Chapter 25:  Pathogens, Infection, and Innate Immunity

Pathogens Have Evolved Specific Mechanisms for Interacting with Their Hosts

The human body is a complex and thriving ecosystem. It contains about 10¹³ human cells and also about 10¹⁴ bacterial, fungal, and protozoan cells, which represent thousands of microbial species. These microbes, called the normal flora, are usually limited to certain areas of the body, including the skin, mouth, large intestine, and vagina. In addition, humans are always infected with viruses, most of which rarely, if ever, become symptomatic. If it is normal for us to live in such close intimacy with a wide variety of microbes, how is it that some of them are capable of causing us illness or death?

Pathogens are usually distinct from the normal flora. Our normal microbial inhabitants only cause trouble if our immune systems are weakened or if they gain access to a normally sterile part of the body (for example, when a bowel perforation enables the gut flora to enter the peritoneal cavity of the abdomen, causing peritonitis). In contrast, dedicated pathogens do not require that the host be immunocompromised or injured. They have developed highly specialized mechanisms for crossing cellular and biochemical barriers and for eliciting specific responses from the host organism that contribute to the survival and multiplication of the pathogen.

In order to survive and multiply in a host, a successful pathogen must be able to: (1) colonize the host; (2) find a nutritionally compatible niche in the host body; (3) avoid, subvert, or circumvent the host innate and adaptive immune responses; (4) replicate, using host resources; and (5) exit and spread to a new host. Under severe selective pressure to induce only the correct host cell responses to accomplish this complex set of tasks, pathogens have evolved mechanisms that maximally exploit the biology of their host organisms. Many of the pathogens we discuss in this chapter are skillful and practical cell biologists. We can learn a great deal of cell biology by observing them.

The Signs and Symptoms of Infection May Be Caused by the Pathogen or by the Host's Responses

Although we can easily understand why infectious microorganisms would evolve to reproduce in a host, it is less clear why they would evolve to cause disease. One explanation may be that, in some cases, the pathological responses elicited by microorganisms enhance the efficiency of their spread or propagation and hence clearly have a selective advantage for the pathogen. The virus-containing lesions on the genitalia caused by herpes simplex infection, for example, facilitate direct spread of the virus from an infected host to an uninfected partner during sexual contact. Similarly, diarrheal infections are efficiently spread from patient to caretaker. In many cases, however, the induction of disease has no apparent advantage for the pathogen.

Many of the symptoms and signs that we associate with infectious disease are direct manifestations of the host's immune responses in action. Some hallmarks of bacterial infection, including the swelling and redness at the site of infection and the production of pus (mainly dead white blood cells), are the direct result of immune system cells attempting to destroy the invading microorganisms. Fever, too, is a defensive response, as the increase in body temperature can inhibit the growth of some microorganisms. Thus, understanding the biology of an infectious disease requires an appreciation of the contributions of both pathogen and host.

Pathogens Are Phylogenetically Diverse

Many types of pathogens cause disease in humans. The most familiar are viruses and bacteria. Viruses cause diseases ranging from AIDS and smallpox to the common cold. They are essentially fragments of nucleic acid (DNA or RNA) instructions, wrapped in a protective shell of proteins and (in some cases) membrane (Figure 25-2A). They use the basic transcription and translation machinery of their host cells for their replication.

Of all the bacteria we encounter in our lives, only a small minority are dedicated pathogens. Much larger and more complex than viruses, bacteria are usually free-living cells, which perform most of their basic metabolic functions themselves, relying on the host primarily for nutrition (Figure 25-2B).

Some other infectious agents are eucaryotic organisms. These range from single-celled fungi and protozoa (Figure 25-2C), through large complex metazoa such as parasitic worms. One of the most common infectious diseases on the planet, shared by about a billion people at present, is an infestation in the gut by Ascaris lumbricoides. This nematode closely resembles its cousin Caenorhabditis elegans, which is widely used as a model organism for genetic and developmental biological research (discussed in Chapter 21). C. elegans, however, is only about 1 mm in length, whereas Ascaris can reach 30 cm (Figure 25-2D).

Some rare neurodegenerative diseases, including mad cow disease, are caused by an unusual type of infectious particle called a prion, which is made only of protein. Although the prion contains no genome, it can nevertheless replicate and kill the host.

Even within each class of pathogen, there is striking diversity. Viruses vary tremendously in their size, shape, and content (DNA versus RNA, enveloped or not, and so on), and the same is true for the other pathogens. The ability to cause disease (pathogenesis) is a lifestyle choice, not a legacy shared only among close relatives (Figure 25-3).

Each individual pathogen causes disease in a different way, which makes it challenging to understand the basic biology of infection. But, when considering the interactions of infectious agents with their hosts, some common themes of pathogenesis emerge. These common themes are the focus of this chapter. First, we introduce the basic features of each of the major types of pathogens that exploit features of host cell biology. Then, we examine in turn the mechanisms that pathogens use to control their hosts and the innate mechanisms that hosts use to control pathogens.

Bacterial Pathogens Carry Specialized Virulence Genes

Bacteria are small and structurally simple, compared to the vast majority of eucaryotic cells. Most can be classified broadly by their shape as rods, spheres, or spirals and by their cell-surface properties. Although they lack the elaborate morphological variety of eucaryotic cells, they display a surprising array of surface appendages that enable them to swim or to adhere to desirable surfaces (Figure 25-4). Their genomes are correspondingly simple, typically on the order of 1,000,000–5,000,000 nucleotide pairs in size (compared to 12,000,000 for yeast and more than 3,000,000,000 for humans).

As emphasized above, only a minority of bacterial species have developed the ability to cause disease in humans. Some of those that do cause disease can only replicate inside the cells of the human body and are called obligate pathogens. Others replicate in an environmental reservoir such as water or soil and only cause disease if they happen to encounter a susceptible host; these are called facultative pathogens. Many bacteria are normally benign but have a latent ability to cause disease in an injured or immunocompromised host; these are called opportunistic pathogens.

Some bacterial pathogens are fastidious in their choice of host and will only infect a single species or a group of related species, whereas others are generalists. Shigella flexneri, for example, which causes epidemic dysentery (bloody diarrhea) in areas of the world lacking a clean water supply, will only infect humans and other primates. By contrast, the closely related bacterium Salmonella enterica, which is a common cause of food poisoning in humans, can also infect many other vertebrates, including chickens and turtles. A champion generalist is the opportunistic pathogen Pseudomonas aeruginosa, which is capable of causing disease in plants as well as animals.

The significant differences between a virulent pathogenic bacterium and its closest nonpathogenic relative may result from a very small number of genes. Genes that contribute to the ability of an organism to cause disease are called virulence genes. The proteins they encode are called virulence factors. Virulence genes are frequently clustered together, either in groups on the bacterial chromosome called pathogenicity islands or on extrachromosomal virulence plasmids (Figure 25-5). These genes may also be carried on mobile bacteriophages (bacterial viruses). It seems therefore that a pathogen may arise when groups of virulence genes are transferred together into a previously avirulent bacterium. Consider, for example, Vibrio cholerae—the bacterium that causes cholera. Several of the genes encoding the toxins that cause the diarrhea in cholera are carried on a mobile bacteriophage (Figure 25-6). Of the hundreds of strains of Vibrio cholerae found in lakes in the wild, the only ones that cause human disease are those that have become infected with this virus.

Many virulence genes encode proteins that interact directly with host cells. Two of the genes carried by the Vibrio cholerae phage, for example, encode two subunits of cholera toxin. The B subunit of this secreted, toxic protein binds to a glycolipid component of the plasma membrane of the epithelial cells in the gut of a person who has consumed Vibrio cholerae in contaminated water. The B subunit transfers the A subunit through the membrane into the epithelial cell cytoplasm. The A subunit is an enzyme that catalyzes the transfer of an ADP-ribose moiety from NAD to the trimeric G protein G_s, which normally activates adenylyl cyclase to make cyclic AMP (discussed in Chapter 15). ADP-ribosylation of the G protein results in an overaccumulation of cyclic AMP and an ion imbalance, leading to the massive watery diarrhea associated with cholera. The infection is then spread by the fecal-oral route by contaminated food and water.

Some pathogenic bacteria use several independent mechanisms to cause toxicity to the cells of their host. Anthrax, for example, is an acute infectious disease of sheep, cattle, other herbivores, and occasionally humans. It is usually caused by contact with spores of the Gram-positive bacterium, Bacillus anthracis. Unlike cholera, anthrax has never been observed to spread directly from one infected person to another. Dormant spores can survive in soil for long periods of time and are highly resistant to adverse environmental conditions, including heat, ultraviolet and ionizing radiation, pressure, and chemical agents. After the spores are inhaled, ingested, or rubbed into breaks in the skin, the spores germinate, and the bacteria begin to replicate. Growing bacteria secrete two toxins, called lethal toxin and edema toxin. Either toxin alone is sufficient to cause signs of infection. Like the A and B subunits of cholera toxin, both toxins are made of two subunits. The B subunit is identical between lethal toxin and edema toxin, and it binds to a host cell-surface receptor to transfer the two different A subunits into host cells. The A subunit of edema toxin is an adenylyl cyclase that directly converts host cell ATP into cyclic AMP. This causes an ion imbalance that can lead to accumulation of extracellular fluid (edema) in the infected skin or lung. The A subunit of lethal toxin is a zinc protease that cleaves several members of the MAP kinase kinase family (discussed in Chapter 15). Injection of lethal toxin into the bloodstream of an animal causes shock and death. The molecular mechanisms and the sequence of events leading to death in anthrax remain uncertain.

These examples illustrate a common theme among virulence factors. They are frequently either toxic proteins (toxins) that directly interact with important host structural or signaling proteins to elicit a host cell response that is beneficial to pathogen colonization or replication, or they are proteins that are needed to deliver such toxins to their host cell targets. One common and particularly efficient delivery mechanism, called the type III secretion system, acts like a tiny syringe that injects toxic proteins from the cytoplasm of an extracellular bacterium directly into the cytoplasm of an adjacent host cell (Figure 25-7). There is a remarkable degree of structural similarity between the type III syringe and the base of a bacterial flagellum (see Figure 15-67), and many of the proteins in the two structures are clearly homologous.

Because bacteria form a kingdom distinct from the eucaryotes they infect (see Figure 25-3), much of their basic machinery for DNA replication, transcription, translation, and fundamental metabolism is quite different from that of their host. These differences enable us to find antibacterial drugs that specifically inhibit these processes in bacteria, without disrupting them in the host. Most of the antibiotics that we use to treat bacterial infections are small molecules that inhibit macromolecular synthesis in bacteria by targeting bacterial enzymes that are either distinct from their eucaryotic counterparts or that are involved in pathways, such as cell wall biosynthesis, that are absent in humans (Figure 25-8 and Table 6-3).

Fungal and Protozoan Parasites Have Complex Life Cycles with Multiple Forms

Pathogenic fungi and protozoan parasites are eucaryotes. It is therefore more difficult to find drugs that will kill them without killing the host. Consequently, antifungal and antiparasitic drugs are often less effective and more toxic than antibiotics. A second characteristic of fungal and parasitic infections that makes them difficult to treat is the tendency of the infecting organisms to switch among several different forms during their life cycles. A drug that is effective at killing one form is often ineffective at killing another form, which therefore survives the treatment.

The fungal branch of the eucaryotic kingdom includes both unicellular yeasts (such as Saccharomyces cerevisiae and Schizosaccharomyces pombe) and filamentous, multicellular molds (like those found on moldy fruit or bread). Most of the important pathogenic fungi exhibit dimorphism—the ability to grow in either yeast or mold form. The yeast-to-mold or mold-to-yeast transition is frequently associated with infection. Histoplasma capsulatum, for example, grows as a mold at low temperature in the soil, but it switches to a yeast form when inhaled into the lung, where it can cause the disease histoplasmosis (Figure 25-9).

Protozoan parasites have more elaborate life cycles than do fungi. These cycles frequently require the services of more than one host. Malaria is the most common protozoal disease, infecting 200–300 million people every year and killing 1–3 million of them. It is caused by four species of Plasmodium, which are transmitted to humans by the bite of the female of any of 60 species of Anopheles mosquito. Plasmodium falciparum—the most intensively studied of the malaria-causing parasites—exists in no fewer than eight distinct forms, and it requires both the human and mosquito hosts to complete its sexual cycle (Figure 25-10). Gametes are formed in the bloodstream of infected humans, but they can only fuse to form a zygote in the gut of the mosquito. Three of the Plasmodium forms are highly specialized to invade and replicate in specific tissues—the insect gut lining, the human liver, and the human red blood cell.

Because malaria is so widespread and devastating, it has acted as a strong selective pressure on human populations in areas of the world that harbor the Anopheles mosquito. Sickle cell anemia, for example, is a recessive genetic disorder caused by a point mutation in the gene that encodes the hemoglobin β chain, and it is common in areas of Africa with a high incidence of the most serious form of malaria (caused by Plasmodium falciparum). The malarial parasites grow poorly in red blood cells from either homozygous sickle cell patients or healthy heterozygous carriers, and, as a result, malaria is seldom found among carriers of this mutation. For this reason, malaria has maintained the sickle cell mutation at high frequency in these regions of Africa.

Viruses Exploit Host Cell Machinery for All Aspects of Their Multiplication

Bacteria, fungi, and eucaryotic parasites are cells themselves. Even when they are obligate parasites, they use their own machinery for DNA replication, transcription, and translation, and they provide their own sources of metabolic energy. Viruses, by contrast, are the ultimate hitchhikers, carrying little more than information in the form of nucleic acid. The information is largely replicated, packaged, and preserved by the host cells (Figure 25-11). Viruses have a small genome, made up of a single nucleic acid type—either DNA or RNA—which, in either case, may be single-stranded or double-stranded. The genome is packaged in a protein coat, which in some viruses is further enclosed by a lipid envelope.

Viruses replicate in various ways. In general, replication involves (1) disassembly of the infectious virus particle, (2) replication of the viral genome, (3) synthesis of the viral proteins by the host cell translation machinery, and (4) reassembly of these components into progeny virus particles. A single virus particle (a virion) that infects a single host cell can produce thousands of progeny in the infected cell. Such prodigious viral multiplication is often enough to kill the host cell: the infected cell breaks open (lyses) and thereby allows the progeny viruses access to nearby cells. Many of the clinical manifestations of viral infection reflect this cytolytic effect of the virus. Both the cold sores formed by herpes simplex virus and the lesions caused by the smallpox virus, for example, reflect the killing of the epidermal cells in a local area of infected skin.

Viruses come in a wide variety of shapes and sizes, and, unlike cellular life forms, they cannot be systematically classified by their relatedness into a single phylogenetic tree. Because of their tiny sizes, complete genome sequences have been obtained for nearly all clinically important viruses. Poxviruses are among the largest, up to 450 nm long, which is about the size of some small bacteria. Their genome of double-stranded DNA consists of about 270,000 nucleotide pairs. At the other end of the size scale are parvoviruses, which are less than 20 nm long and have a single-stranded DNA genome of under 5000 nucleotides (Figure 25-12). The genetic information in a virus can be carried in a variety of unusual nucleic acid forms (Figure 25-13).

The capsid that encloses the viral genome is made of one or several proteins, arranged in regularly repeating layers and patterns. In enveloped viruses, the capsid itself is enclosed by a lipid bilayer membrane that is acquired in the process of budding from the host cell plasma membrane (Figure 25-14). Whereas nonenveloped viruses usually leave an infected cell by lysing it, an enveloped virus can leave the cell by budding, without disrupting the plasma membrane and, therefore, without killing the cell. These viruses can cause chronic infections, and some can help transform an infected cell into a cancer cell.

Despite this variety, all viral genomes encode three types of proteins: proteins for replicating the genome, proteins for packaging the genome and delivering it to more host cells, and proteins that modify the structure or function of the host cell to suit the needs of the virus (Figure 25-15). In the second section of this chapter, we focus primarily on this third class of viral proteins.

Since most of the critical steps in viral replication are performed by host cell machinery, the identification of effective antiviral drugs is particularly problematic. Whereas the antibiotic tetracycline specifically poisons bacterial ribosomes, for example, it will not be possible to find a drug that specifically poisons viral ribosomes, as viruses use the ribosomes of the host cell to make their proteins. The best strategy for containing viral diseases is to prevent them by vaccination of the potential hosts. Highly successful vaccination programs have effectively eliminated smallpox from the planet, and the eradication of poliomyelitis is imminent (Figure 25-16).

Prions Are Infectious Proteins

All information in biological systems is encoded by structure. We are used to thinking of biological information in the form of nucleic acid sequences (as in our description of viral genomes), but the sequence itself is a shorthand code for describing nucleic acid structure. The replication and expression of the information encoded in DNA and RNA are strictly dependent on the structure of these nucleic acids and their interactions with other macromolecules. The propagation of genetic information primarily requires that the information be stored in a structure that can be duplicated from unstructured precursors. Nucleic acid sequences are the simplest and most robust solution that organisms have found to the problem of faithful structural replication.

Nucleic acids are not the only solution, however. Prions are infectious agents that are replicated in the host by copying an aberrant protein structure. They can occur in yeasts, and they cause various neurodegenerative diseases in mammals. The most well-known infection caused by prions is bovine spongiform encephalopathy (BSE, or mad cow disease), which occasionally spreads to humans who eat infected parts of the cow (Figure 25-17). Isolation of the infectious prions that cause the disease scrapie in sheep, followed by years of painstaking laboratory characterization of scrapie-infected mice, eventually established that the protein itself is infectious.

Intriguingly, the infectious prion protein is made by the host, and its amino acid sequence is identical to a normal host protein. Moreover, the prion and normal forms of the protein are indistinguishable in their posttranslational modifications. The only difference between them appears to be in their folded three-dimensional structure. The misfolded prion protein tends to aggregate, and it has the remarkable capacity to cause the normal protein to adopt its misfolded prion conformation and thereby to become infectious (see Figure 6-89). This ability of the prion to convert the normal host protein to misfolded prion protein is equivalent to the prion's having replicated itself in the host. If eaten by another susceptible host, these newly-misfolded prions can transmit the infection.

It is not known how normal proteins are usually able to find the single, correct, folded conformation, among the billions of other possibilities, without becoming stuck in dead-end intermediates (discussed in Chapters 3 and 6). Prions are a good example of how protein folding can go dangerously wrong. But, why are the prion diseases so uncommon? What are the constraints that determine whether a misfolded protein will behave like a prion, or simply get refolded or degraded by the cell that made it? We do not yet have answers to these questions, and the study of prions remains an area of intense research.

Summary

Infectious diseases are caused by pathogens, which include bacteria, fungi, protozoa, worms, viruses, and even infectious proteins called prions. Pathogens of all classes must have mechanisms for entering their host and for evading immediate destruction by the host immune system. Most bacteria are not pathogenic. Those that are contain specific virulence genes that mediate interactions with the host, eliciting particular responses from the host cells that promote the replication and spread of the pathogen. Pathogenic fungi, protozoa, and other eucaryotic parasites typically pass through several different forms during the course of infection; the ability to switch among these forms is usually required for the parasites to be able to survive in a host and cause disease. In some cases, such as malaria, parasites must pass sequentially through several host species to complete their life cycles. Unlike bacteria and eucaryotic parasites, viruses have no metabolism of their own and no intrinsic ability to produce the proteins encoded by their DNA or RNA genomes. They rely entirely on subverting the machinery of the host cell to produce their proteins and to replicate their genomes. Prions, the smallest and simplest infectious agents, contain no nucleic acid; instead, they are rare, aberrantly folded proteins that happen to catalyze the misfolding of proteins in the host that share their primary amino acid sequence.

Pathogens Cross Protective Barriers to Colonize the Host

The first step in infection is for the pathogen to colonize the host. Most parts of the human body are well-protected from the environment by a thick and fairly tough covering of skin. The protective boundaries in some other human tissues (eyes, nasal passages and respiratory tract, mouth and digestive tract, urinary tract, and female genital tract) are less robust. For example, in the lungs and small intestine where oxygen and nutrients, respectively, are absorbed from the environment, the barrier is just a single monolayer of epithelial cells.

Skin and many other barrier epithelial surfaces are usually densely populated by normal flora. Some bacterial and fungal pathogens also colonize these surfaces and attempt to outcompete the normal flora, but most of them (as well as all viruses) avoid such competition by crossing these barriers to gain access to unoccupied niches within the host.

Wounds in barrier epithelia, including the skin, allow pathogens direct access to the interior of the host. This avenue of entry requires little in the way of specialization on the part of the pathogen. Indeed, many members of the normal flora can cause serious illness if they enter through such wounds. Anaerobic bacteria of the genus Bacteroides, for example, are carried as harmless flora at very high density in the large intestine, but they can cause life-threatening peritonitis if they enter the peritoneal cavity through a perforation in the intestine caused by trauma, surgery, or infection in the intestinal wall. Staphylococcus from the skin and nose, or Streptococcus from the throat and mouth, are also responsible for many serious infections resulting from breaches in epithelial barriers.

Dedicated pathogens, however, need not wait for a well-timed wound to allow them access to their host. A particularly efficient way for a pathogen to cross the skin is to catch a ride in the saliva of a biting insect. Many arthropods nourish themselves by sucking blood, and a diverse group of bacteria, viruses, and protozoa have developed the ability to survive in the arthropod so that they can use these biting animals as vectors to spread from one mammalian host to another. As discussed earlier, the Plasmodium protozoan that causes malaria develops through several forms in its life cycle, including some that are specialized for survival in a human and some that are specialized for survival in a mosquito (see Figure 25-10). Viruses that are spread by insect bites include the causative agents for several types of hemorrhagic fever, including yellow fever and Dengue fever, as well as the causative agents for many kinds of viral encephalitis (inflammation of the brain). All these viruses have acquired the ability to replicate in both insect cells and mammalian cells, as is required for a virus to be transmitted by an insect vector. Bloodborne viruses such as HIV that are not capable of replicating in insect cells are rarely, if ever, spread from insect to human.

The efficient spread of a pathogen via an insect vector requires that individual insects consume blood meals from numerous mammalian hosts. In a few striking cases, the pathogen appears to alter the behavior of the insect so that its transmission is more likely. Like most animals, the tsetse fly (whose bite spreads the protozoan parasite Trypanosoma brucei which causes sleeping sickness in Africa) stops eating when it is satiated. But tsetse flies carrying trypanosomes bite much more frequently and ingest more blood than do uninfected flies. The presence of trypanosomes impairs the function of the insect mechanoreceptors that measure blood flow through the gullet to assess the fullness of the stomach, effectively fooling the tsetse fly into thinking that it is still hungry. The bacterium Yersinia pestis, which causes bubonic plague, uses a different mechanism to ensure that a flea carrying it bites repeatedly: it multiplies in the flea's foregut to form aggregated masses that eventually enlarge and physically block the digestive tract. The insect is then unable to feed normally and begins to starve. During repeated attempts to satisfy its appetite, some of the bacteria in the foregut are flushed into the bite site, thus transmitting plague to a new host (Figure 25-18).

Pathogens That Colonize Epithelia Must Avoid Clearance by the Host

Hitching a ride through the skin on an insect proboscis is just one strategy that pathogens use to pass through the initial barriers of host defense. Whereas many barrier zones such as the skin, mouth, and large intestine, are densely populated by normal flora, others including the lower lung, the small intestine, and the bladder, are normally kept nearly sterile, despite relatively direct access to the environment. The epithelium in these zones actively resists bacterial colonization. As discussed in Chapter 22, the respiratory epithelium is covered with a layer of protective mucus, and the coordinated beating of cilia sweeps the mucus and trapped bacteria and debris up and out of the lung. The epithelia lining the bladder and the upper gastrointestinal tract also have a thick layer of mucus, and these organs are periodically flushed by micturition and peristalsis, respectively, to wash away undesirable microbes. The pathogenic bacteria and parasites that infect these epithelial surfaces have specific mechanisms for overcoming these host cleaning mechanisms. Those that infect the urinary tract, for example, resist the washing action of urine by adhering tightly to the bladder epithelium via specific adhesins, proteins or protein complexes that recognize and bind to host cell-surface molecules. An important group of adhesins in uropathogenic E. coli strains are components of the P pili (see Figure 25-4E) that help the bacteria infect the kidney. These surface projections can be several micrometers long and are thus able to span the thickness of the protective mucus layer. At the tip of each pilus is a protein that binds tightly to a particular disaccharide linked to a glycolipid that is found on the surface of cells in the bladder and kidney (Figure 25-19)

One of the hardest organs for a microbe to colonize is the stomach. Besides peristaltic washing and protection by a thick layer of mucus, the stomach is filled with acid (average pH ~2). This extreme environment is lethal to almost all bacteria ingested in food. Nonetheless, it is colonized by the hardy and enterprising bacterium Helicobacter pylori, which was recognized only recently as a major causative agent of stomach ulcers and possibly stomach cancer. Although the older treatments for ulcers (acid-reducing drugs and bland diets) are still used to reduce inflammation, a short and relatively cheap course of antibiotics can now effectively cure a patient of recurrent stomach ulcers. The hypothesis that stomach ulcers could be caused by a persistent bacterial infection of the stomach lining initially met with considerable skepticism. The point was finally proven by the young Australian doctor who made the initial discovery: he drank a flask of a pure culture of H. pylori and developed a typical ulcer. One way that H. pylori survives in the stomach is by producing the enzyme urease, which converts urea to ammonia and carbon dioxide; in this way, the bacterium surrounds itself with a layer of ammonia, which neutralizes stomach acid in its immediate vicinity. The bacteria also express at least five types of adhesins, which enable them to adhere to the stomach epithelium, and they produce several cytotoxins that destroy the stomach epithelial cells, creating painful ulcers. The resulting chronic inflammation promotes cell proliferation and thus predisposes the infected individual to stomach cancer.

A more extreme example of active colonization is provided by Bordetella pertussis, the bacterium that causes whooping cough. The first step in a B. pertussis infection is colonization of the respiratory epithelium. The bacteria circumvent the normal clearance mechanism (the mucociliary escalator described in Chapter 22) by binding tightly to the surface of ciliated cells and multiplying on them. B. pertussis expresses at least four types of adhesins that bind tightly to particular glycolipids on the ciliated cells. The adherent bacteria produce various toxins which eventually kill the ciliated cells, compromising the host's ability to clear the infection. The most familiar of these is pertussis toxin, which—like cholera toxin—is an ADP-ribosylating enzyme. It ADP-ribosylates the α subunit of the G protein G_i, causing deregulation of the host cell adenylyl cyclase and overproduction of cyclic AMP (discussed in Chapter 15). Not content with this, B. pertussis also produces an adenylyl cyclase of its own, which is inactive unless bound to the eucaryotic Ca²⁺-binding protein calmodulin. The bacterially-produced enzyme is therefore active only in the cytoplasm of a eucaryotic cell. Although both B. pertussis and V. cholerae have the similar effect of drastically raising cAMP levels in the host cells to which they adhere, the symptoms of the diseases are very different because the two bacteria colonize different sites in the host: B. pertussis colonizes the respiratory tract and causes paroxysmal coughing, whereas V. cholerae colonizes the gut and causes watery diarrhea.

Not all examples of specific colonization require that the bacterium express adhesins that bind to host cell glycolipids or proteins. Enteropathogenic E. coli, which causes diarrhea in young children, instead uses a type III secretion system (see Figure 25-7) to deliver its own bacterially-expressed receptor protein (called Tir) into its host cell (Figure 25-20A). After Tir is inserted into the host cell membrane, a bacterial surface protein binds to the extracellular domain of Tir, triggering a remarkable series of events inside the host cell. First, the Tir receptor protein is phosphorylated on tyrosine residues by a host protein tyrosine kinase. Unlike eucaryotic cells, bacteria generally do not phosphorylate tyrosine residues, yet Tir contains a peptide domain that is a specific recognition motif for a eucaryotic tyrosine kinase. The phosphorylated Tir then is thought to recruit a member of the Rho family of small GTPases, which promotes actin polymerization through a series of intermediate steps (discussed in Chapter 16). The polymerized actin forms a unique cell surface protrusion, called a pedestal, that pushes the tightly adherent bacteria up about 10 μm from the host cell surface (Figure 25-20B, C).

These examples of host colonization illustrate the importance of host-pathogen communication in the infection process. Pathogenic organisms have acquired genes that encode proteins that interact specifically with particular molecules of the host cells. In some cases, such as the B. pertussis adenylyl cyclase, an ancestor of the pathogen may have acquired the gene from its host, whereas in others, such as Tir, random mutation may have given rise to protein motifs that are recognized by a eucaryotic protein partner.

Intracellular Pathogens Have Mechanisms for Both Entering and Leaving Host Cells

Many pathogens, including V. cholerae and B. pertussis, infect their host without entering host cells. Others, however, including all viruses and many bacteria and protozoa, are intracellular pathogens. Their preferred niche for replication and survival is within the cytoplasm or intracellular compartments of particular host cells. This strategy has several advantages. The pathogens are not accessible to antibodies (discussed in Chapter 24), and they are not easy targets for phagocytic cells (discussed below). This lifestyle, however, does require that the pathogen develop mechanisms for entering host cells, for finding a suitable subcellular niche where it can replicate, and for exiting the infected cell to spread the infection. In the remainder of this section, we consider some of the myriad ways that individual intracellular pathogens exploit and modify host cell biology to satisfy these requirements.

Viruses Bind to Molecules Displayed on the Host Cell Surface

The first step for any intracellular pathogen is to bind to the surface of the host target cell. For viruses, binding is accomplished through the association of a viral surface protein with a specific receptor on the host cell surface. Of course, no host cell receptor evolved for the sole purpose of allowing a pathogen to bind to it; these receptors all have other functions. The first such “virus receptor” identified was the E. coli surface protein that allows the bacteriophage lambda to bind to the bacterium. Its normal function is as a transport protein responsible for the uptake of maltose.

Viruses that infect animal cells generally use cell-surface receptor molecules that are either very abundant (such as sialic-acid-containing oligosaccharides, which are used by the influenza virus) or uniquely found on those cell types in which the virus can replicate (such as the nerve growth factor receptor, the nicotinic acetylcholine receptor, or the cell-cell adhesion protein N-CAM, all of which are used by the rabies virus to specifically infect neurons). Often, a single type of receptor is used by many types of virus, and some viruses can use several different receptors. Moreover, different viruses that infect the same cell type may each use a different receptor. Hepatitis, for example, is caused by at least six viruses, all of which preferentially replicate in liver cells. Receptors for four of the hepatitis viruses have been identified, and they are all different. Receptors need not be proteins; herpes simplex virus, for example, binds to heparan sulfate proteoglycans through specific viral membrane proteins.

Frequently, viruses require both a primary receptor and a secondary co-receptor for efficient attachment and entry into host cells. An important example is HIV. Its primary receptor is CD4, a protein involved in immune recognition which is found on the surface of many T cells and macrophages (discussed in Chapter 24). Viral entry also requires the presence of a co-receptor, either CCR5 (a receptor for β-chemokines) or CXCR4 (a receptor for α-chemokines), depending on the particular variant of the virus (Figure 25-21). Macrophages are susceptible only to HIV variants that use CCR5 for entry, whereas T cells are most efficiently infected by variants that use CXCR4. The viruses that are found within the first few months after HIV infection almost invariably require CCR5, which presumably explains why individuals who carry a defective ccr5 gene are not susceptible to HIV infection. In the later stages of infection, viruses may either switch to use the CXCR4 co-receptor or adapt to use both co-receptors; in this way, the virus can change the cell types it infects as the disease progresses.

Viruses Enter Host Cells by Membrane Fusion, Pore Formation, or Membrane Disruption

After recognition and attachment to the host cell surface, the virus must next enter the host cell and release its nucleic acid genome from its protective protein coat or lipid envelope. In most cases, the liberated nucleic acid remains complexed with some viral proteins. Enveloped viruses enter the host cell by fusing either with the plasma membrane or with the endosomal membrane following endocytosis (Figure 25-22A,B). Fusion is thought to proceed via a mechanism similar to SNARE-mediated fusion of vesicles during normal intracellular vesicular traffic (discussed in Chapter 13).

Fusion is regulated both to ensure that virus particles fuse only with the appropriate host cell membrane and to prevent virus particles from fusing with one another. For viruses such as HIV that fuse at neutral pH at the plasma membrane, binding to receptors or co-receptors usually triggers a conformational change in the viral envelope protein to expose a normally buried fusion peptide (see Figure 13-16). Other enveloped viruses, such as influenza, postpone fusion until after endocytosis; in this case, it is frequently the acid environment in the early endosome that triggers the conformational change in a viral surface protein that exposes the fusion peptide (Figure 25-23). The H⁺ pumped into the early endosome enters the influenza particle through an ion channel and triggers the uncoating of the viral nucleic acid, which is directly released into the cytosol as the virus fuses with the endosomal membrane. For some viruses, uncoating occurs after release into the cytosol. In the case of Semliki forest virus, for example, the binding of host ribosomes to the capsid causes the capsid proteins to separate from the viral genome.

It is more difficult to envision how nonenveloped viruses enter host cells, as it is not obvious how large assemblies of protein and nucleic acid can cross the plasma or endosomal membrane. Where the entry mechanism is understood, nonenveloped viruses generally either form a pore in the cell membrane to deliver the viral genome into the cytoplasm or they disrupt the endosomal membrane after endocytosis.

Poliovirus uses the first strategy. Binding of poliovirus to its receptor triggers both receptor-mediated endocytosis and a conformational change in the viral particle. The conformational change exposes a hydrophobic projection on one of the capsid proteins, which apparently inserts into the endosomal membrane to form a pore. The viral genome then enters the cytoplasm through the pore, leaving the capsid either in the endosome or on the cell surface, or in both places (see Figure 25-22C).

Adenovirus uses the second strategy. It is initially taken up by receptor-mediated endocytosis. As the endosome matures and becomes more acidic, the virus undergoes multiple uncoating steps in which structural proteins are sequentially removed from the capsid. Some of these steps require the action of a viral protease, which is inactive in the extracellular virus particle (probably because of intrachain disulfide bonds) but which is activated in the reducing environment of the endosome. One of the proteins released from the capsid lyses the endosomal membrane, releasing the remainder of the virus into the cytosol. This trimmed-down virus then docks onto the nuclear pore complex, and the viral DNA genome is released through the pore into the nucleus, where it is transcribed (see Figure 25-22D).

In these various entry strategies, viruses exploit a variety of host cell molecules and processes, including cell-surface components, receptor-mediated endocytosis, and endosomal maturation steps. These strategies again illustrate the sophisticated ways that pathogens have evolved to utilize the basic cell biology of their hosts.

Bacteria Enter Host Cells by Phagocytosis

Bacteria are much larger than viruses, and they are too large to be taken up by receptor-mediated endocytosis. Instead, they enter host cells through phagocytosis. Phagocytosis of bacteria is a normal function of macrophages. They patrol the tissues of the body and ingest and destroy unwanted microbes. Some pathogens, however, have acquired the ability to survive and replicate within macrophages after they have been phagocytosed.

Tuberculosis, a serious lung infection that is widespread in some urban populations, is caused by one such pathogen, Mycobacterium tuberculosis. This bacterium is usually acquired by inhalation into the lungs, where it is phagocytosed by alveolar macrophages. Although the microbe can survive and replicate within macrophages, the macrophages of most healthy individuals, with the help of the adaptive immune system, contain the infection within a lesion called a tubercle. In most cases, the lesion becomes walled off within a fibrous capsule that undergoes calcification, after which it can easily be seen on an X-ray of the lungs of an infected person. An unusual feature of M. tuberculosis is its ability to survive for decades within macrophages contained in such lesions. Later in life, especially if the immune system becomes weakened by disease or drugs, the infection may be reactivated, spreading in the lung and even to other organs.

Tuberculosis has been an obvious presence in human populations for thousands of years, but another bacterium that lives within alveolar macrophages was first recognized as a human pathogen only in 1976. Legionella pneumophila is normally a parasite of freshwater amoebae, which take it up by phagocytosis. When droplets of water containing L. pneumophila or infected amoebae are inhaled into the lung, the bacteria can invade and live inside alveolar macrophages (Figure 25-24), which, to the bacteria, must seem just like large amoebae. This infection leads to the type of pneumonia known as Legionnaire's disease. The pathogen can be efficiently spread by central air conditioning systems, as the amoebae that are the bacterium's normal host are particularly adept at growing in air-conditioning cooling towers; moreover, these cooling systems produce microdroplets of water that are easily inhaled. The incidence of Legionnaire's disease has increased dramatically in recent decades, and outbreaks are frequently traced to the air conditioning systems in office buildings, hospitals, and hotels. Other forms of modern aerosolization, including decorative fountains and produce sprayers in supermarkets, have also been implicated in outbreaks of this disease.

Some bacteria invade cells that are normally nonphagocytic. One way that bacteria can induce such a cell to phagocytose them is by expressing an adhesin that binds with high affinity to a cell adhesion protein that the cell normally uses to adhere to another cell or to the extracellular matrix (discussed in Chapter 19). For example, a bacterium that causes diarrhea, Yersinia pseudotuberculosis (a close relative of the plague bacterium Yersinia pestis), expresses a protein called invasin that binds to β1 integrins, and Listeria monocytogenes, which causes a rare but serious form of food poisoning, expresses a protein that binds to E-cadherin. Binding to these transmembrane adhesion proteins fools the host cell into attempting to form a cell junction, and it begins moving actin and other cytoskeletal components to the site of bacterial attachment. Since the bacterium is small relative to the host cell, the host cell's attempt to spread over the adhesive surface of the bacterium results in the phagocytic uptake of the bacterium—a process known as the zipper mechanism of invasion (Figure 25-25A). The similarity of this form of invasion to the natural process of cell adhesion was underscored by the determination of the three-dimensional structure of invasin. This bacterial protein has an RGD motif, the structure of which is almost identical to that of the normal β1-integrin-binding site in laminin (discussed in Chapter 19).

A second pathway by which bacteria can invade nonphagocytic cells is known as the trigger mechanism (Figure 25-25B). It is used by various pathogens, including Salmonella enterica, which causes food poisoning. This dramatic form of invasion is initiated when the bacterium injects a set of effector molecules into the host cell cytoplasm through a type III secretion system. Some of these effector molecules activate Rho-family GTPases, which stimulate actin polymerization (discussed in Chapter 16). Others interact with cytoskeletal elements more directly, severing actin filaments and causing the rearrangement of cross-linking proteins. The net effect is to cause dramatic localized ruffling on the surface of the host cell (Figure 25-25C), which throws up large actin-rich protrusions that fold over and trap the bacterium within large endocytic vesicles called macropinosomes. The overall appearance of cells being invaded by the trigger mechanism is similar to the dramatic ruffling induced by some growth factors, suggesting that similar intracellular signaling cascades are activated in both cases.

Intracellular Parasites Actively Invade Host Cells

The uptake of viruses by receptor-mediated endocytosis and bacteria by phagocytosis requires energy provided by the host cell. The pathogen is a relatively passive participant, usually providing a trigger to initiate the invasion process, but not contributing any metabolic energy. In contrast, the invasion of intracellular eucaryotic parasites, which are typically much larger than bacteria, proceeds through a variety of complex pathways that usually require significant energy expenditure on the part of the parasite.

Toxoplasma gondii, the cat parasite that also causes occasional serious human infections, is an instructive example. When this protozoan contacts a host cell, it protrudes a microtubule-ribbed structure called a conoid and then reorients so that the conoid is touching the host cell surface. The parasite then slowly pushes its way into the host cell. The energy for invasion seems to come entirely from the parasite, and the process can be disrupted by depolymerizing the parasite's—but not the host's—actin cytoskeleton. As the parasite moves into the host cell, it secretes lipids from a pair of specialized organelles, generating a vacuolar membrane that is made primarily of parasite lipids and proteins. In this way, the organism protects itself in a membrane-enclosed compartment within the host cell that does not participate in host cell membrane trafficking pathways and does not fuse with lysosomes (Figure 25-26). The specialized vacuolar membrane allows the parasite to take up metabolic intermediates and nutrients from the host cell cytosol but excludes larger molecules.

An entirely different, but no less peculiar, invasion strategy is used by the protozoan Trypanosoma cruzi, which causes a multiorgan disease (Chagas disease) mainly in Mexico and Central and South America. After attachment to β1 integrins on the host cell surface, this parasite induces a local elevation of Ca²⁺ in the host cell cytoplasm. The Ca²⁺ signal recruits lysosomes to the site of parasite attachment, which then fuse with the plasma membrane, allowing the parasite to enter a vacuole derived almost entirely from lysosomal membrane (Figure 25-27). As we discuss below, most intracellar pathogens go to great lengths to avoid exposure to the hostile, proteolytic environment of the lysosome, but this trypanosome uses the lysosome as its means of entry. In the vacuole, the parasite secretes a transsialidase enzyme that removes sialic acid from lysosomal glycoproteins and transfers it to its own surface molecules, thereby coating itself with host cell sugars. Next, the parasite secretes a pore-forming toxin that lyses the vacuolar membrane, releasing the parasite into the host cell cytosol, where it replicates.

Many Pathogens Alter Membrane Traffic in the Host Cell

The two examples of intracellular parasites discussed in the preceding section raise a general problem that faces all pathogens with an intracellular lifestyle, including viruses, bacteria, and parasites. They must deal in some way with membrane traffic in the host cell. After endocytosis by a host cell, they usually find themselves in an endosomal compartment that normally would fuse with lysosomes. They therefore must either modify the compartment to prevent its fusion, escape from the compartment before getting digested, or find ways to survive in the hostile environment of the phagolysosome (Figure 25-28).

Most pathogens use either the first or second strategy. It is much less common to find pathogens that can survive in the phagolysosome. As we have seen, Trypanosoma cruzi uses the escape route, as do essentially all viruses (see Figure 25-22). The bacterium Listeria monocytogenes also uses this strategy. It is taken up into cells via the zipper mechanism discussed earlier and secretes a protein called hemolysin that forms large pores in the phagosomal membrane, eventually disrupting the membrane and releasing the bacteria into the cytosol. Once in the cytosol, the bacteria continue to secrete hemolysin, but it does not destroy the plasma membrane. The hemolysin selectively destroys only the phagosomal membrane for two reasons: first, it is ten times more active at the acidic pH found in the phagosome than at neutral pH found in the cytosol, and second, the hemolysin contains a PEST sequence (a peptide motif rich in proline, glutamate, serine, and threonine), which is recognized by the protein degradation machinery of the host cell, leading to the rapid degradation of the bacterial hemolysin in the proteasome (see Figure 6-86). The hemolysin is stable in the phagosome, as the degradation machinery does not have access to it there (Figure 25-29). The hemolysin secreted by L. monocytogenes is closely related to hemolysins secreted by other bacteria that are not intracellular pathogens. These related hemolysins, however, all lack PEST sequences. It seems that the L. monocytogenes hemolysin has acquired an essentially eucaryotic protein domain expressly to allow its activity to be regulated in the host cell.

By far the most common strategy used by intracellular bacteria and parasites is to modify the endosomal compartment so that they can remain there and replicate. They must modify the compartment in at least two ways: first, they must prevent lysosomal fusion, and second, they must provide a pathway for importing nutrients from the host cytosol. Different pathogens have distinct strategies for doing this. As we have seen, Toxoplasma gondii creates its own membrane-enclosed compartment that does not participate in normal host cell membrane traffic and allows nutrient transport. This option is not open to bacteria, which are too small to carry in their own supply of membrane. Mycobacterium tuberculosis somehow prevents the very early endosome that contains it from maturing, so that it never acidifies or acquires the characteristics of a late endosome. Endosomes containing Salmonella enterica, in contrast, do acidify and acquire markers of late endosomes, but they arrest their maturation at a stage prior to lysosomal fusion. Other bacteria seem to find shelter in intracellular compartments that are completely distinct from the usual endocytic pathway. Legionella pneumophila, for example, replicates in compartments that are wrapped in layers of rough endoplasmic reticulum. Chlamydia trachomatis, a sexually transmitted bacterial pathogen that can cause sterility and blindness, replicates in a compartment that seems to be most similar to part of the exocytic pathway (Figure 25-30). The mechanisms used by these organisms to alter their membrane compartments are not yet understood.

Viruses also often alter membrane traffic in the host cell. Enveloped viruses must acquire their membrane from host cell phospholipids. In the simplest cases, virally encoded proteins are inserted into the ER membrane and follow the usual path through the Golgi apparatus to the plasma membrane, undergoing various posttranslational modifications en route. The viral capsid then assembles at the plasma membrane and buds off from the cell surface. This is the mechanism used by HIV. Other enveloped viruses interact in complex ways with membrane trafficking pathways in the host cell (Figure 25-31). Even some nonenveloped viruses alter membrane traffic in the host cell to suit their own purposes. The replication of poliovirus, for example, is carried out by a membrane-associated, virus-encoded RNA polymerase. Replication can proceed more quickly if the surface area of the host cell membranes is increased. To accomplish this, the virus induces increased lipid synthesis in the host cell and blocks secretion from the ER. Intracellular membranes thereby accumulate, expanding the surface area on which RNA replication can occur (Figure 25-32).

Viruses and Bacteria Exploit the Host Cell Cytoskeleton for Intracellular Movement

The cytoplasm of mammalian cells is extremely viscous. It is crowded with organelles and supported by networks of cytoskeletal filaments, all of which inhibit the diffusion of particles the size of a bacterium or a viral capsid. If a pathogen must reach a particular part of the cell to carry out part of its replication cycle, it must move there actively. As with transport of intracellular organelles, pathogens generally use the cytoskeleton for active movement.

Several bacteria that replicate in the host cell cytosol (rather than in membrane-enclosed compartments) have adopted a remarkable mechanism for moving, which depends on actin polymerization. These bacteria, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii (which causes Rocky Mountain spotted fever) induce the nucleation and assembly of host cell actin filaments at one pole of the bacterium. The growing filaments generate substantial force and push the bacteria through the cytoplasm at rates up to 1 μm/sec. New filaments form at the rear of each bacterium and are left behind like a rocket trail as the bacterium advances, depolymerizing again within a minute or so as they encounter depolymerizing factors in the cytosol. When a moving bacterium reaches the plasma membrane, it continues to move outward, inducing the formation of a long, thin protrusion with the bacterium at its tip. This projection is often engulfed by a neighboring cell, allowing the bacterium to enter the neighbor's cytoplasm without exposure to the extracellular environment, thereby avoiding recognition by antibodies produced by the host's adaptive immune system (Figure 25-33).

The molecular mechanism of pathogen-induced actin assembly has been determined for two of these bacteria. The mechanisms are different, suggesting that they evolved independently. Although both make use of the same host cell regulatory pathway that normally controls the nucleation of actin filaments, they exploit different points in the pathway. As discussed in Chapter 16, activation of the small GTPase Cdc42 by growth factors or other external signals causes the activation of a protein called N-WASp, which in turn activates the ARP complex that can nucleate the growth of a new actin filament. An L. monocytogenes surface protein directly binds to and activates the ARP complex to initiate the formation of an actin tail, while an unrelated surface protein on S. flexneri binds to and activates N-WASp, which then activates the ARP complex. Remarkably, vaccinia virus uses yet another mechanism to move intracellularly by inducing actin polymerization, again exploiting this same regulatory pathway (Figure 25-34).

Other pathogens rely primarily on microtubule-based transport to move within the host cell. This movement is particularly well-illustrated by viruses that infect neurons. An important example is provided by the neurotropic alpha herpes viruses, a group that includes the virus that causes chicken pox. Upon infection of sensory neurons, the virus particles are transported to the nucleus by microtubule-based transport, probably mediated by attachment of the capsids to the motor protein dynein. After replication and assembly in the nucleus, the enveloped virus is transported along microtubules away from the neuronal cell body down the axon, presumably by attachment to a kinesin motor protein (Figure 25-35).

One bacterium that is known to associate with microtubules is Wolbachia. This fascinating genus includes many species that are parasites or symbionts of insects and other invertebrates, living in the cytosol of each cell in the animal. The infection is spread vertically from mother to offspring, as Wolbachia are also present in eggs. The bacteria ensure their transmission into every cell by binding to microtubules, and they are therefore segregated by the mitotic spindle simultaneously with chromosome segregation when an infected cell divides (Figure 25-36). As we discuss later, Wolbachia infection can significantly alter the reproductive behavior of its insect hosts.

Viruses Take Over the Metabolism of the Host Cell

Most intracellular bacteria and parasites carry the basic genetic information required for their own metabolism and replication, and they rely on their host cells only for nutrients. Viruses, in contrast, use the basic host cell machinery for most aspects of their reproduction: they all depend on host cell ribosomes to produce their proteins, and some also use host cell DNA and RNA polymerases for replication and transcription, respectively.

Many viruses encode proteins that modify the host transcription or translation apparatus to favor the synthesis of viral proteins over those of the host cell. As a result, the synthetic capability of the host cell is directed principally to the production of new virus particles. Poliovirus, for example, encodes a protease that specifically cleaves the TATA-binding factor component of TFIID (see Figure 6-16), effectively shutting off all host cell transcription via RNA polymerase II. Influenza virus produces a protein that blocks both the splicing and the polyadenylation of mRNA transcripts, which therefore fail to be exported from the nucleus (see Figure 6-40).

Translation initiation of most host cell mRNAs depends on recognition of their 5′ cap by a group of translation initiation factors (see Figure 6-71). Translation initiation of host mRNAs is often inhibited during viral infection, so that the host cell ribosomes can be used more efficiently for synthesis of viral proteins. Some viruses encode endonucleases that cleave the 5′ cap from host cell mRNAs. Some even then use the liberated 5′ caps as primers to synthesize viral mRNAs, a process called cap snatching. Several other viral RNA genomes encode proteases that cleave certain translation initiation factors. These viruses rely on 5′ cap-independent translation of the viral RNA, using internal ribosome entry sites (IRESs) (see Figure 7-102).

A few DNA viruses use host cell DNA polymerase to replicate their genome. These viruses need to solve a problem: DNA polymerase is expressed at high levels only during S phase of the cell cycle, whereas most cells that the viruses infect spend most of their time in G₁ phase. Adenovirus solves this problem by inducing the host cell to enter S phase. Its genome encodes proteins that inactivate both Rb and p53, two key suppressors of cell-cycle progression (discussed in Chapter 17). As might be expected for any mechanism that induces unregulated DNA replication, these viruses are frequently oncogenic.

Pathogens Can Alter the Behavior of the Host Organism to Facilitate the Spread of the Pathogen

As we have seen, pathogens often alter the behavior of the host cell in ways that benefit the survival and replication of the pathogen. Similarly, pathogens often alter the behavior of the whole host organism to facilitate pathogen spread, as we saw earlier for Trypanosoma brucei and Yersinia pestis. In some cases, it is difficult to tell whether a particular host response is more for the benefit of the host or for the pathogen. Pathogens like Salmonella enterica that cause diarrhea, for example, usually produce self-limiting infections because the diarrhea efficiently washes out the pathogen. The bacteria-laden diarrhea, however, can spread the infection to a new host. Similarly, coughing and sneezing help to clear patho-gens from the respiratory tract, but they also spread the infection to new individuals. A person with a common cold may produce 20,000 droplets in a single sneeze, all carrying rhinovirus or corona virus.

A frightening example of a pathogen modifying host behavior is seen in rabies, as first described in Egyptian writings over 3000 years ago. This virus replicates in neurons and causes infected people or animals to become “rabid”: they are unusually aggressive and develop a strong desire to bite. The virus is shed in the saliva and transmitted through the bite wound into the bloodstream of the victim, spreading the infection to a new host.

Toxoplasma gondii, a eucaryotic parasite that forms lesions in muscle and brain tissue, provides an equally remarkable example. It can complete its life cycle only in its normal host—cats. If it infects an intermediate host, such as a rodent or human, the infection is a dead end for the parasite, unless the intermediate host is eaten by a cat. Behavioral studies show that rats infected with T. gondii lose their innate fear of cats and instead preferentially seek out locations perfumed with cat urine over locations perfumed with rabbit urine, exactly the opposite of normal rat behavior.

But the most dramatic example of pathogens modifying host behavior belongs to Wolbachia. These bacteria manipulate the sexual behavior of their host to maximize their dissemination. As described earlier, Wolbachia are passed vertically into offspring through eggs. If they live in a male, however, they hit a dead end, as they are excluded from sperm. In some species of Drosophila, Wolbachia modify the sperm of their host so that they can fertilize the eggs only of infected females. This modification creates a reproductive advantage for infected females over uninfected females, so that the overall proportion of Wolbachia carriers increases. In other host species, a Wolbachia infection kills males but spares females, increasing the number of females in the population and thus the number of individuals that can produce eggs to pass on the infection. In a few types of wasp, Wolbachia infections enable the females to produce eggs that develop parthenogenetically without the need for fertilization by sperm; in this species, males have been completely eliminated. For some of its hosts, Wolbachia has become an indispensable symbiont, and curing the infection causes death of the host. In one case, humans are making use of this dependence: the filarial nematode that causes African river blindness is difficult to kill with antiparasite medications, but when people with river blindness are treated with antibiotics that cure the nematode's Wolbachia infection, the nematode infection is also arrested.

Pathogens Evolve Rapidly

The complexity and specificity of the molecular interactions between pathogens and their host cells might suggest that virulence would be difficult to acquire by random mutation. Yet, new pathogens are constantly emerging, and old pathogens are constantly changing in ways that make familiar infections difficult to treat. Pathogens have two great advantages that enable them to evolve rapidly. First, they replicate very quickly, providing a great deal of material for the engine of natural selection. Whereas humans and chimpanzees have acquired a 2% difference in genome sequences over about 8 million years of divergent evolution, poliovirus manages a 2% change in its genome in 5 days, about the time it takes the virus to pass from the human mouth to the gut. Second, this rapid genetic variation is acted on by powerful selective pressures provided by the host's adaptive immune system and by modern medicine, which destroy pathogens that fail to change.

A small-scale example of the constant battle between infection and immunity is the phenomenon of antigenic variation. An important adaptive immune response against many pathogens is the production of antibodies that recognize specific antigens on the pathogen surface (discussed in Chapter 24). Many pathogens evade complete elimination by antibodies by changing these antigens during the course of an infection. Some parasites, for example, undergo programmed rearrangements of the genes encoding their surface antigens. The most striking example occurs in African trypanosomes such as Trypanosoma brucei, a parasite that causes sleeping sickness and is spread by an insect vector (T. brucei is a close relative of T. cruzi (see Figure 25-27), but it replicates extracellularly rather than inside cells). T. brucei is covered with a single type of glycoprotein, called variant-specific glycoprotein (VSG), which elicits a protective antibody response that rapidly clears most of the parasites. The trypanosome genome, however, contains about 1000 VSG genes, each encoding a VSG with distinct antigenic properties. Only one of these genes is expressed at any one time, by being copied into an active expression site in the genome. The VSG gene expressed can be changed repeatedly by gene rearrangements that copy new alleles into the expression site. In this way, a few trypanosomes with an altered VSG escape the antibody-mediated clearance, replicate, and cause a recurrence of disease, leading to a chronic cyclic infectionFigure 25-37).

A different type of antigenic variation occurs during the course of infection by viruses that have error-prone replication mechanisms. Retroviruses, for example, acquire on average one point mutation every replication cycle, because the viral reverse transcriptase that produces DNA from the viral RNA genome cannot correct nucleotide misincorporation errors. A typical, untreated HIV infection may eventually involve HIV genomes with every possible point mutation. In some ways, the high mutation rate is beneficial for the pathogen. By a microevolutionary process of mutation and selection within each host, the virus can switch from infecting macrophages to infecting T-cells (as described earlier), and, once treatment is begun, it can quickly acquire resistance to drugs. If the reverse transcriptase error rate were too high, however, deleterious mutations might accumulate too rapidly to allow the virus to survive. Furthermore, rapid viral diversification in one host does not necessarily lead to rapid evolution of the virus in the population, as a mutated virus may not be able to infect a new host. For HIV-1, the extent of this constraint can be estimated by examining the sequence diversity among different individuals infected with the virus. Remarkably, only about one-third of the nucleotide positions in the coding sequence of the virus are invariant, and nucleotide sequences in some parts of the genome, such as the env gene, can differ by as much as 30%. This extraordinary genomic plasticity greatly complicates attempts to develop vaccines against HIV, and it can also lead to rapid drug resistance (see below). A further consequence has been rapid diversification and emergence of new HIV strains. Sequence comparisons between various strains of HIV and the very similar simian immunodeficiency virus (SIV) from a variety of different monkey species seem to indicate that the most virulent type of HIV, HIV-1, may have jumped from chimpanzees to humans as recently as 1930Figure 25-38).

Rapid evolution in bacteria frequently takes place by horizontal gene transfer rather than by point mutation. Most of this horizontal gene transfer is mediated by the acquisition of plasmids and bacteriophages. Bacteria readily pick up pathogenicity islands and virulence plasmids (see Figure 25-5) from other bacteria. Once a bacterium has acquired a new set of virulence-related genes, it may quickly establish itself as a new cause of human epidemics. Yersinia pestis, for example, is an infection endemic to rats and other rodents that first appeared in human history in 542 A.D., when the city of Constantinople was devastated by plague. Sequence comparisons of Y. pestis to its close relative Y. pseudotuberculosis, which causes a severe diarrheal disease, suggest that Y. pestis may have emerged as a distinct strain only a few thousand years ago, not long before its debut in the field of urban devastation.

The emergence and evolution of new infectious disease is in many cases exacerbated by changes in human behavior. For example, crowded and filthy living conditions in medieval cities contributed to the spread of plague to humans from the natural rodent host. The tendency of modern humans to live at high population densities in large cities has also created the opportunity for infectious organisms to initiate epidemics, such as influenza, tuberculosis, and AIDS, which could not have spread so rapidly or so far among sparser human populations. Air travel can, in principle, allow an asymptomatic, newly infected host to carry an epidemic to any previously unexposed population within a few hours or days. Modern agricultural practices also foster the emergence of certain types of infectious agents that could not have easily developed in the hunter-gatherer societies of early humans. Influenza viruses, for example, are unusual in that their genome consists of several (usually eight) strands of RNA. When two strains of influenza infect the same host, the strands of the two strains can reshuffle to form a new and distinct type of influenza virus. Prior to 1900, the influenza strain that infected humans caused a very mild disease; a separate influenza strain infected fowl such as ducks and chickens but could not infect humans. Both the avian and the human strains, however, are capable of infecting pigs, and can recombine in a pig host to form new strains that can cause serious human disease. In communities where pigs are farmed together with chickens or turkeys, newly recombined influenza strains periodically emerge and cause worldwide epidemics. The first and most serious of these epidemics was the “Spanish flu” epidemic of 1918, which killed more people than did World War I.

Drug Resistant Pathogens Are a Growing Problem

While some human activities have promoted the spread of certain infectious diseases, advances in public sanitation and in medicine have prevented or alleviated the suffering caused by many others. Effective vaccines and worldwide vaccination programs have eliminated smallpox and severely reduced poliomyelitis, and many deadly childhood infections such as mumps and measles are now rarities in wealthy industrialized nations. However, there are still many widespread and devastating infectious diseases, such as malaria, for which no effective vaccines are available. Of equal importance is the development of drugs that cure rather than prevent infections. The most successful class are the antibiotics, which kill bacteria. Penicillin was one of the first antibiotics used to treat infections in humans, introduced into clinical use just in time to prevent tens of thousands of deaths from infected battlefield wounds in World War II. The rapid evolution of pathogens, however, has enabled targeted bacteria to develop resistance to antibiotics very quickly; the typical lag between introduction of an antibiotic into clinical use and the appearance of resistant strains is only a few years. Similar drug resistance arises rapidly among viruses when infections are treated with antiviral agents. The virus population in an HIV-infected person treated with the reverse transcriptase inhibitor AZT, for example, will acquire complete resistance to the drug within a few months. The current protocol for treatment of HIV infections involves the simultaneous use of three drugs, which helps to minimize the acquisition of resistance.

There are three general strategies by which pathogens develop drug resistance. Pathogens can (1) produce an enzyme that destroys the drug, (2) alter the molecular target of the drug so that it is no longer sensitive to the drug, or (3) prevent access to the target by, for example, actively pumping the drug out of the pathogen. Once a pathogen has chanced upon an effective strategy, the newly acquired or mutated genes that confer that resistance are frequently spread throughout the pathogen population and may even be transferred to pathogens of different species that are treated with the same drug. The highly effective and very expensive antibiotic vancomycin, for example, has been used as a treatment of last resort for many severe hospital-acquired bacterial infections that are already resistant to most other known antibiotics. Vancomycin prevents one step in bacterial cell wall biosynthesis, by binding to part of the growing peptidoglycan chain and preventing it from being cross-linked to other chains. Resistance can arise if the bacterium synthesizes a different type of cell wall, using different subunits that do not bind vancomycin. The most effective type of vancomycin resistance depends on a transposon that encodes seven genes. The products of these genes work together to sense the presence of vancomycin, shut down the normal pathway for bacterial cell wall synthesis, and generate a different type of cell wall. Although the joining of these genes into a single transposon must have been a difficult evolutionary task (it took 15 years for vancomycin resistance to develop, rather than the typical lag of a year or two), the transposon can now be readily transmitted to many other pathogenic bacterial species.

Like most other aspects of infectious disease, the problem of drug resistance has been exacerbated by human behavior. Many patients take antibiotics for viral diseases that are not helped by the drugs, including influenza, colds, and earaches. Persistent and chronic misuse of antibiotics in this way can eventually result in the antibiotic resistance of the normal flora, which can then transfer the resistance to pathogens. Several antibiotic-resistant outbreaks of infectious diarrhea caused by Shigella flexneri, for example, originated in this way. The problem is particularly severe in countries where antibiotics are available without a physician's prescription, as in Brazil, where more than 80% of the strains of S. flexneri found in infected patients are resistant to four or more antibiotics.

Antibiotics are also misused in agriculture, where they are commonly employed as food additives to promote the growth of healthy animals. An antibiotic closely related to vancomycin was commonly added to cattle feed in Europe. Resistance arising in the normal flora of these animals is widely believed to be one of the original sources for the vancomycin resistance that is now seen in life-threatening human infections.

Summary

All pathogens share the ability to interact with host cells in ways that promote replication and spread of the pathogen, but these host-pathogen interactions are diverse. Pathogens often colonize the host by adhering to or invading through the epithelial surfaces that line the lungs, gut, bladder, and other surfaces in direct contact with the environment. Intracellular pathogens, including all viruses and many bacteria and protozoa, replicate inside a host cell, which they invade by one of a variety of mechanisms. Viruses rely largely on receptor-mediated endocytosis for host cell entry, while bacteria exploit cell adhesion and phagocytic pathways. Protozoa employ unique invasion strategies that usually require significant metabolic expense. Once inside, intracellular pathogens seek out a niche that is favorable for their replication, frequently altering host cell membrane traffic and exploiting the cytoskeleton for intracellular movement. Besides altering the behavior of individual host cells, pathogens frequently alter the behavior of the host organism in ways that favor spread to a new host. Pathogens evolve rapidly, so new infectious diseases frequently emerge, and old diseases acquire new ways to evade human attempts at treatment, prevention, and eradication.

Epithelial Surfaces Help Prevent Infection

In vertebrates, the skin and other epithelial surfaces, including those lining the lung and gut Figure 25-39), provide a physical barrier between the inside of the body and the outside world. Tight junctions (discussed in Chapter 19) between neighboring cells prevent easy entry by potential pathogens. The interior epithelial surfaces are also covered with a mucus layer that protects these surfaces against microbial, mechanical, and chemical insults; many amphibians and fish also have a mucus layer covering their skin. The slimy mucus coating is made primarily of secreted mucin and other glycoproteins, and it physically helps prevent pathogens from adhering to the epithelium. It also facilitates their clearance by beating cilia on the epithelial cells (discussed in Chapter 22).

The mucus layer also contains substances that kill pathogens or inhibit their growth. Among the most abundant of these are antimicrobial peptides, called defensins, which are found in all animals and plants. They are generally short (12–50 amino acids), positively charged, and have hydrophobic or amphipathic domains in their folded structure. They constitute a diverse family with a broad spectrum of antimicrobial activity, including the ability to kill or inactivate Gram-negative and Gram-positive bacteria, fungi (including yeasts), parasites (including protozoa and nematodes), and even enveloped viruses like HIV. Defensins are also the most abundant protein type in neutrophils (see below), which use them to kill phagocytosed pathogens.

It is still uncertain how defensins kill pathogens. One possibility is that they use their hydrophobic or amphipathic domains to insert into the membrane of their victims, thereby disrupting membrane integrity. Some of their selectivity for pathogens over host cells may come from their preference for membranes that do not contain cholesterol. After disrupting the membrane of the pathogen, the positively-charged peptides may also interact with various negatively-charged targets within the microbe, including DNA. Because of the relatively nonspecific nature of the interaction between defensins and the microbes they kill, it is difficult for the microbes to acquire resistance to the defensins. Thus, in principle, defensins might be useful therapeutic agents to combat infection, either alone or in combination with more traditional drugs.

Human Cells Recognize Conserved Features of Pathogens

Microorganisms do occasionally breach the epithelial barricades. It is then up to the innate and adaptive immune systems to recognize and destroy them, without harming the host. Consequently, the immune systems must be able to distinguish self from nonself. We discuss in Chapter 24 how the adaptive immune system does this. The innate immune system relies on the recognition of particular types of molecules that are common to many pathogens but are absent in the host. These pathogen-associated molecules (called pathogen-associated immunostimulants) stimulate two types of innate immune responses—inflammatory responses (discussed below) and phagocytosis by cells such as neutrophils and macrophages. Both of these responses can occur quickly, even if the host has never been previously exposed to a particular pathogen.

The pathogen-associated immunostimulants are of various types. Procaryotic translation initiation differs from eucaryotic translation initiation in that formylated methionine, rather than regular methionine, is generally used as the first amino acid. Therefore, any peptide containing formylmethionine at the N-terminus must be of bacterial origin. Formylmethionine-containing peptides act as very potent chemoattractants for neutrophils, which migrate quickly to the source of such peptides and engulf the bacteria that are producing them (seeFigure 16-96).

In addition, the outer surface of many microorganisms is composed of molecules that do not occur in their multicellular hosts, and these molecules also act as immunostimulants. They include the peptidoglycan cell wall and flagella of bacteria, as well as lipopolysaccharide (LPS) on Gram-negative bacteria (Figure 25-40) and teichoic acids on Gram-positive bacteria (see Figure 25-4D). They also include molecules in the cell walls of fungi such as zymosan, glucan, and chitin. Many parasites also contain unique membrane components that act as immunostimulants, including glycosylphosphatidylinositol in Plasmodium.

Short sequences in bacterial DNA can also act as immunostimulants. The culprit is a “CpG motif”, which consists of the unmethylated dinucleotide CpG flanked by two 5′ purine residues and two 3′ pyrimidines. This short sequence is at least twenty times less common in vertebrate DNA than in bacterial DNA, and it can activate macrophages, stimulate an inflammatory response, and increase antibody production by B cells.

The various classes of pathogen-associated immunostimulants often occur on the pathogen surface in repeating patterns. They are recognized by several types of dedicated receptors in the host, that are collectively called pattern recognition receptors. These receptors include soluble receptors in the blood (components of the complement system) and membrane-bound receptors on the surface of host cells (members of the Toll-like receptor family). The cell-surface receptors have two functions: they initiate the phagocytosis of the pathogen, and they stimulate a program of gene expression in the host cell for stimulating innate immune responses. The soluble receptors also aid in the phagocytosis and, in some cases, the direct killing of the pathogen.

Complement Activation Targets Pathogens for Phagocytosis or Lysis

The complement system consists of about 20 interacting soluble proteins that are made mainly by the liver and circulate in the blood and extracellular fluid. Most are inactive until they are triggered by an infection. They were originally identified by their ability to amplify and “complement” the action of antibodies, but some components of complement are also pattern recognition receptors that can be activated directly by pathogen-associated immunostimulants.

The early complement components are activated first. There are three sets of these, belonging to three distinct pathways of complement activation—the classical pathway, the lectin pathway, and the alternative pathway. The early components of all three pathways act locally to activate C3, which is the pivotal component of complement (Figure 25-41). Individuals with a deficiency in C3 are subject to repeated bacterial infections. The early components and C3 are all proenzymes, which are activated sequentially by proteolytic cleavage. The cleavage of each proenzyme in the series activates the next component to generate a serine protease, which cleaves the next proenzyme in the series, and so on. Since each activated enzyme cleaves many molecules of the next proenzyme in the chain, the activation of the early components consists of an amplifying, proteolytic cascade.

Many of these cleavages liberate a biologically active small peptide fragment and a membrane-binding larger fragment. The binding of the large fragment to a cell membrane, usually the surface of a pathogen, helps to carry out the next reaction in the sequence. In this way, complement activation is confined largely to the particular cell surface where it began. The larger fragment of C3, called C3b, binds covalently to the surface of the pathogen. Once in place, it not only acts as a protease to catalyze the subsequent steps in the complement cascade, but it also is recognized by specific receptors on phagocytic cells that enhance the ability of these cells to phagocytose the pathogen. The smaller fragment of C3 (called C3a), as well as fragments of C4 and C5 (see Figure 25-41), act independently as diffusible signals to promote an inflammatory response by recruiting phagocytes and lymphocytes to the site of infection.

The classical pathway is activated by IgG or IgM antibody molecules (discussed in Chapter 24) bound to the surface of a microbe. Mannan-binding lectin, the protein that initiates the second pathway of complement activation, is a serum protein that forms clusters of six carbohydrate-binding heads around a central collagen-like stalk. This assembly binds specifically to mannose and fucose residues in bacterial cell walls that have the correct spacing and orientation to match up perfectly with the six carbohydrate-binding sites, providing a good example of a pattern recognition receptor. These initial binding events in the classical and lectin pathways cause the recruitment and activation of the early complement components. In the alternative pathway, C3 is spontaneously activated at low levels, and the resulting C3b covalently attaches to both host cells and pathogens. Host cells produce a series of proteins that prevent the complement reaction from proceeding on their cell surfaces. Because pathogens lack these proteins, they are singled out for destruction. Activation of the classical or lectin pathways also activates the alternative pathway through a positive feedback loop, amplifying their effects.

Membrane-immobilized C3b, produced by any of the three pathways, triggers a further cascade of reactions that leads to the assembly of the late components to form membrane attack complexes (Figure 25-42). These complexes assemble in the pathogen membrane near the site of C3 activation and have a characteristic appearance in negatively stained electron micrographs, where they are seen to form aqueous pores through the membrane (Figure 25-43). For this reason, and because they perturb the structure of the bilayer in their vicinity, they make the membrane leaky and can, in some cases, cause the microbial cell to lyse, much like the defensins mentioned earlier.

The self-amplifying, inflammatory, and destructive properties of the complement cascade make it essential that key activated components be rapidly inactivated after they are generated to ensure that the attack does not spread to nearby host cells. Deactivation is achieved in at least two ways. First, specific inhibitor proteins in the blood or on the surface of host cells terminate the cascade, by either binding or cleaving certain components once they have been activated by proteolytic cleavage. Second, many of the activated components in the cascade are unstable; unless they bind immediately to either an appropriate component in the cascade or to a nearby membrane, they rapidly become inactive.

Toll-like Proteins Are an Ancient Family of Pattern Recognition Receptors

Many of the mammalian cell-surface pattern recognition receptors responsible for triggering host cell gene expression in response to pathogens are members of the Toll-like receptor (TLR) family. Drosophila Toll is a transmembrane protein with a large extracellular domain consisting of a series of leucine-rich repeats (see Figure 15-76). It was originally identified as a protein involved in the establishment of dorso-ventral polarity in developing fly embryos (discussed in Chapter 21). It is also involved, however, in the adult fly's resistance to fungal infections. The intracellular signal transduction pathway activated downstream of Toll when a fly is exposed to a pathogenic fungus leads to the translocation of the NF-κB protein (discussed in Chapter 15) into the nucleus, where it activates the transcription of various genes, including those encoding antifungal defensins. Another member of the Toll family in Drosophila is activated by exposure to pathogenic bacteria, leading to the production of an antibacterial defensin.

Humans have at least ten TLRs, several of which have been shown to play important parts in innate immune recognition of pathogen-associated immunostimulants, including lipopolysaccharide, peptidoglycan, zymosan, bacterial flagella, and CpG DNA. As with Drosophila Toll family members, the different human TLRs are activated in response to different ligands, although many of them use the NF-κB signaling pathway (Figure 25-44). In mammals, TLR activation stimulates the expression of molecules that both initiate an inflammatory response (discussed below) and help induce adaptive immune responses. TLRs are abundant on the surface of macrophages and neutrophils, as well as on the epithelial cells lining the lung and gut. They act as an alarm system to alert both the innate and adaptive immune systems that an infection is brewing.

Molecules related to Toll and TLRs are apparently involved in innate immunity in all multicellular organisms. In plants, proteins with leucine-rich repeats and with domains homologous to the cytosolic portion of the TLRs are required for resistance to fungal, bacterial, and viral pathogens (Figure 25-45). Thus, at least two parts of the innate immune system—the defensins and the TLRs—seem to be evolutionarily very ancient, perhaps predating the split between animals and plants over a billion years ago. Their conservation during evolution indicates the importance of these innate responses in the defense against microbial pathogens.

Phagocytic Cells Seek, Engulf, and Destroy Pathogens

In all animals, invertebrate as well as vertebrate, the recognition of a microbial invader is usually quickly followed by its engulfment by a phagocytic cell. Plants, however, lack this type of innate immune response. In vertebrates, macrophages reside in tissues throughout the body and are especially abundant in areas where infections are likely to arise, including the lungs and gut. They are also present in large numbers in connective tissues, the liver, and the spleen. These long-lived cells patrol the tissues of the body and are among the first cells to encounter invading microbes. The second major family of phagocytic cells in vertebrates, the neutrophils, are short-lived cells, which are abundant in blood but are not present in normal, healthy tissues. They are rapidly recruited to sites of infection both by activated macrophages and by molecules such as formylmethionine-containing peptides released by the microbes themselves.

Macrophages and neutrophils display a variety of cell-surface receptors that enable them to recognize and engulf pathogens. These include pattern recognition receptors such as TLRs. In addition, they have cell-surface receptors for the Fc portion of antibodies produced by the adaptive immune system, as well as for the C3b component of complement. Ligand binding to any of these receptors induces actin polymerization at the site of pathogen attachment, causing the phagocyte's plasma membrane to surround the pathogen and engulf it in a large membrane-enclosed phagosome (Figure 25-46).

Once the pathogen has been phagocytosed, the macrophage or neutrophil unleashes an impressive armory of weapons to kill it. The phagosome is acidified and fuses with lysosomes, which contain lysozyme and acid hydrolases that can degrade bacterial cell walls and proteins. The lysosomes also contain defensins, which make up about 15% of the total protein in neutrophils. In addition, the phagocytes assemble an NADPH oxidase complex on the phagosomal membrane that catalyzes the production of a series of highly toxic oxygen-derived compounds, including superoxide (O₂ ^-), hypochlorite (HOCl, the active ingredient in bleach), hydrogen peroxide, hydroxyl radicals, and nitric oxide (NO). The production of these toxic compounds is accompanied by a transient increase in oxygen consumption by the cells, called the respiratory burst. Whereas macrophages will generally survive this killing frenzy and continue to patrol tissues for other pathogens, neutrophils usually die. Dead and dying neutrophils are a major component of the pus that forms in acutely infected wounds. The distinctive greenish tint of pus is due to the abundance in neutrophils of the copper-containing enzyme myeloperoxidase, which is one of the components active in the respiratory burst.

If a pathogen is too large to be successfully phagocytosed (if it is a large parasite such as a nematode, for example), a group of macrophages, neutrophils, or eosinophils (discussed in Chapter 22) will gather around the invader. They will secrete their defensins and other lysosomal products by exocytosis and will also release the toxic products of the respiratory burst (Figure 25-47). This barrage is generally sufficient to destroy the pathogen.

Many pathogens have developed strategies that allow them to avoid being ingested by phagocytes. Some Gram-positive bacteria coat themselves with a very thick, slimy polysaccharide coat, or capsule, that is not recognized by complement or any phagocyte receptor. Other pathogens are phagocytosed but avoid being killed; as we saw earlier, Mycobacterium tuberculosis prevents the maturation of the phagosome and thereby survives. Some pathogens escape the phagosome entirely, and yet others secrete enzymes that detoxify the products of the respiratory burst. For such wily pathogens, these first lines of defense are insufficient to clear the infection, and adaptive immune responses are required to contain them.

Activated Macrophages Recruit Additional Phagocytic Cells to Sites of Infection

When a pathogen invades a tissue, it almost always elicits an inflammatory response. This response is characterized by pain, redness, heat, and swelling at the site of infection, all caused by changes in local blood vessels. The blood vessels dilate and become permeable to fluid and proteins, leading to local swelling and an accumulation of blood proteins that aid in defense, including the components of the complement cascade. At the same time, the endothelial cells lining the local blood vessels are stimulated to express cell adhesion proteins (discussed in Chapter 19) that facilitate the attachment and extravasion of white blood cells, including neutrophils, lymphocytes, and monocytes (the precursors of macrophages).

The inflammatory response is mediated by a variety of signaling molecules. Activation of TLRs results in the production of both lipid signaling molecules such as prostaglandins and protein (or peptide) signaling molecules such as cytokines (discussed in Chapter 15), all of which contribute to the inflammatory response. The proteolytic release of complement fragments also contribute. Some of the cytokines produced by activated macrophages are chemoattractants (known as chemokines). Some of these attract neutrophils, which are the first cells recruited in large numbers to the site of the new infection. Others later attract monocytes and dendritic cells. The dendritic cells pick up antigens from the invading pathogens and carry them to nearby lymph nodes, where they present the antigens to lymphocytes to marshal the forces of the adaptive immune system (discussed in Chapter 24). Other cytokines trigger fever, a rise in body temperature. On balance, fever helps the immune system in the fight against infection, since most bacterial and viral pathogens grow better at lower temperatures, whereas adaptive immune responses are more potent at higher temperatures.

Some proinflammatory signaling molecules stimulate endothelial cells to express proteins that trigger blood clotting in local small vessels. By occluding the vessels and cutting off blood flow, this response can help prevent the pathogen from entering the bloodstream and spreading the infection to other parts of the body.

The same inflammatory responses, however, which are so effective at controlling local infections, can have disastrous consequences when they occur in a disseminated infection in the bloodstream, a condition called sepsis. The systemic release of proinflammatory signaling molecules into the blood causes dilation of blood vessels, loss of plasma volume, and widespread blood clotting, which is an often fatal condition known as septic shock. Inappropriate or overzealous inflammatory responses are also associated with some chronic conditions, such as asthma (Figure 25-48).

Just as with phagocytosis, some pathogens have developed mechanisms to either prevent the inflammatory response or, in some cases, take advantage of it to spread the infection. Many viruses, for example, encode potent cytokine antagonists that block aspects of the inflammatory response. Some of these are simply modified forms of cytokine receptors, encoded by genes acquired by the viral genome from the host. They bind the cytokines with high affinity and block their activity. Some bacteria, such as Salmonella, induce an inflammatory response in the gut at the initial site of infection, thereby recruiting macrophages and neutrophils that they then invade. In this way, the bacteria hitch a ride to other tissues in the body.

Virus-Infected Cells Take Drastic Measures to Prevent Viral Replication

The pathogen-associated immunostimulants on the surface of bacteria and parasites that are so important in eliciting innate immune responses are generally not present on the surface of viruses. Viral proteins are constructed by the host cell ribosomes, and the membranes of enveloped viruses are composed of host cell lipids. The only unusual molecule associated with viruses is the double-stranded RNA (dsRNA) that is an intermediate in the life cycle of many viruses. Host cells can detect the presence of dsRNA and initiate a program of drastic responses in attempt to eliminate it.

The program occurs in two steps. First, the cells degrade the dsRNA into small fragments (about 21–25 nucleotide pairs in length). These fragments bind to any single-stranded RNA (ssRNA) in the host cell with the same sequence as either strand of the dsRNA fragment, leading to the destruction of the ssRNA. This dsRNA-directed ssRNA destruction is the basis of the technique of RNA interference (RNAi) that is used by researchers to block specific gene expression (discussed in Chapter 8). Second, the dsRNA induces the host cell to produce and secrete two cytokines—interferon α (IFN-α) and interferon β (IFN-β), which act in both an autocrine fashion on the infected cell and a paracrine fashion on uninfected neighbors. The binding of the interferons to their cell-surface receptors stimulates specific gene transcription by the Jak/STAT intracellular signaling pathway (see Figure 15-63), leading to the activation of a latent ribonuclease, which nonspecifically degrades ssRNA. It also leads to the activation of a protein kinase that phosphorylates and inactivates the protein synthesis initiation factor eIF-2, shutting down most protein synthesis in the embattled host cell. Apparently, by destroying most of the RNA it contains and transiently halting most protein synthesis, the cell inhibits viral replication without killing itself. In some cases, however, a cell infected with a virus is persuaded by white blood cells to destroy itself to prevent the virus from replicating.

Natural Killer Cells Induce Virus-Infected Cells to Kill Themselves

Another way that the interferons help vertebrates defend themselves against viruses is by stimulating both innate and adaptive cellular immune responses. In Chapter 24, we discuss how interferons enhance the expression of class I MHC proteins, which present viral antigens to cytotoxic T lymphocytes (see Figure 24-48). Here, we consider how interferons enhance the activity of natural killer cells (NK cells), which are part of the innate immune system. Like cytotoxic T cells, NK cells destroy virus-infected cells by inducing the infected cell to kill itself by undergoing apoptosis. Unlike T cells, however, NK cells do not express antigen-specific receptors. How, then, do they distinguish virus-infected cells from uninfected cells?

NK cells monitor the level of class I MHC proteins, which are expressed on the surface of most vertebrate cells. The presence of high levels of these proteins inhibits the killing activity of NK cells, so that the NK cells selectively kill cells expressing low levels, including both virally-infected cells and some cancer cells (Figure 25-49). Many viruses have developed mechanisms to inhibit the expression of class I MHC molecules on the surface of the cells they infect, in order to avoid detection by cytotoxic T lymphocytes. Adenovirus and HIV, for example, encode proteins that block class I MHC gene transcription. Herpes simplex virus and cytomegalovirus block the peptide translocators in the ER membrane that transport proteasome-derived peptides from the cytosol into the lumen of the ER; such peptides are required for newly-made class I MHC proteins to assemble in the ER membrane and be transported through the Golgi apparatus to the cell surface (see Figure 24-58). Cytomegalovirus causes the retrotranslocation of class I MHC proteins from the ER membrane into the cytosol, where they are rapidly degraded by proteasomes. Proteins encoded by still other viruses prevent the delivery of assembled class I MHC proteins from the ER to the Golgi apparatus, or from the Golgi apparatus to the plasma membrane. By evading recognition by cytotoxic T cells in these ways, however, a virus incurs the wrath of NK cells. The local production of IFN-α and IFN-β activates the killing activity of NK cells and also increases the expression of class I MHC proteins in uninfected cells. The cells infected with a virus that blocks class I MHC expression are thereby exposed and become the victims of the activated NK cells. Thus, it is difficult or impossible for viruses to hide from both the innate and adaptive immune systems simultaneously.

Both NK cells and cytotoxic T lymphocytes kill infected target cells by inducing them to undergo apoptosis before the virus has had a chance to replicate. It is not surprising, then, that many viruses have acquired mechanisms to inhibit apoptosis, particularly early in infection. As discussed in Chapter 17, apoptosis depends on an intracellular proteolytic cascade, which the cytotoxic cell can trigger either through the activation of cell-surface death receptors or by injecting a proteolytic enzyme into the target cell (see Figure 24-46). Viral proteins can interfere with nearly every step in these pathways. In some cases, however, viruses encode proteins that act late in their replication cycle to induce apoptosis in the host cell, thereby releasing progeny virus that can infect neighboring cells.

The battle between pathogens and host defenses is remarkably balanced. At present, humans seem to be gaining a slight advantage, using public sanitation measures, vaccines, and drugs to aid the efforts of our innate and adaptive immune systems. However, infectious and parasitic diseases are still the leading cause of death worldwide, and new epidemics such as AIDS continue to emerge. The rapid evolution of pathogens and the almost infinite variety of ways that they can invade the human body and elude immune responses will prevent us from ever winning the battle completely.

Summary

The innate immune responses are the first line of defense against invading pathogens. They are also required to initiate specific adaptive immune responses. Innate immune responses rely on the body's ability to recognize conserved features of pathogens that are not present in the uninfected host. These include many types of molecules on microbial surfaces and the double-stranded RNA of some viruses. Many of these pathogen-specific molecules are recognized by Toll-like receptor proteins, which are found in plants and in invertebrate and vertebrate animals. In vertebrates, microbial surface molecules also activate complement, a group of blood proteins that act together to disrupt the membrane of the microorganism, to target microorganisms for phagocytosis by macrophages and neutrophils, and to produce an inflammatory response. The phagocytic cells use a combination of degradative enzymes, antimicrobial peptides, and reactive oxygen species to kill the invading microorganisms. In addition, they release signaling molecules that trigger an inflammatory response and begin to marshal the forces of the adaptive immune system. Cells infected with viruses produce interferons, which induce a series of cell responses to inhibit viral replication and activate the killing activities of natural killer cells and cytotoxic T lymphocytes.