We now know that the indigenous microbial organisms living in close association with almost all animals and plants are organized into complex communities that strongly modulate overall host physiology, including the ability of pathogenic microbes to establish themselves in or on host surfaces. The sheer numbers of these microbes and their genomic variability often exceed the numbers of host cells and the variability of host genes in a typical animal. Changes and differences in microbiomes within and between individuals, currently characterized by high-throughput DNA sequencing techniques and bioinformatic analysis, impact such diverse conditions as obesity; type 1 diabetes; cognition; neurologic states; autoimmune diseases; skin, gastrointestinal, respiratory, and vaginal infectious diseases; and development and control of the immune system. It has been difficult to directly associate specific types of microbiomes with pathophysiologic states, and our understanding of the degree to which microbial species are conserved or variable within human and other animal microbiotas is evolving. Experimental studies in laboratory animals, particularly in germ-free mammals, show the potent ability of changes in the microbiota to manipulate health status and outcomes. One of the clearest functions of the microbiota is to influence and mature the cells of the immune system, thereby exerting a major effect on susceptibility and resistance to microbial infection. The degree to which studies of the microbiome will translate into strategies for the management of human health and disease (e.g., the use of fecal transplants to treat and prevent recurrences of serious Clostridium difficile infection) is still an open question. For the moment, defining clusters of organisms associated with diseases may be more feasible than identifying single organisms or microbial molecules. Results from the Human Microbiome Project suggest a high level of variability among individuals in microbiome components, although many individuals appear to maintain a fairly conserved microbiome throughout their lives. In the context of infectious diseases, changes and disruptions of the indigenous microbiome—i.e., alterations of the normal flora due to antibiotic and immunosuppressive drug use, environmental changes, and the effects of microbial virulence factors used to displace the indigenous microbial flora and thus to facilitate pathogen colonization—have a strong and often fundamental impact on the progression of infection. While the technology for defining and understanding the microbiome is still quite young, there is little doubt that the resulting data will markedly affect our concepts of and approaches to microbial pathogenesis and infectious disease treatment.
A microbial pathogen can potentially enter any part of a host organism. In general, the type of disease produced by a particular microbe is often a direct consequence of its route of entry into the body. The most common sites of entry are mucosal surfaces (the respiratory, alimentary, and urogenital tracts) and the skin. Ingestion, inhalation, and sexual contact are typical routes of microbial entry. Other portals of entry include sites of skin injury (cuts, bites, burns, trauma) along with injection via natural (e.g., vector-borne) or artificial (e.g., needlestick injury) routes. A few pathogens, such as Schistosoma species, can penetrate unbroken skin. The conjunctiva can serve as an entry point for pathogens of the eye, which occasionally spread systemically from that site.
Microbial entry usually relies on the presence of specific factors needed for persistence and growth in a tissue. Fecal–oral spread via the alimentary tract requires a biologic profile consistent with survival in the varied environments of the gastrointestinal tract (including the low pH of the stomach and the high bile content of the intestine) as well as in contaminated food or water outside the host. Organisms that gain entry via the respiratory tract survive well in small moist droplets produced during sneezing and coughing. Pathogens that enter by venereal routes often survive best in the warm moist environment of the urogenital mucosa and have restricted host ranges (e.g., Neisseria gonorrhoeae, Treponema pallidum, and HIV).
The biology of microbes entering through the skin is highly varied. Some of these organisms can survive under a broad range of environmental conditions, such as those in the salivary glands or alimentary tracts of arthropod vectors, the mouths of larger animals, soil, and water. A complex biology allows protozoan parasites such as Plasmodium, Leishmania, and Trypanosoma species to undergo morphogenic changes that permit transmission of the organism to mammalian hosts during insect feeding for blood meals. Plasmodia are injected as infective sporozoites from the salivary glands during mosquito feeding. Leishmania parasites are regurgitated as promastigotes from the alimentary tract of sandflies and injected by bite into a susceptible host. Trypanosomes are first ingested from infected hosts by reduviid bugs; the pathogens then multiply in the gastrointestinal tract of the insects and are released in feces onto the host’s skin during subsequent feedings. Most microbes that land directly on intact skin are destined to die, as survival on the skin or in hair follicles requires resistance to fatty acids, low pH, and other antimicrobial factors on the skin. Once it is damaged (and particularly if it becomes necrotic), the skin can be a major portal of entry and growth for pathogens and elaboration of their toxic products. Burn wound infections and tetanus are clear examples. After animal bites, pathogens resident in the animal’s saliva gain access to the victim’s tissues through the damaged skin. Rabies is the paradigm for this pathogenic process; rabies virus grows in striated muscle cells at the site of inoculation.
Once in or on a host, many microbes must situate themselves favorably to avoid clearance mechanisms, in part by microbial anchoring to a tissue or tissue factor. (One possible exception is an organism that directly enters the bloodstream and multiplies there.) Because most host cells—responding to activation of innate immunity (see “Avoidance of Innate Host Defenses,” below)—express multiple surface and cytoplasmic molecules that detect pathogens and pathogen factors, a complex interplay ensues and determines whether the microbe will avoid host clearance and remain in a tissue. Viruses and intracellular pathogens like Mycobacterium tuberculosis must bind to cells and enter them, whereas common extracellular bacterial pathogens of the human respiratory tract survive better if they avoid binding to pulmonary epithelial cells.
Specific ligands or adhesins for host receptors constitute a major area of study in microbial pathogenesis. Adhesins comprise a wide range of surface structures, anchoring the microbe to a tissue and promoting cellular entry as well as eliciting host responses critical to innate immunity (Table 116-1). Most microbes produce multiple adhesins specific for multiple host receptors that often are redundant, are serologically variable, and act additively or synergistically with other microbial factors to promote sticking to host tissues. In addition, some microbes adsorb host proteins onto their surface and use the natural host protein receptor for binding and entry into cells. While it is clear that, for some pathogenic organisms, blocking adherence can be a means to prevent infection, for others it could have unintended consequences, decreasing the innate host response that facilitates elimination of the infecting microbe.
TABLE 116-1Examples of Microbial Ligand–Receptor Interactions ||Download (.pdf) TABLE 116-1 Examples of Microbial Ligand–Receptor Interactions
|MICROORGANISM ||TYPE OF MICROBIAL LIGAND ||HOST RECEPTOR |
|Viral Pathogens |
|Influenza virus ||Hemagglutinin ||Sialic acid |
|Measles virus || || |
| Vaccine strain ||Hemagglutinin ||CD46/moesin/signaling lymphocytic activation molecule (SLAM)/nectin-4 |
| Wild-type strains ||Hemagglutinin |
|Human herpesvirus type 6A ||Glycoprotein complex gH/gL/gQ1/gQ2 ||CD46 |
|Herpes simplex virus ||Glycoprotein C ||Heparan sulfate |
|HIV ||Surface glycoprotein ||CD4 and chemokine receptors (CCR5 and CXCR4) |
|Epstein-Barr virus ||Envelope protein ||CD21 (CR2) |
|Adenovirus ||Fiber protein ||Coxsackie-adenovirus receptor (CAR) |
|Coxsackievirus ||Viral coat proteins ||CAR and major histocompatibility class I antigens |
|Bacterial Pathogens |
|Neisseria spp. ||Pili ||Membrane cofactor protein (CD46) |
|Pseudomonas aeruginosa || |
Pili and flagella
Cystic fibrosis transmembrane conductance regulator (CFTR)
|Escherichia coli ||Pili ||Ceramides/mannose and digalactosyl residues |
|Streptococcus pyogenes ||Hyaluronic acid capsule ||CD44 |
|Yersinia spp. ||Invasin/accessory invasin locus ||β1 Integrins |
|Bordetella pertussis ||Filamentous hemagglutinin ||CR3 |
|Legionella pneumophila ||Adsorbed C3bi ||CR3 |
|Mycobacterium tuberculosis ||Adsorbed C3bi ||CR3; DC-SIGN |
|Fungal Pathogens |
|Blastomyces dermatitidis ||WI-1 ||Possibly matrix proteins and integrins |
|Candida albicans ||Int1p ||Extracellular matrix proteins |
|Protozoal Pathogens |
|Plasmodium vivax ||Merozoite form ||Duffy Fy antigen |
|Plasmodium falciparum ||Erythrocyte-binding protein 175 (EBA-175) ||Glycophorin A |
|Entamoeba histolytica ||Surface lectin ||N-Acetylglucosamine |
All viral pathogens must bind to host cells, enter them, and replicate within them. Viral coat proteins serve as ligands for cellular entry, and more than one ligand–receptor interaction may be needed. In some types of viruses, such as lipid bilayer–encapsulated Retroviridae or Rhabdoviridae, a single protein mediates both viral binding and entry via fusion with the host cell membrane. In other cases, a second viral fusion protein is needed to complete viral entry. HIV uses its envelope glycoprotein (gp) 120 to enter host cells by binding to both CD4 and one of two receptors for chemokines (CCR5 or CXCR4). Measles virus requires two proteins for cellular entry: the hemagglutinin (H) glycoprotein of wild-type measles virus binds to the signaling lymphocytic activation molecule (SLAM or CD150) on macrophages and dendritic cells, where the virus initially replicates, and also to nectin-4 on respiratory epithelial cells, where later replication occurs. The vaccine strain of measles virus binds to both CD46 and SLAM. For full cellular entry, however, measles virus requires a second fusion (F) protein. The gB and gC proteins on herpes simplex virus bind to heparan sulfate, although this adherence is not essential for entry but rather serves to concentrate virions close to the cell surface; this step is followed by attachment to mammalian cells mediated by the viral gD protein, with subsequent formation of a homotrimer of viral gB protein or a heterodimer of viral gH and gL proteins that permits fusion of the viral envelope with the host cell membrane. Herpes simplex virus can use a number of eukaryotic cell-surface receptors for entry, including the herpesvirus entry mediator, members of the immunoglobulin superfamily, the proteins nectin-1 and nectin-2, and modified heparan sulfate.
Among the adhesins studied in greatest detail are bacterial pili and flagella (Fig. 116-1). Pili or fimbriae are commonly used by gram-negative bacteria for attachment to host cells and tissues; similar factors are produced by gram-positive organisms such as group B streptococci. In electron micrographs, these hairlike projections (up to several hundred per cell) may be confined to one end of the organism (polar pili) or distributed more evenly over the surface. An individual cell may have pili with a variety of functions. Most pili are made up of a major pilin protein subunit (17,000–30,000 Da) that polymerizes to form the pilus. Many strains of Escherichia coli isolated from urinary tract infections express a mannose-binding type 1 pilus that attaches to the uroplakins coating the cells in the bladder epithelium. Other strains produce the Pap (pyelonephritis-associated) or P pilus adhesin that mediates binding to digalactose (gal-gal) residues on globosides of the human P blood groups. Both of these pili have proteins located at the tips of the main pilus unit that are critical to the binding specificity of the whole pilus unit. E. coli cells causing diarrheal disease express pilus-like receptors for enterocytes on the small bowel, along with other receptors termed colonization factors.
Bacterial surface structures. A and B. Traditional electron micrographic images of fixed cells of Pseudomonas aeruginosa. Flagella (A) and pili (B) project out from the bacterial poles. C and D. Atomic force microscopic image of live P. aeruginosa freshly planted onto a smooth mica surface. This technology reveals the fine, three-dimensional detail of the bacterial surface structures.
The type IV pili found in Neisseria species, Moraxella species, Vibrio cholerae, Legionella pneumophila, Salmonella enterica serovar Typhi, enteropathogenic E. coli, and Pseudomonas aeruginosa often mediate adherence of organisms to target surfaces. Type IV pili tend to have a relatively conserved amino-terminal region and a more variable carboxyl-terminal region. For some species (e.g., N. gonorrhoeae, Neisseria meningitidis, and enteropathogenic E. coli), the pili are critical for attachment to mucosal epithelial cells. For others, such as P. aeruginosa, the pili may inhibit colonization; recent studies of P. aeruginosa colonization showed that, in a bank of mutants in which all nonessential genes were interrupted, those unable to produce type IVa pili were actually better able to colonize the gastrointestinal and lung mucosa of mice. V. cholerae cells appear to use two different types of pili for intestinal colonization. Whereas interference with this stage of colonization would appear to be an effective antibacterial strategy, attempts to develop pilus-based vaccines against human diseases have not been highly successful to date.
Flagella are long appendages attached at one or both ends of the bacterial cell (polar flagella) or distributed over the entire cell surface (peritrichous flagella). Flagella, like pili, are composed of a polymerized or aggregated basic protein. In flagella, the protein subunits form a tight helical structure and vary serologically with the species. Spirochetes such as T. pallidum and Borrelia burgdorferi have axial filaments similar to flagella running down the long axis of the center of the cell, and they “swim” by rotation around these filaments. Some bacteria can glide over a surface in the absence of obvious motility structures.
Other bacterial structures involved in adherence to host tissues include staphylococcal and streptococcal proteins that bind to human extracellular matrix proteins such as fibrin, fibronectin, fibrinogen, laminin, and collagen. Fibronectin is a commonly used receptor for various pathogens; a particular amino acid sequence in fibronectin, Arg-Gly-Asp or RGD, is a conserved target used by bacteria to bind to host tissues. Binding of the Staphylococcus aureus surface protein clumping factor A (ClfA) to fibrinogen has been implicated in many aspects of pathogenesis. The conserved outer-core portion of the lipopolysaccharide (LPS) of P. aeruginosa mediates binding to the cystic fibrosis transmembrane conductance regulator (CFTR) on airway epithelial cells—an event that appears to be critical for normal host resistance to infection, initiating recruitment of polymorphonuclear neutrophils (PMNs) to the lung mucosa to kill the cells via opsonophagocytosis. A large number of microbial pathogens encompassing major gram-positive bacteria (staphylococci and streptococci), gram-negative bacteria (major enteric species and coccobacilli), fungi (Candida, Fusobacterium, Aspergillus), and even eukaryotic pathogens (Trichomonas vaginalis and Plasmodium falciparum) express a surface polysaccharide composed of β-1-6-linked-poly-N-acetyl-d-glucosamine (PNAG). One of its functions is to promote binding to synthetic materials used in catheters and other types of implanted devices. This polysaccharide may be a critical factor in the establishment of device-related infections by pathogens such as staphylococci and E. coli. High-powered imaging techniques (e.g., atomic force microscopy) have revealed that bacterial cells have a nonhomogeneous surface that is probably attributable to different concentrations of cell surface molecules, including microbial adhesins, at specific locations (Fig. 116-1, panels C and D).
Fungi produce adhesins that mediate colonization of epithelial surfaces, adhering particularly to structures like fibronectin, laminin, and collagen. The Candida albicans INT1 protein bears similarity to mammalian integrins that bind to extracellular matrix proteins. The agglutinin-like sequence (ALS) adhesins are large cell-surface glycoproteins mediating adherence of pathogenic Candida to host tissues. These adhesins possess a conserved three-domain structure composed of an N-terminus that mediates adherence to host tissue receptors, a central motif consisting of a number of repeats of a conserved sequence of 36 amino acids, and a C-terminal domain that varies in length and sequence and contains a glycosylphosphatidylinositol (GPI) anchor addition that allows the adhesins to bind to the fungal cell wall. Variability in the number of central domains characterizes different ALS proteins with specificity for different host receptors. The ALS adhesins are expressed under certain environmental conditions and are crucial for pathogenesis of fungal infections.
For several respiratory fungal pathogens, the inoculum is ingested by alveolar macrophages in which the fungal cells transform to pathogenic phenotypes. Like C. albicans, Blastomyces dermatitidis produces a 120-kDa surface protein, designated WI-1, that binds to CD11b/CD18 integrins as well as to CD14 on macrophages. An unidentified factor on Histoplasma capsulatum also mediates binding to the integrin surface proteins.
Eukaryotic Pathogen Adhesins
Eukaryotic parasites use complicated surface glycoproteins as adhesins, some of which are lectins (proteins that bind to specific carbohydrates on host cells). Plasmodium vivax, one of six Plasmodium species causing malaria, binds (via Duffy-binding protein) to the Duffy blood group carbohydrate antigen Fy on erythrocytes. Entamoeba histolytica, the third leading cause of death from parasitic diseases, expresses two proteins that bind to the disaccharide galactose/N-acetyl galactosamine. Children with mucosal IgA antibody to one of these lectins are resistant to reinfection with virulent E. histolytica. A major surface glycoprotein (gp63) of Leishmania promastigotes is needed for these parasites to enter human macrophages—the principal target cell of infection. This glycoprotein promotes complement binding but inhibits complement lytic activity, allowing the parasite to use complement receptors for entry into macrophages; gp63 also binds to fibronectin receptors on macrophages. As part of hepatic granuloma formation, Schistosoma mansoni expresses a carbohydrate epitope related to the Lewis X blood group antigen that promotes adherence of helminthic eggs to vascular endothelial cells under inflammatory conditions.
Host receptors are found both on target cells (such as epithelial cells lining mucosal surfaces) and within the mucus layer covering these cells. Microbial pathogens bind to a wide range of host receptors to establish infection (Table 116-1). Selective loss of host receptors for a pathogen may confer natural resistance to an otherwise susceptible population. For example, 70% of individuals in western Africa lack Fy antigens and are resistant to P. vivax infection. S. enterica serovar Typhi, the etiologic agent of typhoid fever, produces a pilus protein that binds to CFTR to enter the gastrointestinal submucosa after being ingested by enterocytes. As homozygous mutations in CFTR are the cause of the life-shortening disease cystic fibrosis, heterozygote carriers (e.g., 4–5% of individuals of European ancestry) may have had a selective advantage due to decreased susceptibility to typhoid fever.
Numerous virus–target cell interactions have been described, and it is now clear that different viruses can use similar host cell receptors for entry. The list of certain and likely host receptors for viral pathogens is long. Among the host membrane components that can serve as receptors for viruses are sialic acids, gangliosides, glycosaminoglycans, integrins and other members of the immunoglobulin superfamily, histocompatibility antigens, and regulators and receptors for complement components. An example of the effect of host receptors on the pathogenesis of infection has emerged from studies comparing the binding of avian influenza A virus subtype H5N1 with that of influenza A strains expressing the H1 hemagglutinin subtype. These subtypes are highly pathogenic and transmissible from human to human, and they bind to a receptor composed of two sugar molecules: sialic acid linked α-2-6 to galactose. This receptor is expressed at high levels in the human airway epithelium; when virus is shed from this surface, its transmission via coughing and aerosol droplets is facilitated. In contrast, the H5N1 avian influenza virus binds to sialic acid linked α-2-3 to galactose, and this receptor is expressed at high levels on cells in the terminal bronchioles, including type II pneumocytes, alveolar macrophages, and nonciliated cuboidal epithelial cells. Infection at these sites is thought to underlie the high mortality rate associated with avian influenza but also the low interhuman transmissibility of this strain, which is not readily transported to the airways from which it can be expelled by coughing. Nonetheless, it has been shown that H5 hemagglutinins can acquire mutations leading to binding to α-2-6-linked sialic acids that increase their human transmissibility but retain their high level of lethality.