Life cycle

Enter

Disseminate

and exit

Colonize

Replicate

Persist

Figure 32-3. The life cycle of a pathogen. For a pathogen to survive it needs to invade the host, colonize the site of infection while resisting expulsion from the host, replicate, then exit to disseminate and find another host that would supply a new replication site and source of nutrients. Courtesy of Stanley Falkow.

or expression levels of flagellin when crossing epithelial cell barriers7; these cells express, on their basolateral surface, TLR5, a PRR that senses flagellin and guards against pathogen invasion.8,9

For intracellular pathogens, the challenge is of a different nature. They need to enter host cells, replicate without being recognized and degraded, and then exit the cell. A common strategy of intracellular pathogens is subversion of the endocytic machinery to their advantage and hiding in endosomal compartments. For instance, Brucella spp., as well as Legionella pneumophila, the causative agent of Legionnaire’s disease, surround their phagosomes with an endoplasmic reticulum–derived membrane that inhibits phagosomelysosome fusion, establishing an ideal niche for replication (Figure 32-4c).10 After replication, pathogens break out of their hiding places into the cytosol, where they induce cell death to exit the cell and disseminate. Pathogens secrete enzymes and toxins that allow their spread in tissues. Enteric pathogens induce death of intestinal epithelial cells or break their tight junctions to reach the submucosa. Various microbial toxins – such as nigericin, maitotoxin, aerolysin, gramicidin, pneumolysin, α-hemolysin, and α-toxin – form pores on the surface of host cells leading to their death. Some toxins derange the plasma membrane or the cytoskeleton, whereas others interfere with signal transduction pathways.11

3. HOST DEFENSE

inducible defense mechanisms. In mammals, constitutive defenses include the normal microbial flora that compete with pathogens, physical barriers of the skin and internal epithelial layers, mechanical defenses of the mucus and cilia, and chemical defenses such as the acidic pH of the stomach. For a long time, innate immunity was considered nonspecific. However, in the late 1980s, Janeway proposed that detection of pathogens by PRRs was key to specific activation of immune responses.12 Various classes of innate immunity recognition systems have been discovered. Some are soluble molecules such as mannose-binding lectins, ficolins, and collectins, whereas others are confined to cells, most notably, C-type lectins, Toll-like receptors (TLRs), nucleotide binding and oligomerization domain (Nod)– like receptors (NLRs), dsRNA helicase–like receptors (RIG-I and Mda5), and the cytoplasmic dsDNA receptors ZBP1-DAI and AIM2. PRRs recognize PAMP “signatures” displayed by microorganisms but not found on host cells. They also sense alterations in the host cellular environment arising indirectly from the infection, referred to as danger-associated molecular patterns (DAMPs), or alarm signals. Decrease in intracellular K+ levels, which occurs in response to pore-forming toxins, accumulation of reactive oxygen species (ROS), or release of “alarmins” in the extracellular space are examples of alarm signals or DAMPs that activate PRRs. Alarmins are unrelated host proteins with distinct cellular functions that acquire the ability to signal tissue damage and trigger an inflammatory response when secreted in the extracellular milieu in response to infection or cell death. Most notable are antimicrobial peptides, heat shock proteins, the non-histone chromatin-binding protein HMGB1, and extracellular matrix degradation products such as hyaluronan.13 Activation of PRRs is transduced via a plethora of cellular proteins (kinases, ubiquitin ligases, adaptors, proteases, transcription factors), which induce a large array of interconnected and synergistic defense mechanisms aimed at killing the pathogen while preserving host cell integrity. These defenses include the production of antimicrobial peptides by epithelial cells and neutrophils, elicitation of an inflammatory response necessary for the activation of phagocytes, and the death of infected cells that, in certain instances, limits the spread of pathogens to surrounding tissues.

3.1. Antimicrobial peptides

The host evolved means to detect pathogens’ intrusion and mechanisms to restrict their growth. Firstline defenses are provided by the innate immune system, which is armed by constitutive as well as

Antimicrobial peptides (AMPs) are endogenous antibiotics that have been established as an essential part of innate immunity. They bind to a wide variety of pathogens, including Gram-negative and Gram-positive

Legionella

phagosome

Endoplasmic reticulum

Ribosome

(c) Sec61 complex

bacteria, fungi, and some viruses, and disrupt their cytoplasmic membrane. In addition to their direct role in killing microbes, they act as immunostimulants that modulate the inflammatory response and chemoattract and activate antigen-presenting cells (APCs). They are small, generally cationic peptides with spaced hydrophobic and charged regions and are synthesized as prepropeptides with an N-terminal signal sequence, an anionic pro segment, and a C-terminal cationic AMP domain that gains biological activity after processing. In humans, AMPs are subgrouped into three classes based on structural characteristics: the defensins (α and β subfamilies), cathelicidins, and histatins (Figure 32-5).

Histatins (His-1 and His-3) are his- tidine-rich, mainly antifungal peptides found in the saliva. Defensins and cathelicidins are, on the other hand, expressed in neutrophils, keratinocytes, and epithelial cells either constitutively or on induction by bacteria and cytokines. More than 300 defensins have been identified so far in many organisms, including mammals, birds, invertebrates, and plants (http:// defensins.bii.a-star.edu.sg). In humans, there are six α-defensins. α-defensins 1 through 4, also known as human neutrophil peptides (HNP1–4), are stored as mature peptides in neutrophil azurophilic granules and contribute to nonoxidative killing of phagocytized pathogens. Human α-defensins 5 and 6 (HD-5 and -6) are primarily produced by epithelial cells and require processing upon release. In rodents, α-defensins are termed cryptdins, as they are mainly found in Paneth cells of the small intestinal crypts.

matrilysin; MMP7−/– mice lack mature cryptdins and are susceptible to oral infection with Salmonella.14 Unlike in mice, matrilysin is not found in the small intestine of humans; the digestive enzyme trypsin was found to be the cleaving enzyme for HD5.15 The question of why mice and humans use different enzymes to process Paneth cells defensins remains unclear. β- defensins (hBD1, hBD2, hBD3, and hBD4) are primarily produced by

epithelial cells. The processing of β-defensins is thought to occur in a similar fashion to that of α-defensins; however, the convertases involved remain unknown. α and β defensin subfamilies are characterized by three intramolecular disulfide bonds mediated by six conserved cysteine residues and differ by the cysteine pairing and the length of peptides between the cysteines (Figure 32-5). Structurally, they are folded in a characteristic three-stranded β sheet. θ-defensins, which form a third defensin subfamily, are structurally unrelated to α and β defensins and are only found in nonhuman primates. Experiments with genetically modified mice were most informative in confirming