Lecture-8: Mechanism of Bacterial Pathogenesis PDF

Lecture-8 Mechanism of Bacterial Pathogenesis Molecular Koch’s postulate Interaction of pathogen with host Mechanism / strategies used by bacteria to colonize and subvert killing by host immune systems Tissue damage Dr. Manish Kumar  Learning ob...

Lecture-8 Mechanism of Bacterial Pathogenesis Molecular Koch’s postulate Interaction of pathogen with host Mechanism / strategies used by bacteria to colonize and subvert killing by host immune systems Tissue damage Dr. Manish Kumar  Learning objectives Bacterial pathogens have different mechanisms to overcome host immune system to ensure survival, long-term persistence in some cases. The main goal is to understand the mechanisms used by bacteria pathogens to subvert killing by the host immune system. Koch’s Postulates : Classical version Remain “gold standard” in medical microbiology, but not always possible to satisfy all postulates for every infectious disease.  Molecular Koch Postulates The gene or its products should be found in only in strains that causes disease Gene should be isolated by cloning. Disruption of gene should should reduce or attenuate its virulence. Gene is expressed by bacterium at some point during infection.  The fifth Postulate Elimination of a disease causing microbe or prevention should eliminate or prevent disease. Examples: – Antibiotic therapy – Improvements in sanitation to prevent cholera – Treatment of H. pylori – Vaccination approaches (Streptococcus pneumoniae)  Virulence Virulence: is a measure of the how pathogenic a bacterium is; pathogenicity refers to the capacity of a microbe to produce disease Virulence factors (or determinants): Gene products that allow a microorganism to establish itself within or on a host and enhance the potential to cause disease  Measuring Virulence: Animal Models Ethical concerns What is an ideal animal model? Ferret Guinea Chinchillas Armadillos Rabbit Zebra fish pig H. Pylori TB H. Influ & S. Leprosy Ocular Streptoccoci, pneumo. ear infections necrotizing infections fasciitis Mycobacterium infections Rodent models of infection: – Why are they used widely and what are their benefits and disadvantages – Knockout and knock-in mice; economics of use etc..  A simple animal model: the basics Infect Treat or Sacrifice & plate with monitor bacteria for CFU bacteria response  Survival analysis Disease progression and survival of mice infected with three different strains. The rel gene is involved in the bacterial stringent response Kazmierczak et al 2009, Mol Micro 72:590-611  LD50 and ID50 values LD50 refers to 50% lethal dose; referring to dose where 50% of animals are moribund. ID50 refers to 50% infectious dose; referring to the number of bacteria required to infect 50% of animals The graph showed the number of bacteria and percentage of animal infected for two strains (A &B). Which one is more virulent?  Competition assays A way to make LD50 and ID50 experiments more sensitive is to use competition assays – In these experiments mutant and wild- type compete for resources, including colonization sites Alternate animal model C. elegans  To Cause a Disease, Microorganism must: 1. Find and enter the host 2. Colonize the host 3. Resist host defenses 4. Cause damage to, or malfunction of host tissue  Portals of Entry Ingestion (fecal-oral, contaminated food or water), GI and oral diseases Respiratory (aerosols, contaminated hands etc) Wounds (scratches, bites, etc) STD (sexual activity) Medical devices (catheters, contact lenses, etc) Normal flora movement or altered growth  Adhesion and Colonization E. Coli pili Pili and Membrane Vesicles on N. meningitidis Parker et al., J. Bact. 174: 2525, 1992 In UTI by non pilliated E. coli is McGee et al., Infect. Immun. not able to attach to host tissues 24: 194 and produces mild symptoms. Piliated gonococci attached more compared to non-piliated  Adhesion - continued Pili – specific adhesion (often involve lectins) Extracellular matrix Flagella Type 4 pili (twitching motility) Capsule (non-specific adhesion, protection) Lam et al., Infect. Immun. 28: M protein of 546, 1980 Streptococcus  Host Factors Involved in Colonization (adhesins) Anatomic (pregnancy UTI) Metabolic (diabetes) Neoplastic (cancer) Therapeutic (anti-rejection drugs, radiation) Ischemia (tissue death due to loss of blood) Substance abuse (contaminated needles, etc) Primary infection (opportunistic infection)  Resisting Host Defenses B – Bacterial antigen 2. Overproduction of bacterial ag, bind up host ab B B B B B B B B B B B B B B B B B B B 1. Loss of ag Super-antigen Capsul e B B B B 3. Capsule binds with serum protein H and inhibit complement activation. B B  Resisting Host Defenses 4. b – Altered bacterial ag 5. Mimic of host b b antigen H H b H b H b b H H (antigenic variation) molecular mimicry e.g. Salmonella use this technique e.g. H pylori LPS O antigen to switch between different types of contains carbohydrates the protein flagellin Similar to host H – Host ag  Resisting Host Defenses 6. Resisting Antibodies (Immunoglobulins) Protein A of S. aureus and Protein G of S. pyogenes binds with Fc region and prevent opsonization Ig protease Bind host materials  Escaping phagocytosis L. pneumophila No fusion of phagosome and lysosome Mycobacteria Escape from phagosome & cell-cell spread Evades phagolysosomal fusion after internalization by escaping the phagosome through secretion of listeriolysin O (LLO) and two phospholipases, PlcA and PlcB Once in the cytoplasm, L. monocytogenes replicates and becomes motile by using actin 'comet tails' generated by the effector ActA LLO functional at pH5.5 is creates pores in phagosomal membrane as early as 5 mins. Penetration Strategies – Salmonella and Shigella Salmonella serovar Thyphimurium delivers effector proteins into host cell to protect vacuole and evade host response. Phagolysosomal maturation is stalled and vacuoles do not contain mannose-6-phosphate receptor and lysosomal hydrolytic enzymes. Invasion – property of pathogen that enables it to invade (get  Other strategies Host Factors Bacteria Blood clots Degrade clots (fibrinolysin) Mucus Enzymes to degrade mucus Ability to compete Normal flora Signal disruption Host signals Siderophores, Fe Lactoferrin, transferrin acquisition Adverse pH, low Habitat selection, use of nutrients alternate nutrients  Toxins Based on the site of action on host cells, toxins may be further classified, as Type I to III toxins – Type I toxins bind to targets at the cell surface and are not translocated into the cell e.g. Superantigens (SAg) that affect macrophages and T cells, altering their functions and causing copious cytokine release – Type II toxins act on the cell membranes (e.g. phospholipases or pore-forming cytotoxins) – Type III toxins, typically A-B toxins, carrying two functional components i.e. an enzymatic domain (A) that inactivates some intracellular target and a binding component (B) that recognizes a surface receptor and mediates the uptake of the A component  Endotoxin Non-protein toxin Gram-ve LPS Mycolactone (cytotoxic ) Buruli ulcer in Peru  Super antigens (greatly enhance T-cell stimulation) Type-I Toxins: Exotoxin, (protein), Toxic shock syndrome Staphylococcus aureus : Tampon recall Membrane Disruption (type II toxin) (α toxin S. aureus) Insert itself in host cell membrane and forms pore.  Membrane Disruption (phospholipase C and hemolysins) Remove the polar head group and destabilize the membrane  Type III (A-B toxins) The simplest A-B toxin is a synthesized as single polypeptide chain These domains are separated during processing by proteolysis The detachment of A from B is necessary for optimal activity of A  Toxin Export Toxins produced in cytoplasm but act externally Protein secretion mechanisms – General secretory system (sec) – Types I – VII secretion; export system varies with organism and toxin  General Secretory System (sec) Occurs in G+ and G- Protein synthesized with 15-26 aa signal sequence (leader peptide) After export, signal peptidase cleaves signal sequence  Secretion systems Type I- to type VII – Specific to Gram-ves: T2SS, T5SS (sec dependent) T1SS T3SS, T4SS, T6SS (sec independent) - Specific to Gram+ve Cytolysin mediated translocation (CMT) e.g. Streptolysin O ExPortal e.g B. subtillis T7SS e.g. Mycobacteria  Secretion systems Sec dependent Tseng et al. BMC Microbiology 2009  Type III secretion First described in Y. pestis (plague) Similarities to flagella synthesis Toxins can be injected directly into host cell https://www.youtube.com/watch?v=gpLUQza4uWw Disease is more of an exception than rule The iceberg concept of infectious diseases