T10 Bacterial Pathogenesis

Learning Objectives for This Topic

1. Define pathogenicity and virulence and distinguish between them.

2. Explain how to measure virulence with LD50 (Lethal Dose 50) and ID50 (Infectious Dose 50); be able to calculate the values given a graph or table.

3. Describe the course of infection of a pathogen.

4. Identify and explain strategies used by pathogens to adhere to host cells; provide specific examples for non-specific and specific adhesion.

5. Distinguish between the 3 major mechanisms for intracellular invasion.

6. Compare and contrast endotoxins and exotoxins in terms of substance type, bacteria that produces them, release methods, and biological responses caused.

7. Distinguish between different Biosafety levels and the types of pathogens in each category.

Pathogenicity

• Definition: The generation of a “pathos” or set of symptoms; anything that can cause disease.

• Factors influencing pathogenicity include genetics (e.g., cancer, cystic fibrosis), environmental factors (e.g., heart disease, Type II diabetes, cancer), and infectious agents (pathogens) such as viruses, fungi, bacteria, and even commensal flora that can become harmful.

Definitions of Pathogenicity and Virulence

• Pathogenicity: Indicates whether an organism can cause disease (Yes/No).

• Virulence: The relative ability of a pathogen to cause disease; can be categorized as highly virulent, moderately virulent, or weakly virulent.

Measuring Virulence/ Quantitative Metric

• LD50 (Lethal Dose 50): Dose at which 50% of hosts die; usually determined experimentally. Mostly done on mouse

• ID50 (Infectious Dose 50): The number of bacteria/viruses required to cause symptoms in 50% of hosts.

• Both metrics are important for assessing the Biosafety Level status of organisms and are expressed in colony-forming units (CFUs).

CFUs vs Cells

• CFUs refer to viable(living) cells, critical for understanding bacterial population assays.

• Living cells can form colonies and induce pathogens

How to estimate the LD50 of a pathogen with a graph

• look for the 50% then track the line of interest horizontally until it intersects with the corresponding dose on the x-axis, which will indicate the lethal dose that kills 50% of the host population.

• Also based on table data, if its ID50 it wont be deadly but if its LD50 then yes but the lower the number, the most deadly it is.

Types of Pathogens

• Extracellular Pathogens: Grow and replicate outside host cells in areas such as epithelial surfaces, interstitial spaces, blood, and lymph.

  • Obligate extracellular pathogens: These pathogens are entirely dependent on the extracellular environment (can only grow outside host cells), often causing diseases through toxins and enzymes that disrupt host functions.

• Intracellular Pathogens: Survive inside host cells and may be either

  • obligate intracellular pathogen: can’t reproduce outside the cell (requiring a host)

  • Facultative pathogen: can do both.

Course of Infection

1. Exposure: Entry points for bacteria include the mouth, respiratory tract, gastrointestinal tract, wounds, urinary tract, and reproductive tract. Specific bacteria for these pathways include:

   • Skin: Best protective agent, barrier for pathogens

   • Mouth: Streptococcus pyogenes, Porphyromonas gingivalis

   • Respiratory Tract: Bordetella pertussis, Mycobacterium tuberculosis

   • Gastrointestinal Tract: Salmonella enterica, Vibrio cholerae, and E. coli

   • Wound Entry: Clostridium tetani

   • Reproductive Tract: Neisseria gonorrhoeae, Chlamydia trachomatis

   • Pathogens are selective for the hostand may exploit specific entry points, utilizing various mechanisms to adhere, invade, and evade the host's immune response.

2. Adherence to Skin or Mucosa: Initial contact point.

3. Colonization and growth:

4. Invasion through epithelium:

   • Intracellular pathogens: into the cell through the apical side (free exposed surface) and out the basal side (bottom surface) of the epithelial cell and then hit the basement membrane.

   • Extracellular pathogens: get through in between the epithelial cells in order to penetrate further . Can bud off from the host cell to spread to adjacent cells or lyse epithelial cells. Break the tight junctions and cellular cements that bond epithelial layers.

5. Toxicity:

6. Dissemination:

7. Tissue damage, disease and transmission:

Host Tissues involved in Infection

• Epithelium: Membranous tissue covering internal and external surfaces of the body; it acts as the primary physical barrier against pathogens. They are involved in critical functions such as protection, secretion, excretion, absorption, and sensation. Pathogens often target epithelial cells for initial adherence and invasion.

• Basement Membrane: A thin, non-cellular layer made of specialized proteins and fibers (e.g., type IV collagen, laminin, proteoglycans, glycosaminoglycans) located at the base of epithelial layers, providing structural support and acting as an anchoring point for epithelial cells. It serves as a selective filter and a crucial barrier that pathogens must breach to reach deeper tissues. Breakdown of the basement membrane by pathogen-secreted enzymes is a key step in tissue invasion.

• Extracellular Matrix (ECM): A complex network of macromolecules secreted by cells that provides structural and biochemical support to surrounding cells and tissues. It primarily consists of fibrous proteins or non-cellular components like collagen, elastin, fibronectin, and laminin, as well as proteoglycans, glycosaminoglycans, heparan sulfate and hyaluronic acid. These components give tissues their strength and elasticity. Pathogens often target ECM components for adhesion and degradation (e.g., using enzymes like collagenase, hyaluronidase, elastase) to facilitate their spread. Also contains cellular components such as fibroblasts and immune cells.

Adhesion Mechanisms

• Mediated by Adhesins: Either specific (ligand-binding) or non-specific (generally sticky).

  • ex of non-specific: Capsule production in Streptococci, a polymer of hyaluronic acid from the ECM, mediates adhesion and evading host recognition. So capsule or slime layer (type of common sugar coats)

  • ex of specific adhesin: Neisseria gonorrhoeae uses Type IV pili to bind specifically to urethral/cervical epithelium. Pili has a “grappling hook” mechanism of attachment.

• Host Specificity: Certain pathogens show specificity towards human hosts (e.g., Neisseria gonorrhoeae).

• Tissue Specificity: Preference for specific tissues; for example, Streptococcus mutants binds specifically to dental plaque.

Bacterial Adhesins Table

Why does colonization not grow out of control?

• some nutrients aren’t available (irons-siderophores) - does not restrict growth of intra pathogens

• oxygen availability- does not restrict growth of intra pathogens

• physical space - does restrict growth of intra pathogens

• adverse conditions (low pH, hot temp) - does not restrict growth of intra pathogens

• predation by immune system or viruses - does not restrict growth of intra pathogens

• competition from normal flora - does not restrict growth of intra pathogens

• antibiotic treatments   - does not restrict growth of intra pathogens

Only extracellular pathogens form biofilms inside the body on the epithelial layer to combat these limitations - 60% infections are caused by biofilm

Pathogen Penetration Mechanisms

• Mechanisms for intracellular invasion:

  • Zipper Mechanism: This mechanism involves a direct interaction between bacterial surface proteins (invasins) and host cell receptors, such as integrins or cadherins. The binding initiates a signaling cascade within the host cell, leading to localized rearrangements of the host cytoskeleton (actin filaments) directly beneath the bacterium. The host cell membrane then wraps tightly around the bacterium, progressively engulfing it into a membrane-bound vacuole without significant generalized membrane ruffling. The bacterium essentially "sinks" into the cell, much like a zipper closing, making it a more subtle and specific entry method.

  • Trigger Mechanism: In contrast to the zipper mechanism, the trigger mechanism induces significant and massive rearrangements of the host cell actin cytoskeleton, leading to dramatic membrane ruffling or pedal-like projections that engulf the bacterium. This process is often mediated by bacterial effector proteins injected into the host cell cytoplasm via a Type III or Type IV secretion system. These effectors manipulate host cell signaling pathways, particularly those controlling actin polymerization, causing the host cell to actively "trigger" its own internalization of the pathogen. A prime example is Shigella flexneri, which uses Ipa (invasion plasmid antigens) proteins to trigger extensive membrane ruffling and uptake.

  • Coiling Phagocytosis: This unique form of intracellular invasion is characteristic of pathogens like Legionella pneumophila. Instead of uniform engulfment, the host cell plasma membrane extends long, thin, pseudopod-like coils or loops that slowly wrap around the bacterium, leading to its internalization into a spacious, ribosome-studded vacuole. This process is distinct from conventional phagocytosis and is believed to enable Legionella to evade immediate lysosomal fusion and establish a replicative niche within alveolar macrophages.

  • Actin rearrangements in the host cell have to be triggered by the pathogen via a secretion system known as a Type III Secretion System (T3SS). Good hallmarks of bacterial pathogens

Invasive Factors

• Invasins are bacterial surface-associated proteins that directly promote the entry (internalization) of pathogens into non-phagocytic host cells. They manipulate the host cell's cytoskeleton to facilitate uptake. They are crucial virulence factors for many intracellular bacteria.

• Most characterized in gram-negative ente////ric pathogens

• Invasins are secreted by organisms such as Salmonella enterica, leading to host cell internalization during infection.

  • Salmonella utilizes a two Type III secretion systems to inject effector proteins/toxins that interact with host cytoskeleton or cellular GTPases, one for invading GI cells and one for breaking free of endosome, which then trigger membrane ruffling and subsequent uptake.

Extracellular invasins

• Enzymes that disrupt occludin

  • LasB

  • HtrA

• Enzymes that disrupt E-cadherin

The Importance of Biofilm Formation

• Up to 60% of infections relate to biofilms, particularly in chronic infections, potentially up to 80%.

• Understanding biofilm formation is crucial for addressing persistent infections.

Spreading Factors

• Enzymes like hyaluronidase, collagenase, and neuraminidase facilitate invasion by degrading tissue structures, enabling pathogens to spread more easily through host tissues:

  • Hyaluronidase: This enzyme degrades hyaluronic acid, a major component of the extracellular matrix (ECM) and connective tissues, which acts as a molecular glue. By breaking down hyaluronic acid, hyaluronidase reduces the viscosity of the ECM, allowing the pathogen to move more freely between host cells and facilitating their dissemination through connective tissues. For example, Streptococcus pyogenes produces hyaluronidase, contributing to its invasive capacity.

  • Collagenase: Collagen is the most abundant protein in the human body, providing structural integrity to tissues. Collagenase enzymes break down collagen fibers, which are crucial components of the extracellular matrix and basement membranes. This degradation allows pathogens, such as Clostridium perfringens (the causative agent of gas gangrene), to penetrate deeper into tissues, especially muscle tissue, by destroying the structural barriers.

  • Neuraminidase: This enzyme degrades neuraminic acid (also known as sialic acid), which is often found as an intercellular cement binding epithelial cells, particularly on mucosal surfaces like the intestinal lining. By cleaving these glycoproteins, neuraminidase weakens the integrity of epithelial barriers, making it easier for pathogens to spread across or through these layers.

  • Hemolysin: Many bacteria produce hemolysins, which are a diverse group of phospholipases, lecithinases, or channel-forming proteins. These toxins destroy red blood cells (erythrocytes) by lysing their membranes, often by forming pores or by enzymatic degradation of membrane lipids. This action not only releases nutrients for bacterial growth (e.g., iron from hemoglobin) but can also damage other host cells, contributing to tissue destruction and facilitating pathogen spread within the host. Examples include streptolysin O from Streptococcus pyogenes and alpha-toxin from Staphylococcus aureus.

Host Immune Evasion Strategies

Pathogens develop strategies to avoid immune detection:

• Hide from immune system

  • Example: Treponema pallidum coats itself in host proteins (just hides).

  • polysaccharide capsules of streptococi blocking phagocytosis.

• Survive inside phagocyte

  • waxy, hydrophobic cell wall containing mycolic acids and other lipids creates a protective barrier

• Kill the immune cells

  • Streptococcus pyogenes producing Streptolysin O binds to cholesterol in membranes and causes lysosomal granules to explode

• Hijack host cells

Production of Toxins

• Toxins: Potent poisonous substances produced by living organisms that can either cause disease directly (toxicity) or indirectly (by triggering harmful host responses resulting from infection).

• Endotoxin: This refers to lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria (only). LPS is not actively secreted but is released from the bacterial cell upon lysis (e.g., by antibiotics or immune responses) or during rapid bacterial growth and shedding of outer membrane vesicles.

  • Structure: LPS consists of three main parts: Lipid A (the toxic component), a core polysaccharide, and a variable O-antigen (polysaccharide chain).

  • Properties: Endotoxins are heat-stable and cannot be converted to toxoids.

  • Mechanism: When released, Lipid A is recognized by host immune cells (specifically, Toll-like receptor 4, or TLR4), leading to a strong inflammatory response.

  • Biological Effects: High levels of endotoxin can cause systemic inflammation characterized by fever, hypotension (reduced blood pressure), disseminated intravascular coagulation (DIC), and can ultimately lead to septic shock and multi-organ failure. The effects are systemic and less specific than exotoxins.

• Exotoxin: Soluble poisonous proteins actively produced and secreted by living bacteria during their growth. These proteins are diverse in structure and function, acting on specific host cellular targets to cause pathology.

  • Properties: Exotoxins are generally heat-labile (destroyed by heat) and can often be inactivated to form toxoids, which retain antigenicity but lose toxicity, making them useful in vaccines

  • Biological Effects: Exotoxins often have highly specific mechanisms of action, causing a wide range of localized and systemic diseases depending on the target cells and molecular pathways affected.

  • There are three major types of exotoxins:

    • Type I Toxins (Superantigens):

      • Mechanism: These toxins bind simultaneously to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and the T-cell receptor (TCR) on T lymphocytes, but outside the conventional antigen-binding site. This cross-linking activates a large, non-specific population of T cells (up to 25% of all T cells) without requiring specific antigen processing and presentation.

      • Effects: This massive, uncontrolled T-cell activation leads to an overwhelming release of pro-inflammatory cytokines (a 'cytokine storm'), causing systemic inflammation, high fever, hypotension, rash, and potentially severe conditions like toxic shock syndrome.

      • Example: Staphylococcus aureus toxic shock syndrome toxin 1 (TSST-1), Streptococcus pyogenes pyrogenic exotoxins (SPEs).

    • Type II Toxins (Membrane-Damaging Toxins):

      • Mechanism: These toxins act on the host cell membrane, either by forming pores (pore-forming toxins) or by enzymatically degrading membrane phospholipids. They disrupt the integrity of the cell membrane.

      • Effects: Damage to the cell membrane leads to cell lysis, leakage of intracellular components, and disruption of cellular functions. These toxins often target red blood cells (hemolysins) but can also affect other cell types (cytolysins), contributing to tissue damage and facilitating pathogen spread.

      • Example: Streptolysin O (produced by Streptococcus pyogenes), Alpha-toxin (lecithinase, produced by Clostridium perfringens and Staphylococcus aureus), which can cause gas gangrene and damage various host cells.

    • Type III Toxins (Intracellular-Acting Toxins / A-B Toxins):

      • Structure: Most Type III toxins are composed of two distinct subunits: an 'A' (active or enzymatic) subunit and a 'B' (binding or delivery) subunit. The B subunit is responsible for binding to specific host cell receptors and facilitating the entry of the A subunit into the cell cytoplasm.

      • Mechanism: After binding, the toxin is internalized (often via endocytosis). The A subunit is then released into the cytoplasm, where it exerts its enzymatic activity on a specific intracellular target, interfering with crucial cellular functions.

      • Effects: These toxins are highly specific and powerful. They can inhibit protein synthesis (e.g., Diphtheria toxin, Shiga toxin), activate or inactivate host signaling pathways (e.g., Cholera toxin, Pertussis toxin), disrupt cytoskeleton integrity, or modify host DNA, leading to severe cellular dysfunction or cell death.

      • Example: Anthrax toxin (a tripartite toxin with Protective Antigen, Edema Factor, and Lethal Factor), Cholera toxin (Vibrio cholerae), Diphtheria toxin (Corynebacterium diphtheriae), Botulinum toxin (Clostridium botulinum), Tetanus toxin (Clostridium tetani).

Comparative Analysis of Toxins

Horizontal Gene Transfer

• The mechanism by which virulence factors, including toxin production (primarily exotoxins), can be exchanged between bacterial strains.

• Important for understanding antibiotic resistance spread.

Importance of Poly-Microbial Infections

• Co-infections with viruses, bacteria, fungi, and parasites are increasingly recognized, involving synergistic or antagonistic relationships.

• Examples include abscesses, AIDS-related opportunistic infections, and respiratory diseases.

Summary of Learning Objectives Revisited

1. Define pathogenicity and virulence; distinguish between them.

2. Explain measurement of virulence using LD50 and ID50; practice calculations.

3. Describe infection course and mechanisms of pathogen attachment.

4. Understand intracellular invasion mechanisms and their implications.

5. Compare and contrast different toxin functions and mechanisms.

6. Identify various biosafety levels regarding pathogen categorization.