Success for an extracellular pathogen – measured in terms of passing genetic info to progeny

• necessary due to host surface Brownian motion, epithelial sloughing as well as mucus and liquid bulk flow often mediated by peristalsis or ciliated epithelium
• some pathogens use chemotaxis to increase the probability of finding the correct host cell surface
• Basic Mechanisms: (each pathogen has unique variations tailored to its specific situation.)
• Adhesin molecules which mediate adherence by binding a receptor on the host cell.
• Often found on pili of Gram Negative bacteria.
• One of these pili class (N-methylphenylalanine) is found in greater concentration at poles of bact. Others are scattered
• Types of Pili
• (1) "Common" or "Type I" Pilus found on most E. Coli, and mediates adherence to a variety of eukaryotic cells. Mechanism: interacts with D-mannose containing COHs on the surface of the bug. Problem: In the presence of free mannose the pilus won’t be able to bind the host because its binding sites will be filled with the free mannose. Mannose-resistant adherence = adherence mechanism not involving common pilus.
• (2) "Pap" or "P" pilli on uropathogenic E. Coli bind digalactoside moieties characteristically found on surface of urinary epithelium. (example of mannose-resistant adherence).
• (3) CFA or "colonization factor" pilus, on enterotoxigenic E. Coli (ETEC), allows adherence to the small intestine. (another example of mannose-resistant adherence).
• Why do many bacteria present adhesins on tips of pili?
• Hypothesis #1: the length of the pilus allows host and bug to interact at a distnace that attenuates the charge repulsion between the negatively charged surfaces of host and bug.
• Hypothesis #2: pili are used for initial binding, which is followed by tighter binding using (non-pili) cell surface interacting proteins.
• Example: Enteropathogenic E. Coli (EPEC): Initial contact made by "bundle forming pilus" (BFP) bringing cells close enough to support protein secretion into the eukaryotic cell. One of the secreted bug proteins, Tir, becomes phosphorylated in the euk cell and inserts into its membrane where it serves as the receptor for an outer membrane protein of the bug called intimin. Thus, EPEC provides both the adhesin (intimin) and the target ligand (Tir) involved in this interaction.
• Nonpili adhesins are exemplified by the diagram of : Streptococcus pyogenes. Previous dogma = belief that lipoteichoic acid (LTA) Þ specific attachment to nasopharengeal (& other) cells. However, it is now known that adherence results from the bacteiral integral membrane F protein binding cell surface fibronectin.
• Biofilms are mats of bacteria colonizing by attaching to large rafts of bacteria (see figure)

• a specialized strategy for survival and multiplication employed only by some pathogens who either enter professional phagocytes (neutrophils and macrophages) or non professional phagocytes (epithelial and endothelial cells)
• specialized function of salmonella, shigella, legionella, listeria, yersinia, E. coli
• intestinal lining has examples of the two types of epithelial target cells
• (1) Enterocytes – absorptive cells with apical brush border that normally will not phagositize but can be induced to do so
• (2) M-cells are interspersed within enterocytes and serve as phagocitic antigen sampling cells to underlying GALT (gut –associated lymphoid tissue: i.e. Peyer’s patch)
• Examples of invasion:
• (1) Yerisina pseudotuberculosis: 2 pathways. Best understood path uses protein called invasin – a outer membrane bacterial protein with 200 residue C-terminus domain that is sufficient to Þ binding by and internalization into target cell.
• Euk. Receptor = b 1 integrins. Increased binding Þ eukariotic membrane zipping bacteria up until enclosed within new vesicle
• Vesicle migrates to basal surface and endocytotically releases bact. to the interstitial space
• Binding with a macrophage Þ Yersinia Type III secretion of protein that inhibits macrophage phagcytosis
• Forms micro colonies, thus avoiding macrophage phagocytosis as well
• (2) Salmonella typhimurium: invasion requires > 24 genes, which code for Type III secreted protein and secretion apparatus needed for transfer to euk cytoplasm. These genes are clustered in chromosomal segment = pathogenicity island.
• This segment is missing in closely related E coli but is in other pathogens so it was likely acquired as unit (perhaps a transposition!)
• contact with microvili Þ dissolution of microvilli in a zone of 5 um radius around bacteria, cytoskeletal rearrangements, Tyr Phosphorylation (host cell proteins) and temporary appearance of invasomes ( short filament appendages) on bacteria
• Levels of regulation:
• (1) Contact Þ erection of invasomes
• (2) Right pH, Osm, pO₂ Þ Ý [HilA proteins]Þ Ý [proteins for invasion]
• Fate after being internalized by membrane cell is same as Yerisina except Salmonella is macrophagocitized without being destroyed and transported to distant tissues like liver and spleen.
• (3) Shigella: Also related to the non-pathogenic E coli except secretes toxin.
• Gains entry through similar mechanism described above
• Once internalized, breaks up vesicle with in minutes and attaches to host cytoskeleton in order to provide propulsive mechanism that allows it to protrude into the lateral membrane of cell and adjacent cell Þ access to the adjacent cell within a double membrane.
• Principal invasion may be through M-cell and lateral spread is route to enterocytes
• Non Invasive Adherence: Vibrio cholera, EPEC, EHEC etc only adhere and have non invasive strategies
• Therapies: targeting these mechanisms include disrupting adhesion by promoting leakage of adhesins (penicillin for S. pyogenes)
• suppressing adherins expression (streptomycin for E coli) - potentially: competitive receptor substitution

Host Defense and Virulence Factors – the host comes up with ways to kill the bacteria and the bacteria use Virulence Factors to overcome these efforts. The number and effectiveness of such tricks define the Relative Virulence of the organism for its host.

• (1) Deprive the bacteria of a nutrient that it needs to survive. Hosts sequester iron with lactoferrin and transferrin so there is very little unbound Fe for the bacteria to use.
• Bacteria use (build a better chelator)
• Sideropores (secreted molecules with high Fe binding capacities) to bind Fe and bring it through the cell membrane via Ton B (a Proline rich transporter that harnesses the electro-chemical gradient for the process)
• Many bacteria use receptors for Transferrin and Lactoferrin
• Other bacteria lyse RBC’s to harvest Fe (plasmid mediated Fe uptake systems); surface proteins regulated by Fe
• bacteria may have a 19 base palindromic sequence called an "Iron Box" in front of iron related genes. This is inhibited by the fur-Fe complex; so when there is iron there is not expression of the iron obtaining genes
• (2) Surface Immunoglobulins bathe the mucous membranes of the respiratory and GI tracts, inhibiting adherence, neutralizing toxins, and excluding antigens. Secreted antibodies can opsonize the bacteria
• Bacteria use:
• Capsules to mask antigenity Þ mask surface antigen; decrease immunogenicity (streptococci capsules)
• Can change surface antigen and therefore antigenity. Neisseria make multiple pillins (E¹ and E²) by way of a hypervariable region that are silent unless put if front of E¹ or E² by way of transformation (which Neisseria does often). Neisseria also makes an IgA Protease that eats up secreted IgA. Salmonella changes between a H1 and H2 flagellum by way of a Transposase.
• (3) Coat the Bug with Immunoglobulin or complement so opsonization takes place and Phagocytes eat them
• Bacteria use:
• Capsules to block opsonin access to the cell wall and hide the coat from the phagocyte
• Salmonella cuts the binding site of the antibody; Staph. Aureus makes protein A, which binds to the Fc region of IgA or IgG, both mechanisms effectively rendering the bacteria unrecognizable to Phagocytes.
• (4) Phagocytic Defense- phagocytes are chemotactically attracted to the site of the invasion. Migration is an energy dependent process.
• Bacteria use (block phagocytic attachment):
• Decrease activation of complement and thus decrease the generation of chemoattractants by sialic acid containing capsules.
• Inhibiting Phagocyte migration or locomotion. Clostridium produces an exotoxin that depolarizes the actin filaments of phagocytes (the phagocytes are immobilized) – inactive complement
• YOPS (Yersinia outer membrane proteins) are in almost all pathogens
• YOP H – is shot into Phagocytes upon cell to cell contact and removes the phosphate off the tyrosine phosphorylase that is the first signaling step in the phagocyte’s plan for phagocytosis.
• YOP N aims YOP H firing (so it is shot into the cell)
• YOP E is a cytotoxin that depolarizes actin filaments to paralyze phagocytes before contact.
• Lots of toxins screw up G-protein signaling in cells
• (5) Oxidative Burst – phagocytes shoot H₂O₂ and degradative enzymes into endosome with bacteria in it.
• increases oxygen consumption Þ O₂^-, H₂O₂, OH^-, and hypochlorous acid
• Bacteria use:
• Suppression of the respiratory burst
• (1) Peroxide Stress Response- H₂O₂ induces 30 proteins (Catalase, Peroxidase, Alkalating enzymes, etc) to control the level of oxidation in the environment. 10 of which are controlled by the OxyR regulator. (when H₂O₂ binds to OxyR repressor, the genes are expressed)
• (2) Superoxide Response- regulated by SOD RS repressor protein; turns on Superoxide Dismutase, G6P Dehydrogenase (to make NADH + H) and enzymes to repair DNA. Also controls cytochromes and stabilizes Kreb’s cycle intermediates. ( Alkalating enzymes help stabilize the DNA, NADH is needed for DNA repairs)
• Strong cell walls provide resistance to non-oxidative mechanisms
• Inhibition of degranulation by blocking the binding of the lysosome to the endosome.
• Blow up the phagocyte by releasing Leukocidins.

Bacterial Exotoxins – made by bug and secreted into extracellular environment; Generic mechanism: Two component system. First component absorbs to surface of target cell and facilitates transfer of second component – which exerts the toxic effect

• Cholera Toxin
• secreted by Vibrio cholerae (slinky-shaped gram(-) rods)
• Fecal-oral transmission, with resulting toxin causing massive diarrhea. High mortality if no fluid replacement.
• The cholera toxin is composed of multiple subunits. The B subunits bind to the surface of the small intestine and then release the active A₁ subunit which ADP-ribosylates the regulatory G-protein.
• Side note: What is ADP-ribosylation?
• Cholera and Diphtheria have subunits that mediate ADP-ribosylation. This involves movement of an ADP molecule from NAD Þ GTP binding protein. This reaction is mediates by the A subunit.
• (See figure below) The action of cholera toxin. The complete toxin is shown binding to the GM1-ganglioside receptor on the cell membrane via the binding (B) subunits. The active portion (A₁) of the A subunit catalyzes the ADP-ribosylation of the G_s (stimulatory) regulatory protein, "locking" it in the active state. Because the G_s protein acts to return adenylate cyclase from its inactive to active form, the net effect is persistent activation of adenylate cyclase. The increased adenylate cyclase activity results in accumulation of cyclic adenosine 3x, 5x-monophosphate (cAMP) along the cell membrane. The cAMP causes the active secretion of sodium (Na⁺), chloride (Cl^-), potassium (K⁺), bicarbonate (HCO_3-), and water out of the cell into the intestinal lumen.

• (2) Diphtheria Toxin
• secreted by Corynebacterium diptheriae (comma shaped gram positive rods.
• Transmitted in air/water droplets.
• Diphtheria toxin inactivates protein synthesis thus killing eukaryotic cells. It causes such diseases as myocarditis, neuropathies, etc.
• Toxin is composed of A and B subunits. B binds to the host surface, the whole complex is endocytosed, and finally the A subunit mediates its effects by inactivating the EF2 enzyme, which stops the elongation factor and consequent protein translation.
• Elongation factor (tRNA translocase) ?? Needed for extension of polypeptide chains in protein synthesis. Elongation factor-2 is essential for ribosomal reactions at the acceptor and donor sites, which transfer triplet code from messenger RNA (mRNA) to amino acid sequences via tRNA. Inactivation of EF2 stops building of the polypeptide chain.
• (see figure below) Action of diphtheria toxin. The toxin-binding (B) portion attaches to the cell membrane, and the complete molecule enters the cell. In the cell, the A subunit dissociates and catalyzes a reaction that ADP-ribosylates and thus inactivates elongation factor 2 (EF-2). This factor is essential for ribosomal reactions at the acceptor and donor sites, which transfer triplet code from messenger RNA (mRNA) to amino acid sequences via transfer RNA (tRNA). Inactivation of EF-2 stops building of the polypeptide chain.

• family of secreted proteins produced by various strains of S. Aureus that produce toxic effects at sites remote from live staphlococci. Ex: TSST-1. TSST-1 is a superantigen exotoxin which binds to T-cell receptors and stimulates release of cytokines. Get Toxic Shock Syndrome.
• Superantigen - molecules that stimulate a subset of T cells by binding to MHC class II molecules and Vb domains of T-cell receptors, stimulating the activation of T cells expressing particular Vb gene segments. Stimulation results in massive activation of immune system by release of cytokines (IL-1, TNF, etc.), lymphokines (cytokines produced by lymphocytes), etc.
• Symptoms of Toxic Shock Syndrome include fever, chills, nausea, diarrhea, vomiting, sunburn-like rash, red tongue, myalgia, conjunctival infection
• Refractory Hypertension – shock
• Multi-Organ Dysfunction

Bacterial Endotoxins – intrinsic part of bug which is released after its death

• Lipopolysaccharide (LPS) is present in the outer membranes of most gram-negative bugs (see figure)
• LPS Consists of o-antigen polysaccharide, outer and inner core polysaccharides, and the lipid A moiety.
• The Lipid A moiety causes the problem
• Lipid A binds to lipopolysaccharide (LPS), and then binds a binding protein (LBP). The LPS/LBP complex binds to CD14 on cell membranes causing the release of such things as TNF-a Þ sepsis and huge drop in MAP