Virulence Factors of Bacterial and Viral Pathogens

Learning Objectives

  • Explain how virulence factors contribute to signs and symptoms of infectious disease
  • Differentiate between endotoxins and exotoxins
  • Describe the mechanisms viruses use for adhesion and antigenic variation


There are a variety of different types of virulence factors that are produced by many types of microbes.  The types are described below.

Virulence Factors for Adhesion

Adhesins allow a number o fdiffernt bacteria to attache to various body sites.  Table 1 lists common adhesins found in some of the pathogens we have discussed or will be seeing later in this chapter.

Table 1. 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

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. 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  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 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.

A picture of a person with a swollen right hand. Figure 1. This patient has edema in the tissue of the right hand. Such swelling can occur when bacteria cause the release of pro-inflammatory molecules from immune cells and these molecules cause an increased permeability of blood vessels, allowing fluid to escape the bloodstream and enter tissue.


Exoenzymes

Some pathogens produce enzymes that are excreted by cells called, exoenzymes; these enzymes  enable bacteria 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 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, an enzyme produced by pathogens like Staphylococcus aureus, Streptococcus pyogenes, and Clostridium perfringens, degrades the glycoside hyaluronic acid, found in connective tissue (Figure 2). This allows the pathogen to spread to deeper body tissues.  Because of this property, hyaluronidase is sometimes referred to as spreading factor (Figure 2).

a) A diagram of epithelial cells that are connected along their membranes. Hyaluronidases enter at these connection points. B) after the hyaluronidases break down the connections between the cells, bacteria can flow through the openings.Figure 2. (a) Hyaluronic acid is a polymer found in the layers of epidermis that connect adjacent cells. (b) Hyaluronidase produced by bacteria degrades this adhesive polymer in the extracellular matrix, allowing passage between cells that would otherwise be blocked.

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.  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.

Collagenase digests collagen, the dominant protein in connective tissue. 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 3).

A diagram of a tube labeled lumen of blood vessel lined by cells labeled endothelial cells. Outside he cells is dense irregular connective tissue. Collagenase is shown as small dots that break up the connections between the cells. A micrograph of the dense connective tissue shows many red lines making a meshwork. Figure 3. The illustration depicts a blood vessel with a single layer of endothelial cells surrounding the lumen and dense connective tissue (shown in red) surrounding the endothelial cell layer. Collagenase produced by C. perfringens degrades the collagen between the endothelial cells, allowing the bacteria to enter the bloodstream. (credit illustration: modification of work by Bruce Blaus; credit micrograph: Micrograph provided by the Regents of University of Michigan Medical School © 2012)


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 toxins, biological poisons that assist in their ability to invade and cause damage to tissues.

Toxins can be categorized as endotoxins or exotoxins. The lipopolysaccharide (LPS) found on the outer membrane of gram-negative bacteria is called endotoxin (Figure 4). During infection and disease, gram-negatives release endotoxin either when the cell lyses, releasing the LPS. The lipid component of endotoxin, lipid A, is responsible for the toxic properties of the LPS molecule. Lipid A triggers an 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.

Endotoxins are stable at high temperatures; temperatures of 121oC for 45 minutes are required to destroy endotoxins.

A long chain of O antigens is drawn as various geometric shapes in a long row. Next is a core; a shorter region of similar shapes. Next is 2 circles labeled lipid A. Each of these has 2 or 3 long wavy lines projecting from them. Figure 4. Lipopolysaccharide is composed of lipid A, a core glycolipid, and an O-specific polysaccharide side chain. Lipid A is the toxic component that promotes inflammation and fever.


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) are mixed with a patient’s serum. The amebocytes will react to the presence of any endotoxin and clotting reaction can be observed 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 pathogenic bacteria. Most exotoxins are produced by gram-positives. Exotoxins are much more specific in their action and the cells they interact with. EMost exotoxins are heat labile because of their protein structure, and many are denatured (inactivated) at temperatures above 41 °C (106 °F). Very small concentrations of exotoxins can be lethal. For example, botulinum toxin, is 240,000 times more lethal than endotoxin). The keyr differences between exotoxins and endotoxins are summarized  in Table 4

Table 4. 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
Amount required to be lethal High Low
Exotoxins are sometimes named based on the organ system they affect. For example, cholera toxin is an enterotoxin produced by the gram-negative bacterium Vibrio cholerae.  It affects 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.

Click this link to see an animation of how the cholera toxin functions.
Botulinum toxin (also known as botox), produced by the gram-positive bacterium Clostridium botulinum is a neurotoxin, affecting the nervous system.  It is the most acutely toxic substance known to date. Botulinum toxin enters the neuromuscular junction,blocking acetylcholine release, resulting in the inhibition of muscle contraction.  When  respiraroty system muslces are invovled, the result is the individual stops breathing, leading to 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.

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 blocks acetylcholinesterase, 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 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. The gram-positive bacterium Streptococcus pyogenes produces streptolysins, water-soluble hemolysins that bind to the cholesterol in the host cell membrane to form a pore.  Some strains of S. aureus also produce leukocidin called causing edema, erythema (reddening of the skin due to blood vessel dilation), and skin necrosis.

Think about It

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


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 phagocytize the cell. In addition, the capsule makes the bacterial cell much larger, making it harder for immune cells to engulf the pathogen (Figure 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. 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 8).

a) a micrograph showing nonencapsulated cells as blue ovals on a light background. Encapsulated cells have a thick clear ring around the blue cells. B) Antibodies on phagocytic cells bind to antigens on the bacterial cell. Capsules on the bacterial cell cover the antigen and prevent the antibody from binding to the antigen. C) A bacterial cell is releasing small donts labeled proteases that are breaking down an antibody. Figure 8. (a) A micrograph of capsules around bacterial cells. (b) Antibodies normally function by binding to antigens, molecules on the surface of pathogenic bacteria. Phagocytes then bind to the antibody, initiating phagocytosis. (c) Some bacteria also produce proteases, virulence factors that break down host antibodies to evade phagocytosis. (credit a: modification of work by Centers for Disease Control and Prevention)


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 prevents the bacterium from being killed mechanisms within the phagocytic cell.

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. Bacteria release coagulase into the bloodstream, and form a fibin clot coats the bacteria in fibrin, protecting the bacteria from exposure to phagocytes circulating in the bloodstream.

Whereas coagulase causes blood to clot, streptokinases 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, has a surface protein that undergoes antigenic variation. Each time fever occurs, the protein in B. burgdorferi can differ so much that antibodies against previous VlsE sequences are not effective. It is believed that this variation 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, the cause of the sexually transmitted disease gonorrhea, which changes it pili to avoid immune defenses.

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
  • 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, d.


Multiple Choice

Which of the following would be a virulence factor of a pathogen?

  1. a surface protein allowing the pathogen to bind to host cells
  2. a secondary host the pathogen can infect
  3. a surface protein the host immune system recognizes
  4. the ability to form a provirus






You have recently identified a new toxin. It is produced by a gram-positive bacterium. It is composed mostly of protein, has high toxicity, and is not heat stable.  Based on these characteristics, how would you classify this toxin?

  1. superantigen
  2. endotoxin
  3. exotoxin
  4. leukocidin






Which of the following applies to hyaluronidase?

  1. It acts as a spreading factor.
  2. It promotes blood clotting.
  3. It is an example of an adhesin.
  4. It is produced by immune cells to target pathogens.






Phospholipases are enzymes that do which of the following?

  1. degrade antibodies
  2. promote pathogen spread through connective tissue.
  3. degrade nucleic acid to promote spread of pathogen
  4. degrade cell membranes to allow pathogens to escape phagosomes






Fill in the Blank

 

Adhesins are usually located on __________ of the pathogen and are composed mainly of __________ and __________.





Think about It

  1. Two types of toxins are hemolysins and leukocidins.

    1. How are these toxins similar?
    2. How do they differ?




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