williams. “The Mucoid Switch in Pseudomonas aeruginosa Represses Quorum Sensing Systems and Leads to Complex Changes to Stationary Phase Virulence Factor Regulation.”2014. PLoS One 9(5): e96166. DOI: 10.1371/journal.pone.0096166.
  21. ↑Weimer, Kristin E.D., Richard A. Juneau, Kyle A. Murrah, Bing Pang, Chelsie E. Armbruster, Stephen H. Richardson, and W. Edward Swords. “Divergent Mechanisms for Passive Pneumococcal Resistance to b-Lactam Antibiotics in the Presence of Haemophilus influenza.” 2011. The Journal of Infectious Diseases 203(4): 549-555. DOI: 10.1093/infdis/jiq087.
  22. ↑ 22.022.1Khalifa, Anis Ben Haj, Didier Moissenet, Hoang Vu Thien, and Mohamed Khedher. “Les facteurs de virulence de Pseudomonas aeruginosa : mécanismes et modes de régulations” (Virulence factors in Pseudomonas aeruginosa: mechanisms and modes of regulation). 2011. Annales de Biologie Clinique 69(4): 393-403. DOI: 10.1684/abc.2011.0589.
  23. ↑Tamura, Yutaka, Shoko Suzuki, and Takuo Sawada. “Role of elastase as a virulence factor in experimental Pseudomonas aeruginosa infection in mice.” 1991. Microbial Pathogenesis 12(3): 237-244. DOI: 10.1016/0882-4010(92)90058-V.
  24. ↑ Azghani, A.O. "Pseudomonas aeruginosa and epithelial permeability: role of virulence factors elastase and exotoxin A." 1996. American Journal of Respiratory Cell and Molecular Biology 15(1):132-40. DOI: 10.1165/ajrcmb.15.1.8679217
  25. ↑ 25.025.1Jensen, Peter Ø., Thomas Bjarnsholt,2Richard Phipps, Thomas B. Rasmussen,2 Henrik Calum,Lars Christoffersen, laus Moser, Paul Williams, Tacjana Pressler, Michael Givskov, and Niels Høiby. “Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensingcontrolled production of rhamnolipid by Pseudomonas aeruginosa." 2007. Microbiology 153(5): 1329-1338. DOI: 10.1099/mic.0.2006/003863-0.
  26. ↑Zhao, Ge, Marcia L. Usui, Soyeon I. Lippman, Garth A. James, Philip S. Stewart, Philip Fleckman, and John E. Olerud. “Biofilms and Inflammation in Chronic Wounds.” 2013. Advances in Wound Care 2(7): 389-399. DOI: 10.1089/wound.2012.0381.
  27. ↑Wolcott, R.D., K.P. Rumbaugh, G. James, G Schultz, P. Phillips, Q. Yang, C. Watters, P.S. Stewart, and S.E. Dowd. “Biofilm maturity studies indicate sharp debridement opens a time dependent therapeutic window.” 2010. Journal of Wound Care 19 (8): 320-328. DOI: 10.12968/jowc.2010.19.8.77709.
  28. ↑Duplantier, Allen J. and Monique L. Van Hoek. “The human cathelicidin antimicrobial peptide LL-37 as a potential treatment for polymicrobial infected wounds.” 2013. Frontiers in Immunology 4(143): 1-14. DOI: 10.3389/fimmu.2013.00143.

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2016, Kenyon College.


Recombinant DNA Technology and Pharmaceutical Production

​Genetic engineering has provided a way to create new pharmaceutical products called recombinant DNA pharmaceuticals. Such products include antibiotic drugs, vaccines, and hormones used to treat various diseases. Table lists examples of recombinant DNA products and their uses.

For example, the naturally occurring antibiotic synthesis pathways of various Streptomycesspp., long known for their antibiotic production capabilities, can be modified to improve yields or to create new antibiotics through the introduction of genes encoding additional enzymes. More than 200 new antibiotics have been generated through the targeted inactivation of genes and the novel combination of antibiotic synthesis genes in antibiotic-producing Streptomyces hosts. 3

Genetic engineering is also used to manufacture subunit vaccines, which are safer than other vaccines because they contain only a single antigenic molecule and lack any part of the genome of the pathogen (see Vaccines). For example, a vaccine for hepatitis B is created by inserting a gene encoding a hepatitis B surface protein into a yeast the yeast then produces this protein, which the human immune system recognizes as an antigen. The hepatitis B antigen is purified from yeast cultures and administered to patients as a vaccine. Even though the vaccine does not contain the hepatitis B virus, the presence of the antigenic protein stimulates the immune system to produce antibodies that will protect the patient against the virus in the event of exposure. 4 5

Genetic engineering has also been important in the production of other therapeutic proteins, such as insulin, interferons, and human growth hormone, to treat a variety of human medical conditions. For example, at one time, it was possible to treat diabetes only by giving patients pig insulin, which caused allergic reactions due to small differences between the proteins expressed in human and pig insulin. However, since 1978, recombinant DNA technology has been used to produce large-scale quantities of human insulin using E. coli in a relatively inexpensive process that yields a more consistently effective pharmaceutical product. Scientists have also genetically engineered E. coli capable of producing human growth hormone (HGH), which is used to treat growth disorders in children and certain other disorders in adults. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector. Eventually, genetic engineering will be used to produce DNA vaccines and various gene therapies, as well as customized medicines for fighting cancer and other diseases.

​Some Genetically Engineered Pharmaceutical Products and Applications

Recombinant DNA Product

Atrial natriuretic peptide

Treatment of heart disease (e.g., congestive heart failure), kidney disease, high blood pressure

Treatment of viscous lung secretions in cystic fibrosis

Treatment of severe anemia with kidney damage

Prevention of hepatitis B infection

Treatment of growth hormone deficiency, Turner’s syndrome, burns

Treatment of multiple sclerosis, various cancers (e.g., melanoma), viral infections (e.g., Hepatitis B and C)

Tissue plasminogen activator

Treatment of pulmonary embolism in ischemic stroke, myocardial infarction

  • What bacterium has been genetically engineered to produce human insulin for the treatment of diabetes?
  • Explain how microorganisms can be engineered to produce vaccines.

​One of the first steps in any viral infection is adhesion of the virus to specific receptors on the surface of cells. This process is mediated by adhesins that are part of the viral capsid or viral adhesins. The interaction of viral adhesins with specific cell receptors defines thetropism(preferential targeting) of viruses for specific cells, tissues, and organs in the body. The spike protein hemagglutinin found on Influenzavirus is an example of a viral adhesin it allows the virus to bind to thesialic acidon the membrane of host respiratory and intestinal cells. Another viral adhesin is the glycoprotein gp20, found on HIV. For HIV to infect cells of the immune system, it must interact with two receptors on the surface of cells. The first interaction involves binding between gp120 and the CD4 cellular marker that is found on some essential immune system cells. However, before viral entry into the cell can occur, a second interaction between gp120 and one of two chemokine receptors (CCR5 and CXCR4) must occur. Table lists the adhesins for some common viral pathogens and the specific sites to which these adhesins allow viruses to attach.

​Some Viral Adhesins and Their Host Attachment Sites


Results and discussion

General features of the Blastocystisgenome

The genome of a Blastocystis subtype 7 was resolved by pulsed-field gel electrophoresis, and 15 chromosomic bands have been characterized. The final assembled sequence is distributed in 54 scaffolds and the deduced genome is 18.8 Mb in size (16.5-fold sequence coverage), which is much smaller than plant parasite stramenopiles (Phytophthora infestans, 240 Mb P. sojae, 95 Mb Phytophthora ramorum, 65 Mb) and also smaller than free stramenopiles (Phaeodactylum tricornutum, 27.4 Mb T. pseudonana, 34.5 Mb). The reference annotation of the Blastocystis subtype 7 genome contains 6,020 genes, covering about 42% of the genome (Table 1). The average number of exons per gene is 4.6 for multiexonic genes and 929 genes are monoexonic. Compaction in this parasite genome is reflected by the short length of the intergenic regions (1,801 bp), the relatively low repeat coverage (25%) and, more strikingly, by the very short size of introns, with a sharp length distribution of around 32 nucleotides (Figure S1 in Additional file 1). A total of 38 rDNA units organized in transcriptional units, including a small subunit rRNA gene, a 5.8S rRNA gene, and a large subunit rRNA gene in a 5'-3' orientation, have been detected in the genome. The sizes of the small subunit, the large subunit and the 5.8S rRNA gene are 1.8 kb, 2.45 kb and 0.44 kb, respectively. Some units are tandemly duplicated, up to four copies on scaffold 18, and some may also be localized in subtelomeric regions, as revealed by a co-mapping of telomeric sequences and rDNA subunits at scaffold 6 and 9 extremities. These two scaffolds could correspond to entire chromosomes. Due to the sequencing method, some units are incomplete (either truncated or lacking genes). The alignment of 20 complete small subunit rRNA genes shows polymorphism between copies, which is also the case for 29 large subunit rRNA gene copies.

The number of genes in Blastocystis (6,020) is reduced in comparison with other stramenopiles (P. infestans, 17,797 P. sojae, 19,027 P. ramorum, 15,743 P. tricornutum, 10,402 T. pseudonana, 11,776). Surprisingly, a large portion of genes were probably duplicated since 404 clusters of paralogous protein-coding genes were identified, containing 1,141 genes, that is, 19% of Blastocystis genes (see Material and methods). Excluding the large multigenic families (up to 32 genes with a histone-fold domain and 20 genes with a 4Fe-4S ferredoxin domain), most of the duplicated genes are present in only two copies (Figure S2 in Additional file 1). As described in other organisms [27, 28], the duplicated genes are more conserved than single copy genes in Blastocystis sp. Indeed, they have more orthologs (defined as best reciprocal hit (BRH) see Materials and methods) and display higher similarities with their orthologs (Figure S3 in Additional file 1). They also tend to display higher expression levels than single copy genes (Figure S4 in Additional file 1).

We investigated whether these gene duplications could have arisen from a whole genome duplication (WGD) or smaller scale segmental duplications. WGD, the duplication of the entire genome by polyploidization, has been shown to have played a key role in the evolutionary history of several animal and plant lineages [27, 29–31]. Segmental duplications occur continually by several mechanisms that can duplicate parts of genes, entire genes, or several adjacent genes. These mechanisms include unequal crossing over, or gene conversion, and tandem duplication [32–34]. We were able to identify 320 blocks of duplicated genes, that is, paralogous segments of several adjacent genes (see Materials and methods), some of which are very large (up to 100 kb), suggesting a WGD. These blocks cover about 39% of the genome (7.3 out of 18.8 Mb) representing 38% (5.15 out of 13.65 Mb) of the unrepeated fraction of the genome. As shown in Figure 1, each scaffold is a mosaic of blocks of homology with several other scaffolds: scaffolds cannot be grouped by pairs as would be expected from a recent WGD. Additionally, some segments are present in more than two copies in the genome (they appear in black in Figure 1), suggesting that segmental duplications are likely to have played a role in the current duplication pattern. However, the duplicated blocks are not often on the same scaffold, nor in tandem, which rules out the tandem duplication model. The comparison of paralogous copies shows surprisingly high nucleic acid identity rates: on average, 99% in coding regions, 98.4% in untranslated regions, and 97.8% in introns and intergenic regions. Interestingly, those values are homogeneous among all paralogous blocks, suggesting that all blocks were duplicated at the same time.

Blocks of duplicated genes in the Blastocystis sp. genome. For each scaffold (from 0 to 25), the duplicated blocks are displayed with colors corresponding to the scaffolds where the paralogous blocks are located (on scaffolds 0 to 19, 21, 22, 24, 25, 27, 28, 30). Below each scaffold, the repeat density is displayed as a grey scale: 0% (white) to 100% (black) repeats in 10-kb windows.

Two hypotheses could explain the origin of these duplicated blocks. First, the duplicates may have arisen from a whole genome duplication that took place recently (since the copies are still very similar) and was followed by rapid genome rearrangements and losses of gene copies. The high homology between gene copies could also result from a high rate of homogenization through gene conversion driven by the high frequency of rearrangements. The frequent rearrangements in the Blastocystis lineage are probably also the reason why no extensive synteny could be detected between Blastocystis sp. and other stramenopiles. Second, the duplicates could also have occurred through segmental duplications (favored by the high rate of rearrangements), although the relatively uniform divergence between copies is more symptomatic of a single event and would imply a burst of segmental duplications during a short period or a very high rate of homogenization by recombination. The intriguing pattern of gene duplications, likely caused by the high rate of rearrangements in the Blastocystis genome, makes it impossible to determine which scenario is the most likely. It could be interesting to sequence other subtypes to determine whether the high rate of recombination (loss of synteny) and the pattern of duplications observed in subtype 7 is a common feature within this lineage.

Endosymbiotic and horizontal gene transfers in Blastocystissp

Phylogenetic analyses revealed two genes of possible cyanobacterial origin in the genome of Blastocystis, those encoding phosphoglycerate kinase [GenBank:CBK20833] and 6-phosphogluconate dehydrogenase [GenBank:CBK22626] (Figure S5 in Additional file 1). It is important to notice that 6-phosphogluconate dehydrogenase-encoding genes have been identified in non-photosynthetic protists such as Heterolobosea (not shown). This was interpreted as secondary horizontal gene transfer (HGT) from photosynthetic eukaryotes to Heterolobosea [35, 36].

The presence of plastids in various photosynthetic stramenopile lineages (for example, diatoms, chrysophytes, raphidophytes) was interpreted as a secondary endosymbiosis that occurred between a red algae and the ancestor of these groups. By contrast, the evolutionary meaning of the lack of plastids in some heterotrophic stramenopile lineages (for example, oomycetes, bicosoesids) is still under discussion: does it indicate secondary losses of the plastid acquired by the ancestor of all stramenopiles? Or does it reflect the fact that the secondary endosymbiosis at the origin of stramenopile plastids did not occur in their common ancestor but after the divergence of heterotrophic lineages [37]? The presence of genes of cyanobacterial origin in Blastocystis supports the first hypothesis even if we can not rule out possible recent acquisitions of genes of chloroplastic origin from photosynthetic eukaryotes as in the case of Heterolobosea.

HGT is important in evolution as an adaptive mechanism of microbial eukaryotes to environmental conditions [38, 39] and is known to play an important role in stramenopiles. For instance, iron is a limiting nutrient in surface waters for diatoms. Therefore, the likely acquisition of ferritin by HGT from bacteria has permitted some species to acquire this nutrient from the environment [40]. This is also the case for the diatom Phaeodactylum, in which nitrogen metabolism, cell wall silification, DNA replication, genome repair and recombination processes have been shaped by HGT [40, 41]. HGT seems also to play an important role in oomycetes since it may be involved in osmotrophy. Genes involved in absorbing products of degradation of complex nutrients were predicted to be candidates for fungi-to-oomycete HGT [42]. By analyzing the set of predicted genes in Blastocystis sp. that are homologous to bacterial or archaeal genes, we identified 133 candidates for HGT (Table S3 in Additional file 2). In most cases, our phylogenetic analyses confirm the bacterial origin of these genes even if they were not sufficiently resolved to allow the precise identification of the donor, suggesting that these HGT events were ancient and/or that the corresponding genes are rapidly evolving in the genome of Blastocystis sp. Interestingly, in a few cases, even when the transferred gene is of bacterial origin, the Blastocystis sp. copy is closely related to homologues found in pathogenic and/or anaerobic eukaryotes, suggesting that HGT between eukaryotes has played a key role in these organisms too (Figure S6 in Additional file 1).

Some of the genes that originated from HGT possess functions that lead to a better understanding of how this lineage emerged. Three are homologous to the bacterial major facilitator transporter (MFS_1), the first two being nearly identical, and therefore resulting from a recent gene duplication event. MFS proteins form a large and diverse group of secondary transporters, which facilitate the transport across membranes of a variety of substrates, including ions, sugar phosphates, drugs, neurotransmitters, nucleosides, amino acids and peptides [43]. Two Blastocystis MFS genes have closely related homologues in some pathogenic eukaryotes like the Alveolata Perkinsus marinus or fungi such as Gibberella zeae and Verticillium albo atrum, suggesting an acquisition from bacteria followed by HGT between these eukaryotes (Figure S6f in Additional file 1). However, the phylogeny resolution is too low to precisely identify the bacterial donor of these genes. The presence of MSF proteins in Blastocystis sp. may confer the ability to absorb nutrients from the environment to this parasite, particularly in the intestinal lumen or when attacking host tissues. We have also found different HGT genes harboring alcohol deshydrogenase, short-chain dehydrogenase and oxidoreductase domains (Table S3 in Additional file 2) that may be involved in specific fermentations that remain to be characterized. Some of them are closely related to homologues found in anaerobic eukaryotes like Trichomonas vaginalis and Entamoeba histolytica (Figure S6b in Additional file 1) or in the bacteria Legionella pneumophila or Parachlamydia acanthamoebae, which infect or are associated with amoeba [44, 45]. These enzymes may increase the range of Blastocystis sp. metabolic abilities to produce energy in anaerobic environments, as has been observed in Giardia lamblia and E. histolytica [46, 47].

Several genes acquired by HGT may participate in the adhesion of the parasite to the host tissues. Indeed, 26 genes (Table S3 in Additional file 2) encode proteins containing the IPR008009 domain, which is often associated with immunoglobulin domains, a conserved core region of an approximately 90-residue repeat found in several hemagglutinins and other cell surface proteins. Among these 26 Blastocystis sp. proteins, some also contain the IPR015919 domain, which characterizes cadherins, a family of adhesion molecules that mediate Ca 2+ -dependent cell-cell adhesion. Homologous genes are also found in some beta-Proteobacteria or Acidobacteria, but the sequences are very divergent and our phylogenetic analysis did not, therefore, allow firm identification of the bacterial donor. Some hydrolase-encoding genes could also result from the transfer from bacteria to Blastocystis sp. One of them possesses an esterase-lipase (IPR013094) domain (Table S3 in Additional file 2) and may participate in the degradation of host tissue during infection. The closest homologues of this gene are found in the fungus Botryotinia fuckeliana, in Firmicutes and Actinobacteria (Figure S6d in Additional file 1).

Overall, these HGT genes may have allowed flexibility in genome expression, enabling the successful adaptation of Blastocystis sp. to digestive environments through genes encoding proteins that could be involved in osmotrophy (MFS), energy metabolism (dehydrogenases) and adhesion.

Circular genome, predicted proteome and metabolic pathways of the MLOs

Although it lives in anaerobic or microaerophilic conditions, Blastocystis sp. harbors MLOs that present both mitochondrial and hydrogenosomal features [24]. We recently reported that Blastocystis sp. MLOs contain a circular genome, including genes encoding 10 of the 20 complex I subunits, but they lack all genes encoding cytochromes, cytochrome oxidases and ATP synthase subunits [24], unlike mitochondrial DNA from other sequenced stramenopiles, such as Phytophthora sp. [48]. The MLO genome of the Blastocystis subtype 7 is a circular molecule 29,270 bp in size. Two other MLO genomes were then sequenced from isolates belonging to other subtypes [49]: a subtype 1, represented by Blastocystis Nand II, with a 27,719 bp genome and a subtype 4, represented by Blastocystis DMP/02-328, with a 28,382 bp genome. In addition to sequence conservation, these three genomes have many similarities. Their A+T content is around 80%, their gene density is higher than 95% and all three encompass 45 genes: 27 ORFs, 16 tRNAs and 2 rRNA genes. The ORFs consist of NADH subunits, ribosomal proteins and proteins with no similarity in the databases. The synteny between the three MLO genomes is highly conserved: gene order is strictly the same among the three genomes [24, 49].

Through the analysis of a Blastocystis EST database, Stechmann et al. [23] have identified 110 potential proteins associated with mitochondrial pathways, such as the oxidative phosphorylation chain, tricarboxylic acid (TCA) cycle, Fe/S cluster assembly, and amino acid and fatty acid metabolisms. Nonetheless, approximately half of these proteins have an incomplete amino terminus due to EST data, making it difficult to confirm mitochondrial import by algorithms. To clarify the metabolic characteristics of these puzzling organelles, we used data from the whole genome sequence in order to establish the in silico proteome of Blastocystis MLOs. For this purpose, a computational approach based on two different prediction algorithms (MitoProt and MitoPred) for mitochondrial-import proteins was chosen (see Materials and methods for more details). This approach predicted 365 MLO proteins (Table S6 in Additional file 3) whereas Stechmann et al. [23] predicted only 110 proteins. Among these 365 proteins, 299 were predicted to have an amino-terminal extension involved in mitochondrial import, suggesting that an alternative system might exist for the 66 remaining proteins. Of the 299 proteins, 41 remain as 'hypothetical protein' with unknown function and 31 have no homologues in public databases, which raises the question of the existence of undiscovered metabolic processes within these intriguing organelles (Table S6 in Additional file 3). The other proteins are involved in classical mitochondrial core functions, such as oxidative phosphorylation, amino acid metabolism, fatty acid oxidation, iron-sulfur cluster assembly, and mitochondrial import system. Several proteins involved in the translocase of the outer mitochondrial membrane (TOM complex), the translocase of the inner membrane (TIM complex), and the presequence translocase-associated motor (PAM complex), which perform protein transport into the matrix, were identified. Interestingly, the two essential subunits of the mitochondrial processing peptidase heterodimer (MPP α/β), essential for the cleavage of the targeting peptide, were also found [50].

Our analyses revealed that MLOs probably have three ways to make acetyl-CoA from pyruvate, supported by the presence of the pyruvate dehydrogenase complex, pyruvate:ferredoxin oxidoreductase and pyruvate:NADP + oxidoreductase (an amino-terminal pyruvate:ferredoxin oxidoreductase domain fused to a carboxy-terminal NADPH-cytochrome P450 reductase domain) (Figure 2). Euglena gracilis mitochondria include this feature, which provides adaptability to various oxygen levels [51], and this might be to a lesser extent the case for Blastocystis sp. We have also identified the 20 subunits of the Blastocystis sp. MLO complex I (ten are encoded by the MLO genome and ten by nuclear genes). The four nuclear-encoded subunits of the mitochondrial respiratory chain complex II were detected and this complex could function in two ways (via succinate dehydrogenase or fumarate reductase) [52]. We did not identify any genes encoding complexes III and IV subunits or ATP synthase. However, we have found components of the TCA cycle, which was shown to be involved with complex II (fumarate reductase) in fumarate respiration in parasitic helminths [52]. Interestingly, we identified a gene encoding a terminal oxidase, called alternative oxidase (AOX), which could be the terminal electron acceptor of complexes I and II (Figure 2), allowing adaptation to oxygen stress and maintaining the NADH/NAD balance, as has been suggested for Cryptosporidium parvum [53, 54]. These data raise questions about the electron acceptor when complex II has succinate dehydrogenase or fumarate reductase activity, the quinone used in this process and the role of the proton gradient.

In silico reconstruction of metabolic pathways of Blastocystis sp. mitochondria-like organelles. The proteins are predicted from the combined analysis of MitoProt and MitoPred algorithms. Proteins with a predicted amino-terminal extension are outlined by a solid black line, and protein complexes for which mitochondrial presequences for only some of the subunits have been predicted are outlined by a dashed black line. The pathways in purple represent: (1) the conversion of pyruvate into acetyl-CoA by the pyruvate dehydrogenase complex (PDH), pyruvate:ferredoxin oxidoreductase (PFO) or pyruvate:NADP oxidoreductase (PNO) (2) acetyl-CoA is then converted to acetate by acetate:succinate CoA transferase (ASCT) and may allow production of ATP (3). Pyruvate may follow routes that potentially use complexes I and II to produce succinate (and propionate) and certainly participate in maintaining the redox balance. The pathways in green and burgundy correspond to amino acid metabolism and fatty acid metabolism, respectively. Pathways for the assembly of iron-sulfur proteins are represented in blue, and proteins involved in mitochondrial import machinery in orange. Enzymes that may play a role in protection against oxidative stress are indicated in pink (superoxide dismutase (SOD), alternative oxidase (AOX), glutathione reductase (GR) and gluthathione peroxidase (GPx)) the role of glycerol-3-phosphate dehydrogenase (G3PDH) remains to be determined. Abbreviations: 1, acetyl-CoA carboxylase 2, 3-oxoacyl-ACP synthase 3, 3-oxoacyl-ACP reductase 4, 2-enoyl-ACP reductase 5, methylmalonyl-CoA mutase 6, methylmalonyl-CoA epimerase 7, propionyl-CoA carboxylase AAC, ATP/ADP translocator ACP, acyl carrier protein ALAT, alanine aminotransferase BC-AAT, branched-chain amino acid aminotransferase C I, complex I ECH, enoyl-CoA hydratase [Fe]-Hyd, [Fe]-hydrogenase FRD/SDH, fumarate reductase/succinate dehydrogenase activity of complex II FUM, fumarase HCDH, 3-hydroxyacyl-CoA dehydrogenase HICH, 3-hydroxyisobutyryl-CoA hydrolase HID, 3-hydroxyisobutyrate dehydrogenase LC-ACS, long-chain acyl-CoA synthetase MDH, malate dehydrogenase OMC, oxoglutarate/malate carrier protein Pyr C, pyruvate carboxylase SCS, succinyl-CoA synthetase SOD, superoxide dismutase.

We also revealed proteins that can be grouped into essential mitochondrial pathways, like the Fe/S cluster assembly. More precisely, we have identified 11 enzymes (6 of which have predicted mitochondrial import signals), composing the iron-sulfur cluster system responsible for the assembly of mitochondrial Fe/S proteins [55], such as the cysteine desulfurase Nfs1, the scaffold protein Isu1, frataxin, and the P-loop NTPase Ind1, which is required for the assembly of complex I (Figure 2). We also highlighted some proteins involved in mitochondrial fatty acid synthesis type II [56], beta oxidation of fatty acids and amino acid metabolism (Table S6 in Additional file 3).

Taken together, our data confirm the mitochondrial nature of the Blastocystis sp. MLO. The oxygen-poor environment may have driven the selection of these unique organelles, which seemingly represent an intermediate situation between anaerobic mitochondria and hydrogenosomes, arguing for multiple situations arising during organelle evolution. It remains now to describe the metabolism occurring in these unusual organelles more precisely.

Secretome and virulence factors

The persistence of Blastocystis sp. in the host may be due, to some extent, to its ability to override the response of the immune system and to adhere and survive within the intestinal tissue. Manipulation of the host might be facilitated by molecules released at the interface between the host and the parasite [57]. Accordingly, the study of the predicted secretome of Blastocystis sp. is of particular interest. With SIGNALP 3.0, 307 proteins were predicted to be secretory, of which 46 had no sequence similarity in the public nr databases. By sequence homology, 170 proteins that could play a role in host-parasite relationships were selected and submitted to PSORTII for extracellular location. Finally, 75 putative secreted proteins have been classified by putative functions, some of which may have a direct connection with pathogenicity (proteases, hexose digestion enzymes, lectins, glycosyltransferases and protease inhibitors Table S4 in Additional file 2).

Blastocystis can secrete members of the immunophilin family, characterized by peptidyl-propyl cis-trans isomerase activity and disulfide isomerases (Figure 3 Table S4 in Additional file 2). These proteins have key roles in protein folding, but it has also been established that they can have moonlighting functions. In bacteria, they have evolved adhesive properties for the host [58] but they can also modulate host leukocyte function and induce cellular apoptosis [59]. A cyclophilin-like protein from the protozoan parasite Toxoplasma gondii is directly involved in host-parasite crosstalk, as it can modulate protective Th1 responses through its binding to the chemokine receptor CCR5 [60]. It is unclear what role these proteins play in Blastocystis sp., but this illustrates a range of functions for cell stress proteins in host-pathogen interactions.

Secretory proteins and virulence factors identified in the Blastocystis sp. subtype 7 potentially involved in host interaction. Blastocystis sp. may release cysteine proteases, which could be processed by legumain. These proteases may attack intestinal epithelium together with other hydrolases, such as glysoside hydrolases. Protease inhibitors, some of which have been predicted to be secreted, could act on host proteases (digestive enzymes or proteases involved in the immune response). Some as yet uncharacterized secondary metabolites produced by polyketide synthase (PKS) identified in the genome could also participate in host intestinal symptoms. Adhesive candidate proteins (proteins with an immunoglobulin Ig domain) have been found. Finally, drug-resistant isolates of the parasite could be explained by the presence of multidrug resitance (MDR) proteins. Lightning bolts indicate potential toxic effects.

Sugar-binding proteins have an important role through a conserved carbohydrate-recognition domain that could interact with host cell receptors. Such proteins have been characterized in other parasites [61] and it is interesting to note that some sugar-binding proteins are able to inhibit Th1- and Th2-mediated inflammation [62, 63]. Moreover, some specific sugar-binding proteins are also able to suppress regulatory T cells [64]. The binding of these proteins is dependent on their specific sugar motifs, which can be added to N- or O-linked glycans by glycosyltransferases. One carbohydrate-binding protein and eight glycosyltransferases (Table S4 in Additional file 2) have been predicted to be secreted. All these enzymes could allow cross-linking of Blastocystis sp. sugar-binding proteins to host cell receptors.

The parasite likely uses hydrolases to attack host tissues. Fucosidase, hexosaminidase and polygalacturonase have been identified in the predicted secretome and may participate in this process by degrading host glycoproteins (Figure 3 Table S4 in Additional file 2). Proteases have been proposed to be involved in diverse processes, such as host cell invasion, excystation, metabolism, cytoadherence or other virulence functions. A correlation between a high level of protease activity and the virulence of the intestinal parasite E. histolytica was proven by McKerrow et al. [65]. Indeed, cysteine proteases degrade extracellular matrix proteins, cleave immunoglobulin A and G, and are thought to be responsible for the cytopathic effect of different pathogens against in vitro cultured cells [66]. Interestingly, Blastocystis sp. proteolytic enzymes are also able to degrade human secretory immunoglobulin A [67]. All the major classes of proteolytic enzymes were identified in the genome data, including serine, aspartic, and cysteine proteases and metalloproteases. Among the 66 proteases identified, 18 are predicted to be secreted by the parasite (Table S4 in Additional file 2). Within the protease family, cysteine protease-encoding genes are the most represented in Blastocystis sp. genome and 96% of the proteins encoded by these genes are predicted to be secreted. Among the cysteine proteases we have found five legumains and eight cathepsins three cathepsins B contain the IPR015643 domain, which is only present in Blastocystis sp. compared to the other stramenopiles. The IPR015643 domain corresponds to the peptidase C1 cathepsin B domain and has a cysteine type peptidase activity, which was also found in pathogenic protozoa (Leishmania sp. and Trypanosoma sp.) [66]. Cysteine proteases are usually secreted in their inactive form and must be matured, having a prosegment that prevents hydrolysis during protease trafficking and storage. This maturation might result from the activity of the same protease or another, such as asparaginyl endopeptidase (also called legumain) [68]. This endopeptidase cleaves peptide bonds carboxy-terminal to asparagine residues, and may be involved in processing and activating both cathepsins L and B. Legumains have been predicted in the secretome of Blastocystis sp. (Table S4 in Additional file 2) and could be involved in protease processing (Figure 3). As an alternative role, secreted Blastocystis sp. legumains could also participate with other effectors in the alteration of the host intestine [69]. Indeed, it has been shown that legumain can degrade fibronectin, an extracellular matrix glycoprotein [70].

Genes coding for protease inhibitors are also present in the Blastocystis sp. genome, and some are predicted to be secreted. Release of protease inhibitors may weaken the host response as described in nematodes [71]. Blastocystis sp. encodes three protease inhibitors: cystatin, type1-proteinase inhibitor and endopeptidase inhibitor-like protein (Table S4 in Additional file 2). Type1-proteinase inhibitor is similar to chymotrypsin inhibitor, which is known to inactivate intestinal digestive enzymes (trypsin and chymotrypsin) as in Ascaris suum [72], thus protecting the parasite against non-specific digestive defenses. Cystatin, also called stefin, was described in Fasciola gigantica [73] and shown to inhibit mammalian cathepsin B, cathepsin L and other cysteine proteases, including parasite ones. In Blastocystis sp., secreted cystatin could participate in the regulation of parasitic cysteine protease activities. Cystatin can also potentially inhibit host proteases involved in MHC II antigen processing and presentation, including the key enzyme asparaginyl endopeptidase [74] and cathepsin S, the mammalian legumain [73].

Interestingly, a putative type I polyketide synthase (PKS) gene was also found in the Blastocystis sp. genome, potentially originating from HGT. PKS and non-ribosomal peptide synthetase (NRPS) synthesize metabolites like simple fatty acids, but also a myriad of chemical structures that possess important pharmacological activities and environmental impact, such as toxins, antibiotics or antimicrobials. Type I PKS was formerly known only from bacteria and fungi, but recently homologous genes were also discovered in some protists [75]. According to the Database for NRPS and PKS [76], the Blastocystis sp. PKS gene possesses the three essential domains, and three other domains: dehydratase, ketoacyl reductase, and enoyl reductase domains. The presence of these additional domains would permit this organism to synthesize both reduced polyketides and fatty acids. Domain comparison with other type I PKSs suggests that Blastocystis sp. PKS is similar to type I PKS from the ascomycete Cochliobolus heterostrophus, a maize pathogen that produces T toxin [77], a polyketide molecule that disturbs mitochondria by binding a protein of the inner mitochondrial membrane. Searching polyketide-related metabolites in the secretome of Blastocystis sp. would be of interest in order to identify molecules that could have effects on the host (Figure 3).

Antioxidant system and multi drug resistance

Like other anaerobic organisms, Blastocystis sp. has to eliminate reactive oxygen species such as superoxide anions (O2 .- ), hydrogen peroxide (H2O2) and hydroxyl radicals (HO . ) resulting from metabolism. In addition, this microorganism has to cope with the oxidative burst imposed by host immune cell effectors (release of O2 .- subsequently processed to give additional reactive oxygen species). For these reasons, to protect against oxidative injury, Blastocystis species have developed an efficient battery of antioxidant enzymes (Table S5 in Additional file 2). The first lines of defense against oxygen damage are superoxide dismutases (SODs), a family of metalloproteins catalyzing the dismutation of O2 .- to form H2O2 and oxygen. Genome annotation revealed the presence of two genes encoding SODs (SOD1 and SOD2) that exhibit sequence characteristics of dimeric iron-containing SODs [78] and likely protect the cytosol and MLOs, respectively, against O2 .- . Catalase and ascorbate peroxidase are subsequently able to remove H2O2 generated by SODs as well as by NADPH-dependent oxidase. However, genes encoding catalase and ascorbate peroxidase have not been identified in Blastocystis sp. nor in many unicellular parasites, including trypanosomatids and Plasmodium falciparum. Additional enzymes, glutathione peroxidase (Gpx) and thioredoxin-dependent peroxidase (commonly known as peroxyredoxin (Prx)) are able to reduce H2O2 to water as well as other substrates, such as hydroperoxides and peroxinitrite. In most eukaryotes, both enzymes obtain their reducing equivalents from two redox systems, the glutathione (GSH) and the thioredoxin (Trx) systems, respectively. Like P. falciparum [79], Blastocystis sp. cells possess a complete GSH synthesis pathway: the genes encoding γ-glutamylcysteine synthetase, glutathione synthetase (eu-GS group) and a functional GSH/Gpx (nonselenium Gpx belonging to the PHGpx group)/glutathione reductase system have been identified and both Gpx and glutathione reductase are probably located in the MLO. This nearly ubiquitous redox cycle is replaced by the trypanothione system in trypanosomatids [80]. Blastocystis sp. also contains genes encoding the proteins of the Trx/thioredoxin reductase (TrxR)/Prx system. Indeed, two genes encode small proteins homologous to Trx: one cytosolic and another most likely located in the MLO (Table S5 in Additional file 2). Trx is itself reduced by TrxR and three genes encoding cytosolic TrxR have been identified in Blastocystis sp. These proteins clearly belong to the high molecular weight (designated H-TrxR) group of enzymes and are similar to metazoan enzymes, including those of Homo sapiens and Drosophila melanogaster, and to those of the apicomplexan protozoa Plasmodium, Toxoplasma, and Cryptosporidium [81]. Interestingly, in contrast to apicomplexan H-TrxRs, two of the H-TrxR enzymes of Blastocystis are predicted to possess a redox active center in the carboxy-terminal domain composed of a selenocysteine (a rare amino acid encoded by the opal codon TGA, which is not recognized as a stop codon) at the penultimate position and its neighboring cysteine residue as in metazoan enzymes (selenoprotein type H-TrxR). This strongly suggests the presence of the Se-Cys insertion machinery (SECYS elements) in Blastocystis sp. Genes encoding another type of TrxR with low molecular weight (designated L-TrxR) have been identified in parasitic protozoa such as Trichomonas, Entamoeba, and Giardia but not in the genome of Blastocystis sp. These data reinforce the assumption of the exclusive occurrence of either L-TrxR or H-Trxr in genomes and of some disadvantages of possessing both types of TrxR [81]. In Blastocystis sp., at least 11 highly similar gene copies encoding predicted cytosolic Prxs have been found that clearly belong to the typical 2-Cys class of Prx. Whether sequence polymorphism of these enzymes is potentially correlated with diversified expression or even function remains to be explored. Another gene encoding a typical 2-Cys Prx, likely located in the MLO, has been identified in this parasite. Interestingly, like the homologous sequence of another stramenopile, P. infestans, this latter protein is fused to Trx with a WCGKC motif. As described above, Blastocystis sp. possesses a whole array of antioxidant enzymes protecting both the cytosol and MLO. As shown in Table S5 in Additional file 2, these enzymes have distinct phylogenetic origins and most of them probably originate from prokaryote HGT. These antioxidant proteins attract attention in unicellular parasites as they have important functions in host-parasite interactions and constitute new drug targets for the design of inhibitors. Indeed, genetic approaches have undoubtedly shown that some anti-oxidant enzymes are essential for the survival of different parasitic species [82–86].

Some genes coding for multi-drug resistance pump proteins have also been discovered in the Blastocystis sp. genome. There are two classes of multi-drug resistance genes: the first class corresponds to proteins that are energized by ATP hydrolysis the second class includes proteins that mediate the drug efflux reaction with a proton or sodium ion gradient. Among the first class, 24 ABC transporter genes were found. In eukaryotes the main physiological function of ABC transporters is the export of endogenous metabolites and cytotoxic compounds [87] and eight families of ABC transporters (ABC A to H) have been identified. The Blastocystis sp. ABC transporters are included in four of these eight families (five in family A, six in family B, six in family C, three in family F, and four not in any class). The A family is involved in lipid trafficking, and the F family in DNA repair and gene regulation. The other two families are more interesting [87], since in protozoan parasites (Leishmania spp., Trypanosoma spp., Plasmodium spp.) transporters belonging to the B and C families confer resistance to drugs. Metronidazole-resistant strains of Blastocystis sp. could have arisen through the action of these multi-drug resistance proteins (Figure 3).


6 CLINICAL IMPLICATIONS OF GENETIC DATA

Despite the plethora of findings about genetic risk factors, the overall picture is that most of these genes are likely to have a relatively limited impact on infection risk, suggesting that many of them are involved in response to viral infection. This fact makes predicting which people will have a severe reaction to SARS-CoV-2 somewhat difficult. Moreover, relatively little is understood about inter-individual genetic differences in the immune response to this novel coronavirus, or the pleiotropic effects that different variants might confer (Godri Pollitt et al., 2020 ).

Nevertheless, the goal of ongoing COVID-19 research is to identify genetic variation that will improve the clinical management of this disease and foster better patient outcomes (Murray et al., 2020 The COVID-19 Host Genetics Initiative, 2020 ). For example, of the 68 severe COVID-19 risk-associated genes identified by Taylor et al. ( 2020 ), several of them yield protein products that can be targeted for drug development, with drugs for nine of these targets having reached Phase I clinical trials. Others have suggested that “[p]airing HLA typing with COVID-19 testing where feasible could improve assessment of viral severity in the population” (Nguyen et al., 2020 : e00510), thereby allowing prioritization of individuals with high-risk HLA types for vaccination. Likewise, knowing the levels of ACE2 and TRPRSS2 expression in asthma patients who use inhaled corticosteroids and those who do not will help clinicians identify subgroups such as males, African Americans and patients with diabetes that are at higher risk for COVID-19 morbidity (Peters et al., 2020 ). Verdecchia et al. ( 2020 ) further point to the possible use of recombinant ACE2, angiotensin 1-7 and AT1 receptor blockers to help treat patients with SARS-CoV-2 infection.

All of the emerging knowledge about genetic risk factors for infection is fueling global research on vaccines for COVID-19, with over 145 vaccines being developed and 19 currently being used in human clinical trials (Coronavirus Vaccine Tracker, 2020 ). Yet, while an effective and affordable vaccine will certainly go a long way to halting the current pandemic, other more basic public health measures such as mask wearing, social distancing, COVID-19 testing, and contact tracing will be essential for mitigating the impact of coronavirus on the general public for the foreseeable future, and particularly in communities of color who are at much higher risk for viral exposure and severe disease expression (Oppel Jr., Gebeloff, Lai, Wright, & Smith, 2020 ).


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