Unit 7.2: Mechanisms of Disease Part 2: How Pathogens Cause Disease

For most infectious diseases, the ability to accurately identify the causative pathogen is a critical step in finding or prescribing effective treatments. The cause of a disease is known as its etiology. Today’s physicians, patients, and researchers owe a sizable debt to the physician Robert Koch (1843–1910), who devised a systematic approach for confirming causative relationships between diseases and specific pathogens.

Koch’s Postulates: In 1884, Koch published four postulates (Table 7.2) that summarized his method for determining whether a particular microorganism was the cause of a particular disease. Each of Koch’s postulates represents a criterion that must be met before a disease can be positively linked with a pathogen. In order to determine whether the criteria are met, tests are performed on laboratory animals and cultures from healthy and diseased animals are compared (Figure 7.5).

In many ways, Koch’s postulates are still central to our current understanding of the causes of disease. However, advances in microbiology have revealed some important limitations in Koch’s criteria. Koch made several assumptions that we now know are untrue in many cases. The first relates to postulate 1, which assumes that pathogens are only found in diseased, not healthy, individuals. This is not true for many pathogens. For example, H. pylori, described earlier in this chapter as a pathogen causing chronic gastritis, is also part of the normal microbiota of the stomach in many healthy humans who never develop gastritis. It is estimated that upwards of 50% of the human population acquires H. pylori early in life, with most maintaining it as part of the normal microbiota for the rest of their life without ever developing disease.

Koch’s second faulty assumption was that all healthy test subjects are equally susceptible to disease. We now know that individuals are not equally susceptible to disease. Individuals are unique in terms of their microbiota and the state of their immune system at any given time. The makeup of the resident microbiota can influence an individual’s susceptibility to an infection. Members of the normal microbiota play an important role in immunity by inhibiting the growth of transient pathogens. In some cases, the microbiota may prevent a pathogen from establishing an infection; in others, it may not prevent an infection altogether but may influence the severity or type of signs and symptoms.

As a result, two individuals with the same disease may not always present with the same signs and symptoms. In addition, some individuals have stronger immune systems than others. Individuals with immune systems weakened by age or an unrelated illness are much more susceptible to certain infections than individuals with strong immune systems.

Koch also assumed that all pathogens are microorganisms that can be grown in pure culture (postulate 2) and that animals could serve as reliable models for human disease. However, we now know that not all pathogens can be grown in pure culture, and many human diseases cannot be reliably replicated in animal hosts. Viruses and certain bacteria, including Rickettsia and Chlamydia, are obligate intracellular pathogens that can grow only when inside a host cell. If a microbe cannot be cultured, a researcher cannot move past postulate 2. Likewise, without a suitable nonhuman host, a researcher cannot evaluate postulate 2 without deliberately infecting humans, which presents obvious ethical concerns. AIDS is an example of such a disease because the human immunodeficiency virus (HIV) only causes disease in humans.

Lastly, in regards to postulate 3, we now know that several diseases can be caused by different pathogens, common examples include pneumonia and meningitis, both of which have many bacterial and viral causes. In the same vein, we know that certain pathogens are capable of causing several different diseases, such as in the case of S. aureus, which causes a wide range of illnesses from skin disorders to gastrointestinal distress. In these cases, using Koch’s postulates would lead you to the wrong result.

Pathogenicity and Virulence: The ability of a microbial agent to cause disease is called pathogenicity, and the degree to which an organism is pathogenic is called virulence. Virulence is a continuum. On one end of the spectrum are organisms that are avirulent (not harmful) and on the other are organisms that are highly virulent. Highly virulent pathogens will almost always lead to a disease state when introduced to the body, and some may even cause multi-organ and body system failure in healthy individuals. Less virulent pathogens may cause an initial infection, but may not always cause severe illness. Pathogens with low virulence would more likely result in mild signs and symptoms of disease, such as low-grade fever, headache, or muscle aches. Some individuals might even be asymptomatic.

Virulence of a pathogen can be quantified using controlled experiments with laboratory animals. Two important indicators of virulence are the median infectious dose (ID50) and the median lethal dose (LD50), both of which are typically determined experimentally using animal models. The ID50 is the number of pathogen cells or virions required to cause active infection in 50% of inoculated animals. The LD50 is the number of pathogenic cells, virions, or amount of toxin required to kill 50% of infected animals. To calculate these values, each group of animals is inoculated with one of a range of known numbers of pathogen cells or virions. In graphs like the one shown in Figure 7.6, the percentage of animals that have been infected (for ID50) or killed (for LD50) is plotted against the concentration of pathogen inoculated. Figure 7.6 represents data graphed from a hypothetical experiment measuring the LD50 of a pathogen. Interpretation of the data from this graph indicates that the LD50 of the pathogen for the test animals is 104 pathogen cells or virions (depending upon the pathogen studied).

Table 7.3 lists selected foodborne pathogens and their ID50 values in humans (as determined from epidemiologic data and studies on human volunteers). Keep in mind that these are median values. The actual infective dose for an individual can vary widely, depending on factors such as route of entry; the age, health, and immune status of the host; and environmental and pathogen-specific factors such as susceptibility to the acidic pH of the stomach. It is also important to note that a pathogen’s infective dose does not necessarily correlate with disease severity. For example, just a single cell of Salmonella enterica serotype Typhimurium can result in an active infection. The resultant disease,

Salmonella gastroenteritis or salmonellosis, can cause nausea, vomiting, and diarrhea, but has a mortality rate of less than 1% in healthy adults. In contrast, S. enterica serotype Typhi has a much higher ID50, typically requiring as many as 1,000 cells to produce infection. However, this serotype causes typhoid fever, a much more systemic and severe disease that has a mortality rate as high as 10% in untreated individuals.

Primary Pathogens versus Opportunistic Pathogens: Pathogens can be classified as either primary pathogens or opportunistic pathogens. A primary pathogen can cause disease in a host regardless of the host’s resident microbiota or immune system. An opportunistic pathogen, by contrast, can only cause disease in situations that compromise the host’s defenses, such as the body’s protective barriers, immune system, or normal microbiota. Individuals susceptible to opportunistic infections include the very young, the elderly, women who are pregnant, patients undergoing chemotherapy, people with immunodeficiencies (such as acquired immunodeficiency syndrome [AIDS]), patients who are recovering from surgery, and those who have had a breach of protective barriers (such as a severe wound or burn).

An example of a primary pathogen is enterohemorrhagic E. coli (EHEC), which produces a virulence factor known as Shiga toxin. This toxin inhibits protein synthesis, leading to severe and bloody diarrhea, inflammation, and renal failure, even in patients with healthy immune systems. Staphylococcus epidermidis, on the other hand, is an opportunistic pathogen that is among the most frequent cause of hospital acquired disease. S. epidermidis is a member of the normal microbiota of the skin, where it is generally avirulent. However, in hospitals, it can also grow in biofilms that form on catheters, implants, or other devices that are inserted into the body during surgical procedures. Once inside the body, S. epidermidis can cause serious infections such as endocarditis, and it produces virulence factors that promote the persistence of such infections.

Members of the normal microbiota may also cause disease when a shift in the environment of the body leads to overgrowth of a particular microorganism. For example, the yeast Candida is part of the normal microbiota of the skin, mouth, intestine, and vagina, but its population is kept in check by other organisms of the microbiota. If an individual is taking antibacterial medications, however, bacteria that would normally inhibit the growth of Candida can be killed off, leading to a sudden growth in the population of Candida, which is not affected by antibacterial medications because it is a fungus. An overgrowth of Candida can manifest as oral thrush (growth on mouth, throat, and tongue), a vaginal yeast infection, or cutaneous candidiasis. Other scenarios can also provide opportunities for Candida infections. Untreated diabetes can result in a high concentration of glucose in the saliva, which provides an optimal environment for the growth of Candida, resulting in thrush. Immunodeficiencies such as those seen in patients with HIV, AIDS, and cancer also lead to higher incidence of thrush. Vaginal yeast infections can result from decreases in estrogen levels during the menstruation or menopause. The amount of glycogen available to lactobacilli in the vagina is controlled by levels of estrogen; when estrogen levels are low, lactobacilli produce less lactic acid. The resultant increase in vaginal pH allows overgrowth of Candida in the vagina.

Sources of Pathogens: Reservoirs and Carriers: For pathogens to persist over long periods of time they require reservoirs where they normally reside. Reservoirs can be living organisms or nonliving sites. Nonliving reservoirs can include soil and water in the environment. These may naturally harbor the organism because it may grow in that environment. These environments may also become contaminated with pathogens in human feces, pathogens shed by intermediate hosts, or pathogens contained in the remains of intermediate hosts.

Pathogens may have mechanisms of dormancy or resilience that allow them to survive (but typically not to reproduce) for varying periods of time in nonliving environments. For example, Clostridium tetani survives in the soil and in the presence of oxygen as a resistant endospore. Although many viruses are soon destroyed once in contact with air, water, or other non-physiological conditions, certain types are capable of persisting outside of a living cell for varying amounts of time. For example, a study that looked at the ability of influenza viruses to infect a cell culture after varying amounts of time on a banknote showed survival times from 48 hours to 17 days, depending on how they were deposited on the banknote. On the other hand, cold-causing rhinoviruses are somewhat fragile, typically surviving less than a day outside of physiological fluids.

A human acting as a reservoir of a pathogen may or may not be capable of transmitting the pathogen, depending on the stage of infection and the pathogen. An individual capable of transmitting a pathogen without displaying symptoms is referred to as a carrier. A passive carrier is contaminated with the pathogen and can mechanically transmit it to another host; however, a passive carrier is not infected. For example, a health-care professional who fails to wash his hands after seeing a patient harboring an infectious agent could become a passive carrier, transmitting the pathogen to another patient who becomes infected. By contrast, an active carrier is infected by the pathogen, but may transmit the disease during the incubation period (before they show signs and symptoms) or the period of convalescence (after symptoms have subsided). Pathogens such as hepatitis B virus, herpes simplex virus, and HIV are frequently transmitted by asymptomatic carriers.

A pathogen may have more than one living reservoir. In zoonotic diseases, animals act as reservoirs of human disease and transmit the infectious agent to humans through direct or indirect contact. In some cases, the disease also affects the animal, but in other cases the animal is asymptomatic.

Transmission: Regardless of the reservoir, transmission must occur for an infection to spread. First, transmission from the reservoir to the individual must occur. Then, the individual must transmit the infectious agent to other susceptible individuals, either directly or indirectly. Pathogenic microorganisms employ diverse transmission mechanisms.

1. Contact Transmission: Contact transmission includes direct contact or indirect contact. Person-to-person transmission is a form of direct contact transmission. Here the agent is transmitted by physical contact between two individuals (Figure 7.7) through actions such as touching, kissing, sexual intercourse, or droplet sprays. Often, contact between mucous membranes is required for entry of the pathogen into the new host, although skin-to-skin contact can lead to mucous membrane contact if the new host subsequently touches a mucous membrane. Contact transmission may also be site-specific; for example, some diseases can be transmitted by sexual contact but not by other forms of contact.

When an individual coughs or sneezes, small droplets of mucus that may contain pathogens are ejected. This leads to direct droplet transmission, which refers to droplet transmission of a pathogen to a new host over distances of one meter or less. A wide variety of diseases are transmitted by droplets, including influenza and many forms of pneumonia. Transmission over distances greater than one meter is called airborne transmission.

Indirect contact transmission involves inanimate objects called fomites that become contaminated by pathogens from an infected individual or reservoir (Figure 7.8). For example, an individual with the common cold may sneeze, causing droplets to land on a fomite such as a tablecloth or carpet, or the individual may wipe her nose and then transfer mucus to a fomite such as a doorknob or towel. Transmission occurs indirectly when a new susceptible host later touches the fomite and transfers the contaminated material to a susceptible portal of entry. Fomites can also include objects used in clinical settings that are not properly sterilized, such as syringes, needles, catheters, and surgical equipment. Pathogens transmitted indirectly via such fomites are a major cause of healthcare-associated infections.

2. Vehicle Transmission: The term vehicle transmission refers to the transmission of pathogens through vehicles such as water, food, and air. Water contamination through poor sanitation methods leads to waterborne transmission of disease. Waterborne disease remains a serious problem in many regions throughout the world. The World Health Organization (WHO) estimates that contaminated drinking water is responsible for more than 500,000 deaths each year. Similarly, food contaminated through poor handling or storage can lead to foodborne transmission of disease (Figure 7.9).

Dust and fine particles known as aerosols, which can float in the air, can carry pathogens and facilitate the airborne transmission of disease. For example, dust particles are the dominant mode of transmission of hantavirus to humans. Hantavirus is found in mouse feces, urine, and saliva, but when these substances dry, they can disintegrate into fine particles that can become airborne when disturbed; inhalation of these particles can lead to a serious and sometimes fatal respiratory infection.

Although droplet transmission over short distances is considered contact transmission as discussed above, longer distance transmission of droplets through the air is considered vehicle transmission. Unlike larger particles that drop quickly out of the air column, fine mucus droplets produced by coughs or sneezes can remain suspended for long periods of time, traveling considerable distances. In certain conditions, droplets desiccate quickly to produce a droplet nucleus that is capable of transmitting pathogens; air temperature and humidity can have an impact on effectiveness of airborne transmission.

Tuberculosis is often transmitted via airborne transmission when the causative agent, Mycobacterium tuberculosis, is released in small particles with coughs. Because tuberculosis requires as few as 10 microbes to initiate a new infection, patients with tuberculosis must be treated in rooms equipped with special ventilation, and anyone entering the room should wear a mask.

3. Vector Transmission: Diseases can also be transmitted by a mechanical or biological vector, an animal (typically an arthropod) that carries the disease from one host to another. Mechanical transmission is facilitated by a mechanical vector, an animal that carries a pathogen from one host to another without being infected itself. For example, a fly may land on fecal matter and later transmit bacteria from the feces to food that it lands on; a human eating the food may then become infected by the bacteria, resulting in a case of diarrhea or dysentery (Figure 7.10).

Biological transmission occurs when the pathogen reproduces within a biological vector that transmits the pathogen from one host to another (Figure 7.10). Arthropods are the main vectors responsible for biological transmission. Most arthropod vectors transmit the pathogen by biting the host, creating a wound that serves as a portal of entry. The pathogen may go through part of its reproductive cycle in the gut or salivary glands of the arthropod to facilitate its transmission through the bite. For example, hemipterans (called “kissing bugs” or “assassin bugs”) transmit Chagas disease to humans by defecating when they bite, after which the human scratches or rubs the infected feces into a mucous membrane or break in the skin.

Biological insect vectors include mosquitoes, which transmit malaria and other diseases, and lice, which transmit typhus. Other arthropod vectors can include arachnids, primarily ticks, which transmit Lyme disease and other diseases, and mites, which transmit scrub typhus and rickettsial pox. Biological transmission, because it involves survival and reproduction within a parasitized vector, complicates the biology of the pathogen and its transmission. There are also important non-arthropod vectors of disease, including mammals and birds. Various species of mammals can transmit rabies to humans, usually by means of a bite that transmits the rabies virus. Chickens and other domestic poultry can transmit avian influenza to humans through direct or indirect contact with avian influenza virus A shed in the birds’ saliva, mucous, and feces.

Stages of Pathogenesis: To cause disease, a pathogen must successfully achieve four steps or stages of pathogenesis: exposure (contact), adhesion (colonization), invasion, and infection. The pathogen must be able to gain entry to the host, travel to the location where it can establish an infection, evade or overcome the host’s immune response, and cause damage (i.e., disease) to the host. In many cases, the cycle is completed when the pathogen exits the host and is transmitted to a new host.

1. Exposure: An encounter with a potential pathogen is known as exposure or contact. The food we eat and the objects we handle are all ways that we can come into contact with potential pathogens. Yet, not all contacts result in infection and disease. For a pathogen to cause disease, it needs to be able to gain access into host tissue. An anatomic site through which pathogens can pass into host tissue is called a portal of entry. These are locations where the host cells are in direct contact with the external environment. Major portals of entry are identified in Figure 7.11 and include the skin, mucous membranes, and parenteral routes.

Mucosal surfaces are the most important portals of entry for microbes; these include the mucous membranes of the respiratory tract, the gastrointestinal tract, and the genitourinary tract. Although most mucosal surfaces are in the interior of the body, some are contiguous with the external skin at various body openings, including the eyes, nose, mouth, urethra, and anus.

Most pathogens are suited to a particular portal of entry. A pathogen’s portal specificity is determined by the organism’s environmental adaptions and by the enzymes and toxins they secrete. The respiratory and gastrointestinal tracts are particularly vulnerable portals of entry because particles that include microorganisms are constantly inhaled or ingested, respectively.

Pathogens can also enter through a breach in the protective barriers of the skin and mucous membranes. Pathogens that enter the body in this way are said to enter by the parenteral route. For example, the skin is a good natural barrier to pathogens, but breaks in the skin (e.g., wounds, insect bites, animal bites, needle pricks) can provide a parenteral portal of entry for microorganisms.

2. Adhesion: Following the initial exposure, the pathogen adheres at the portal of entry. The term adhesion refers to the capability of pathogenic microbes to attach to the cells of the body using adhesion factors, and different pathogens use various mechanisms to adhere to the cells of host tissues. Molecules (either proteins or carbohydrates) called adhesins are found on the surface of certain pathogens and bind to specific receptors (glycoproteins) on host cells. Adhesins are present on the fimbriae and flagella of bacteria, the cilia of protozoa, and the capsids or membranes of viruses. Protozoans can also use hooks and barbs for adhesion; spike proteins on viruses also enhance viral adhesion. The production of glycocalyces (slime layers and capsules), with their high sugar and protein content, can also allow certain bacterial pathogens to attach to cells.

Biofilm growth can also act as an adhesion factor. A biofilm is a community of bacteria that produce a glycocalyx, known as extrapolymeric substance (EPS), that allows the biofilm to attach to a surface. Persistent Pseudomonas aeruginosa infections are common in patients suffering from cystic fibrosis, burn wounds, and middle-ear infections (otitis media) because P. aeruginosa produces a biofilm. The EPS allows the bacteria to adhere to the host cells and makes it harder for the host to physically remove the pathogen. The EPS not only allows for attachment but provides protection against the immune system and antibiotic treatments, preventing antibiotics from reaching the bacterial cells within the biofilm. In addition, not all bacteria in a biofilm are rapidly growing; some are in stationary phase.

Since antibiotics are most effective against rapidly growing bacteria, portions of bacteria in a biofilm are protected against antibiotics.

3. Invasion: Once adhesion is successful, invasion can proceed. Invasion involves the dissemination of a pathogen throughout local tissues or the body. Pathogens may produce exoenzymes or toxins, which serve as virulence factors that allow them to colonize and damage host tissues as they spread deeper into the body. Pathogens may also produce virulence factors that protect them against immune system defenses. A pathogen’s specific virulence factors determine the degree of tissue damage that occurs. Figure 7.12 shows the invasion of H. pylori into the tissues of the stomach, causing damage as it progresses.

Intracellular pathogens achieve invasion by entering the host’s cells and reproducing. Some are obligate intracellular pathogens (meaning they can only reproduce inside of host cells) and others are facultative intracellular pathogens (meaning they can reproduce either inside or outside of host cells). By entering the host cells, intracellular pathogens are able to evade some mechanisms of the immune system while also exploiting the nutrients in the host cell.

Entry to a cell can occur by endocytosis. For most kinds of host cells, pathogens use one of two different mechanisms for endocytosis and entry. One mechanism relies on effector proteins secreted by the pathogen; these effector proteins trigger entry into the host cell. This is the method that Salmonella and Shigella use when invading intestinal epithelial cells. When these pathogens come in contact with epithelial cells in the intestine, they secrete effector molecules that cause protrusions of membrane ruffles that bring the bacterial cell in. This process is called membrane ruffling. The second mechanism relies on surface proteins expressed on the pathogen that bind to receptors on the host cell, resulting in entry. For example, Yersinia pseudotuberculosis produces a surface protein known as invasin that binds to proteins expressed on the surface of host cells.

Some host cells, such as white blood cells and other phagocytes of the immune system, actively endocytose pathogens in a process called phagocytosis. Although phagocytosis allows the pathogen to gain entry to the host cell, in most cases, the host cell kills and degrades the pathogen by using digestive enzymes. Normally, when a pathogen is ingested by a phagocyte, it is enclosed within a phagosome in the cytoplasm; the phagosome fuses with a lysosome to form a phagolysosome, where digestive enzymes kill the pathogen (see Pathogen Recognition and Phagocytosis). However, some intracellular pathogens have the ability to survive and multiply within phagocytes. Examples include Listeria monocytogenes and Shigella; these bacteria produce proteins that lyse the phagosome before it fuses with the lysosome, allowing the bacteria to escape into the phagocyte’s cytoplasm where they can multiply. Bacteria such as Mycobacterium tuberculosis, Legionella pneumophila, and Salmonella species use a slightly different mechanism to evade being digested by the phagocyte. These bacteria prevent the fusion of the phagosome with the lysosome, thus remaining alive and dividing within the phagosome.

4. Infection: Following invasion, successful multiplication of the pathogen leads to infection. Infections can be described as local, focal, or systemic, depending on the extent of the infection. A local infection is confined to a small area of the body, typically near the portal of entry. For example, a hair follicle infected by Staphylococcus aureus infection may result in a boil around the site of infection, but the bacterium is largely contained to this small location. Other examples of local infections that involve more extensive tissue involvement include urinary tract infections confined to the bladder or pneumonia confined to the lungs.

In a focal infection, a localized pathogen, or the toxins it produces, can spread to a secondary location. For example, a dental hygienist nicking the gum with a sharp tool can lead to a local infection in the gum by Streptococcus bacteria of the normal oral microbiota. These Streptococcus spp. may then gain access to the bloodstream and make their way to other locations in the body, resulting in a secondary infection.

When an infection becomes disseminated throughout the body, we call it a systemic infection. For example, infection by the varicella-zoster virus typically gains entry through a mucous membrane of the upper respiratory system. It then spreads throughout the body, resulting in the classic red skin lesions associated with chickenpox. Since these lesions are not sites of initial infection, they are signs of a systemic infection.

Sometimes a primary infection, the initial infection caused by one pathogen, can lead to a secondary infection by another pathogen. For example, the immune system of a patient with a primary infection by HIV becomes compromised, making the patient more susceptible to secondary diseases like oral thrush and others caused by opportunistic pathogens. Similarly, a primary infection by Influenza virus damages and decreases the defense mechanisms of the lungs, making patients more susceptible to a secondary pneumonia by a bacterial pathogen like Haemophilus influenzae or Streptococcus pneumoniae. Some secondary infections can even develop as a result of treatment for a primary infection. Antibiotic therapy targeting the primary pathogen can cause collateral damage to the normal microbiota, creating an opening for opportunistic pathogens.

5 Leaving the host: For a pathogen to persist, it must put itself in a position to be transmitted to a new host, leaving the infected host through a portal of exit. As with portals of entry, many pathogens are adapted to use a particular portal of exit. Similar to portals of entry, the most common portals of exit include the skin and the respiratory, urogenital, and gastrointestinal tracts. Coughing and sneezing can expel pathogens from the respiratory tract. A single sneeze can send thousands of virus particles into the air. Secretions and excretions can transport pathogens out of other portals of exit. Feces, urine, semen, vaginal secretions, tears, sweat, and shed skin cells can all serve as vehicles for a pathogen to leave the body. Pathogens that rely on insect vectors for transmission exit the body in the blood extracted by a biting insect. Similarly, some pathogens exit the body in blood extracted by needles.

Bacterial Exoenzymes and Toxins as Virulence Factors: In a 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. This virulence might be conferred by features such as having capsules, to evade phagocytosis, having adhesins or fimbriae to aid in attachment, or by living in biofilms. Virulence can also be increased due to molecules produced by the microbes themselves.

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. For example, 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 7.13). 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, multiorgan failure, shock, and death can occur.

1. 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 7.4. 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 7.14). This allows the pathogen to pass through the tissue layers at the portal of entry and disseminate elsewhere in the body (Figure 7.14).

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. 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 (hemolysis), white blood cells, and tissue cells.

Bacterial pathogens also produce various protein-digesting enzymes, or proteases. 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) (Figure 7.15).

2. 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 and the presence of toxin in the blood is known as toxemia. Toxins can be categorized as endotoxins or exotoxins. The lipopolysaccharide (LPS) found on the outer membrane of gram-negative bacteria is called endotoxin (Figure 7.16). 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. 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 (shock), multi-organ failure, and death. Neisseria meningitidis, the bacterium responsible for meningococcal meningitis, contains an endotoxin that results in septic shock and hemorrhage due to the destruction of red blood cells.

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 7.5. 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).

The exotoxins can be grouped into three categories based on their target: intracellular targeting, membrane disrupting, and superantigens. Table 7.6 provides examples of well-characterized toxins within each of these three categories.

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 7.17). 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.

Three unique examples of A-B toxins are the cholera, botulinum, and tetanus toxins. 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 the

“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 7.18 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 also membrane-disrupting toxins that degrade the phospholipid bilayer of cell membranes rather than forming pores and can function as hemolysins.

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.

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 7.19). 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 non-encapsulated 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 7.19).

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.