Premed · Premed · Microbiology
Lecture 23: Bacterial Pathogenesis: Mechanisms
Microbiology
Learning Objectives
By the end of this lecture, students will be able to:
- Define virulence, pathogenicity, and virulence factors
- Describe the stages of bacterial pathogenesis: adhesion, invasion, immune evasion, tissue damage, and transmission
- Explain the mechanisms of bacterial adhesion and colonization
- Distinguish between exotoxins and endotoxin and describe their mechanisms of action
- Describe bacterial strategies for immune evasion
- Explain the role of secretion systems, biofilms, and quorum sensing in pathogenesis
- Discuss the genetic regulation of virulence factors
Lecture Content
I. Fundamental Concepts
Pathogenicity is the ability of a microorganism to cause disease, while virulence refers to the degree or measure of pathogenicity. Virulence is quantified using the ID50, the infectious dose required to infect 50% of exposed hosts, and the LD50, the lethal dose for 50% of hosts. A low ID50 indicates high virulence; for instance, Shigella can cause disease with as few as 10 organisms, whereas Salmonella typically requires approximately 10^5 organisms.
Virulence factors are the specific molecules produced by pathogens that contribute to pathogenicity. These include adhesins, invasins, toxins, capsules, secretion systems, siderophores, and immune evasion molecules. Many virulence factors are encoded on pathogenicity islands (PAIs), which are large chromosomal regions of 10 to 200 kilobases acquired by horizontal gene transfer. PAIs characteristically have a different G+C content from the core genome, are flanked by direct repeats, are often associated with tRNA genes, and may carry integrase genes. Important examples include the locus of enterocyte effacement (LEE) in EHEC and EPEC, and the Salmonella pathogenicity islands SPI-1 and SPI-2.
Koch's molecular postulates, as reformulated by Falkow, provide a framework for establishing the role of specific genes in virulence. First, the virulence trait should be associated with pathogenic strains but absent from non-pathogenic strains. Second, inactivation of the gene should reduce virulence. Third, restoration of the gene should restore virulence.
II. Stages of Bacterial Pathogenesis
A. Adhesion and Colonization
The first critical step in pathogenesis is adhesion to host tissues, which allows the pathogen to resist mechanical forces such as mucus flow, peristalsis, urinary flushing, and ciliary action. Adhesins are bacterial surface molecules that bind specific receptors on host cells.
Fimbriae, also called pili, are hair-like protein structures that mediate adhesion. Type 1 pili in E. coli bear the FimH adhesin, which binds mannose residues on uroepithelial cells and promotes urinary tract infections. P pili on uropathogenic E. coli carry the PapG adhesin that binds globoseries glycolipids on kidney epithelium, promoting pyelonephritis. Type IV pili, found in Neisseria and Pseudomonas, mediate both adhesion and twitching motility; the pili of N. gonorrhoeae undergo antigenic variation to evade immune recognition. Bundle-forming pili on EPEC mediate initial attachment to intestinal epithelium.
Afimbrial adhesins are outer membrane proteins that also mediate attachment. Intimins on EPEC and EHEC bind the Tir receptor, which the bacterium itself injects into the host cell via a type III secretion system, creating intimate attachment and attaching-and-effacing (A/E) lesions. Fibronectin-binding proteins of S. aureus bind host fibronectin to facilitate adhesion to wounds and implanted devices. Invasins of Yersinia bind beta-1 integrins on M cells in the intestinal epithelium.
Biofilm formation represents a specialized mode of colonization in which bacteria organize into structured communities encased in extracellular polymeric substances composed of polysaccharides, proteins, and DNA. Biofilm development proceeds through stages of initial attachment, microcolony formation, maturation, and dispersal. Clinically, biofilms are important in catheter-associated infections, prosthetic joint infections, endocarditis, chronic wound infections, and chronic Pseudomonas aeruginosa lung infections in cystic fibrosis. Bacteria within biofilms are 10 to 1000 times more tolerant to antibiotics and are highly resistant to immune clearance.
B. Invasion
Some pathogens actively invade host cells to access intracellular nutrients, replicate in a protected niche, and evade immune responses. Two principal invasion strategies exist. The trigger mechanism, exemplified by Salmonella, employs a type III secretion system (T3SS) that injects effector proteins directly into host epithelial cells. These effectors cause dramatic actin rearrangement and membrane ruffling, leading to macropinocytosis of the bacterium. The zipper mechanism, used by Listeria and Yersinia, relies on bacterial surface proteins such as internalin A or invasin that bind host receptors like E-cadherin or beta-1 integrins, inducing receptor-mediated endocytosis through a tight, controlled invagination of the membrane.
Once inside host cells, pathogens employ various strategies for intracellular survival. Listeria monocytogenes escapes the phagosome using listeriolysin O (LLO), a pore-forming toxin, combined with phospholipases that lyse the phagosomal membrane. Once free in the cytoplasm, Listeria uses ActA to nucleate actin polymerization, propelling itself through the cytoplasm and into neighboring cells. Mycobacterium tuberculosis inhibits phagosome maturation by blocking Rab7 recruitment and acidification, preventing phagosome-lysosome fusion. Coxiella burnetii has adapted to thrive within the acidic environment of the phagolysosome itself. Salmonella resides in a Salmonella-containing vacuole (SCV) maintained by SPI-2 T3SS effectors, while Chlamydia occupies an inclusion body that intercepts host vesicular trafficking.
<image>A comparison of bacterial invasion strategies. Panel A (Trigger mechanism): Salmonella approaching an intestinal epithelial cell; T3SS needle injecting effector proteins (SipA, SipC, SopB, SopE) into the host cell; actin rearrangement causing dramatic membrane ruffling; bacterium engulfed by macropinocytosis; bacterium residing in a Salmonella-containing vacuole (SCV). Panel B (Zipper mechanism): Listeria monocytogenes surface protein InlA binding E-cadherin on the host cell; tight receptor-mediated invagination of the membrane; internalization into a vacuole; listeriolysin O (LLO) and phospholipases lysing the vacuolar membrane; free bacterium in the cytoplasm; ActA protein nucleating actin (comet tail) propelling the bacterium toward the adjacent cell membrane; formation of a double-membraned protrusion engulfed by the neighboring cell. Panel C (Phagosome-lysosome fusion block): M. tuberculosis inside a macrophage phagosome; arrows showing blocked Rab7 recruitment and blocked V-ATPase-mediated acidification; lysosome unable to fuse. Each panel is clearly labeled.</image>
III. Bacterial Toxins
A. Exotoxins
Exotoxins are proteins secreted by living bacteria, produced by both Gram-positive and Gram-negative organisms. They are highly potent and specific in their actions, often encoded on plasmids, phages, or pathogenicity islands. Exotoxins are generally heat-labile, destroyed at temperatures between 60 and 80 degrees Celsius, and highly immunogenic, which means they can be converted to toxoids for use in vaccines.
Exotoxins are classified by their mechanism of action. The A-B toxins consist of an enzymatically active A subunit and a binding B subunit; the B subunit binds a host receptor and facilitates entry of the A subunit into the cell. Diphtheria toxin, produced by C. diphtheriae and phage-encoded, uses its B subunit to bind the HB-EGF receptor, after which the A subunit ADP-ribosylates elongation factor 2 (EF-2), inhibiting protein synthesis and causing cell death. Cholera toxin, produced by V. cholerae, has a B pentamer that binds GM1 ganglioside; the A subunit ADP-ribosylates the Gs-alpha subunit of a G protein, constitutively activating adenylate cyclase and increasing cAMP, which drives chloride and water secretion into the intestinal lumen, producing the voluminous watery diarrhea characteristic of cholera. Pertussis toxin from B. pertussis ADP-ribosylates the Gi-alpha subunit, preventing inhibition of adenylate cyclase and similarly increasing cAMP; it also promotes lymphocytosis. Shiga toxin from Shigella dysenteriae and Shiga-like toxins (Stx1, Stx2) from EHEC O157:H7 have an A subunit that cleaves 28S rRNA, inhibiting protein synthesis and killing cells, particularly renal endothelial cells, leading to hemolytic uremic syndrome (HUS). Tetanus toxin (tetanospasmin) from C. tetani is a zinc metalloprotease that cleaves VAMP/synaptobrevin in inhibitory interneurons, blocking the release of GABA and glycine and causing spastic paralysis. Botulinum toxin from C. botulinum is a zinc metalloprotease that cleaves SNARE proteins (SNAP-25, VAMP, and syntaxin) at the neuromuscular junction, blocking acetylcholine release and causing flaccid paralysis. Anthrax toxin from B. anthracis is a three-component system consisting of protective antigen (PA, the binding component), edema factor (EF, an adenylate cyclase), and lethal factor (LF, a metalloprotease that cleaves MAPKKs).
Pore-forming toxins (PFTs) insert into host cell membranes to form channels, causing lysis or signaling disruption. Alpha-hemolysin of S. aureus forms a heptameric pore in host cell membranes. Streptolysin O from S. pyogenes is a cholesterol-dependent cytolysin that is oxygen-labile; its antibody (ASO titer) is used diagnostically. Streptolysin S from S. pyogenes is oxygen-stable and responsible for the beta-hemolysis observed on blood agar. Pneumolysin from S. pneumoniae is a cholesterol-dependent cytolysin that activates complement and damages epithelium. Listeriolysin O from L. monocytogenes enables phagosomal escape. Panton-Valentine leukocidin (PVL), found especially in CA-MRSA strains of S. aureus, forms pores in neutrophil membranes and is associated with necrotizing pneumonia and skin abscesses.
Superantigens bind MHC class II molecules on antigen-presenting cells and the V-beta region of the T cell receptor simultaneously, bypassing normal antigen processing. They activate up to 20% of all T cells at once, compared to the 0.001% activated in a normal immune response, triggering a massive "cytokine storm." TSST-1 from S. aureus causes toxic shock syndrome. Streptococcal pyrogenic exotoxins (Spe) from S. pyogenes cause scarlet fever and streptococcal toxic shock syndrome. Staphylococcal enterotoxins from S. aureus cause food poisoning; they are notable for being heat-stable and emetic.
B. Endotoxin (LPS)
Endotoxin is the lipopolysaccharide (LPS) component of the Gram-negative outer membrane, released upon bacterial lysis or during active growth through outer membrane vesicles. The active toxic moiety is lipid A. Unlike exotoxins, endotoxin is heat-stable, only weakly immunogenic, and cannot be converted to a toxoid.
The mechanism of endotoxin action involves LPS binding to LPS-binding protein (LBP) in serum, which transfers LPS to CD14 on macrophages. This complex then signals through the TLR4/MD-2 receptor complex, activating both MyD88 and TRIF signaling pathways and ultimately stimulating NF-kB. The result is massive release of pro-inflammatory cytokines including TNF-alpha, IL-1, IL-6, and IL-8. At low doses, this produces a beneficial inflammatory response with fever and neutrophil activation. At high doses, however, endotoxin triggers septic shock characterized by systemic vasodilation, hypotension, disseminated intravascular coagulation (DIC), multi-organ failure, and death. The Limulus amebocyte lysate (LAL) assay is used to detect endotoxin contamination in pharmaceuticals and medical devices.
<image>A comparison table and diagram of exotoxins versus endotoxin. Left side -- Exotoxins: protein, secreted by living cells, heat-labile, high potency (small amounts cause disease), highly specific targets, strong immunogenicity (toxoid vaccines possible), produced by Gram-positive and Gram-negative bacteria. Right side -- Endotoxin (LPS): lipopolysaccharide (lipid A), part of outer membrane released on lysis, heat-stable, lower potency per unit weight, systemic effects (fever, shock), weak immunogenicity, Gram-negative only. Below the table: a molecular diagram showing LPS structure (lipid A embedded in outer membrane, core polysaccharide, O-antigen extending outward), and the signaling cascade: LPS + LBP -> CD14 -> TLR4/MD-2 -> MyD88 -> NF-kB -> TNF-alpha, IL-1, IL-6 -> fever, inflammation, and at high levels -> septic shock (vasodilation, DIC, organ failure).</image>
IV. Immune Evasion Strategies
Bacteria have evolved numerous strategies to evade host immune defenses. The polysaccharide capsule inhibits phagocytosis by masking surface antigens and preventing deposition of complement C3b. Capsules are critical virulence factors for S. pneumoniae, N. meningitidis, K. pneumoniae, H. influenzae type b, and B. anthracis (which uniquely has a poly-D-glutamic acid capsule). Protein A of S. aureus binds the Fc region of IgG in the wrong orientation, preventing opsonization and complement activation. The M protein of S. pyogenes binds factor H, promoting degradation of C3b deposited on the bacterial surface and thereby inhibiting phagocytosis.
IgA proteases, produced by N. gonorrhoeae, N. meningitidis, H. influenzae, and S. pneumoniae, cleave secretory IgA at mucosal surfaces, undermining a key first-line defense. Antigenic variation allows pathogens to change their surface antigens and evade adaptive immunity. N. gonorrhoeae achieves this through pilin gene recombination, Borrelia burgdorferi through VlsE variation, and the eukaryotic parasite Trypanosoma brucei through switching of variant surface glycoproteins.
Intracellular survival represents another powerful evasion strategy, as bacteria hiding inside host cells are shielded from antibodies and complement. Biofilms provide a physical barrier to both immune cell penetration and antibody access. Some bacteria actively degrade complement components or bind host complement regulatory proteins like factor H. Yersinia species inject the effector protein YopJ via their T3SS, which inhibits both NF-kB and MAPK signaling pathways to suppress the inflammatory response.
V. Bacterial Secretion Systems
Gram-negative bacteria have evolved specialized secretion systems for delivering virulence factors across their complex double-membrane cell envelope. The Type I secretion system (T1SS) transports proteins in a single step from the cytoplasm directly to the exterior using an ABC transporter, as seen with E. coli hemolysin (HlyA). The Type II secretion system (T2SS) operates in two steps: the protein first enters the periplasm via the Sec or Tat pathway, then passes through an outer membrane secretin. Examples include cholera toxin and Pseudomonas exotoxin A.
The Type III secretion system (T3SS), often described as a "molecular syringe" or injectisome, injects effector proteins directly into the host cell cytoplasm. It is essential for pathogenesis in Salmonella (SPI-1 and SPI-2), Shigella, EPEC/EHEC, Yersinia, and Pseudomonas. The Type IV secretion system (T4SS) resembles the conjugation machinery and can transfer both DNA and proteins. It is used by Agrobacterium for T-DNA transfer, H. pylori for CagA injection, Legionella through the Dot/Icm system, and Bordetella for pertussis toxin secretion. The Type V secretion system (T5SS) encompasses the autotransporters, in which the passenger domain is secreted through the protein's own beta-barrel in the outer membrane, as exemplified by the IgA protease of Neisseria. The Type VI secretion system (T6SS) is a contractile injection system that delivers effectors into both eukaryotic cells and competing bacteria for inter-bacterial killing; it is found in V. cholerae and P. aeruginosa.
VI. Quorum Sensing and Virulence Regulation
Quorum sensing (QS) is a mechanism of cell-density-dependent communication in which bacteria produce and respond to small signaling molecules called autoinducers. As the bacterial population grows, autoinducer concentration increases proportionally. When it reaches a threshold level, coordinated changes in gene expression occur across the entire population.
In Gram-negative bacteria, the predominant autoinducers are acyl-homoserine lactones (AHLs), synthesized and detected through LuxI/LuxR-type systems. P. aeruginosa employs the Las and Rhl quorum sensing systems to regulate biofilm formation, elastase production, pyocyanin synthesis, and exotoxin A expression. In Gram-positive bacteria, autoinducing peptides (AIPs) serve as the signaling molecules. The Agr system of S. aureus provides an elegant example: at high cell density, accumulated AIP activates the AgrA response regulator, which upregulates secreted virulence factors such as toxins and proteases while downregulating surface adhesins, effectively coordinating a shift from a colonization phenotype to a dissemination phenotype. The AI-2 system, based on the LuxS pathway first described in Vibrio harveyi, facilitates interspecies communication. Quorum sensing is being explored as a therapeutic target through "quorum quenching" strategies, including enzymatic degradation of autoinducers and receptor antagonists.
<image>A two-panel diagram of quorum sensing systems. Panel A (Gram-negative AHL system): At low cell density, individual bacteria produce low levels of AHL (acyl-homoserine lactone via LuxI synthase); AHL diffuses out; intracellular concentration is too low to activate LuxR receptor. At high cell density, AHL accumulates in the environment; AHL binds LuxR intracellularly; LuxR-AHL complex activates transcription of virulence genes (biofilm, toxins, proteases). Panel B (S. aureus Agr system): At low cell density, bacteria express surface adhesins (protein A, fibronectin-binding proteins) for colonization. At high cell density, autoinducing peptide (AIP) accumulates; binds AgrC histidine kinase; phosphorylation cascade activates AgrA; AgrA upregulates RNAIII which increases secreted virulence factors (alpha-hemolysin, TSST-1, proteases) and decreases surface adhesins. A diagram of the agr locus (agrA, agrB, agrC, agrD, RNAIII) is shown below.</image>
VII. Iron Acquisition
Iron is essential for bacterial growth, yet the host actively sequesters it within proteins such as transferrin, lactoferrin, ferritin, and hemoglobin as a defense mechanism known as nutritional immunity. To overcome this, bacteria produce siderophores, which are small, high-affinity iron-chelating molecules secreted into the environment to scavenge iron. Enterobactin, produced by E. coli, is one of the strongest known iron chelators. Pyoverdine, the fluorescent siderophore of P. aeruginosa, and mycobactin of M. tuberculosis serve similar functions. The host counters with siderocalin (lipocalin-2), which binds enterobactin and prevents bacterial iron uptake. In response, some bacteria such as Salmonella and certain E. coli strains produce salmochelin, a glycosylated form of enterobactin that evades siderocalin.
Beyond siderophores, bacteria employ additional iron acquisition strategies. Hemolysins lyse red blood cells to release hemoglobin and liberate iron. Some organisms, such as Neisseria, express surface receptors that directly bind host iron-containing proteins like transferrin. Iron homeostasis in bacteria is regulated by Fur (ferric uptake regulator): when intracellular iron is abundant, Fur acts as a repressor that shuts down siderophore and virulence gene expression. Under iron-limiting conditions such as those encountered within a host, Fur is inactive and these genes become derepressed, coordinating an upregulation of iron acquisition systems and virulence factors.
