1. Lecture #14 Bio3124 Medical Microbiology Microbial Pathogenicity

2. Pathogens as Parasites pathogens are parasites –organisms that live on or within a host organism, metabolically dependent on the host –Parasitism: Ectoparasite: parasite lives on the host Endoparasite: parasite lives in the host

3. Parasitism and disease Infection –growth and multiplication of parasite on or within host Infectious disease –disease resulting from infection Pathogen: any parasitic organism that causes infectious disease –primary (frank) pathogen – causes disease by direct interaction with healthy host –opportunistic pathogen – part of normal flora, causes disease when gains access to other tissue sites or when host is immunocompromised Pathogenicity –ability of a parasite to cause disease

4. Host-parasite relationship and disease outcome Disease state depends on : –number of organisms present –degree of virulence of pathogen –virulence factors e.g., capsules, pili, toxins –host’s defenses or degree of resistance Virulence: degree/intensity of pathogenicity determined by, –Invasiveness: ability to spread to adjacent tissues –Infectivity: ability to establish focal point of infection –pathogenic potential: degree to which pathogen can cause damage to host Toxigenicity: ability to produce toxins Immunopathogenicity: ability to trigger exaggerated immune responses

5. Measuring virulence lethal dose 50 (LD 50 ) –number of pathogens that will kill 50% of an experimental group of hosts in a specified time Infectious dose 50 (ID 50 ) –number of pathogens that will infect 50% of an experimental group of hosts in a specified time

6. Infection Cycle Mode of entry depends on pathogen Mucosal surfaces, wounds, insect bites Infection cycle Route a pathogen takes to spread Spread via direct contact Indirect contact –Contact with fomites –Horizontal transmission via vectors Mosquitoes—Yellow fever, malaria Reservoir for disease organism –May not show disease symptoms

7. Virulence Factors Virulence genes –Help pathogen to invade host Toxins, attachment proteins, capsules Pathogenicity islands –Section of genome Contain multiple virulence genes –Often encode related functions »protein secretion system, toxin production –Horizontally transmitted Often flanked by tRNA genes; phage or plasmid genes Often have GC content different from rest of genome

8. Virulence Factors Several factors contribute –Protein secretory systems Examples:Type II, type III and type IV –Adhesins: host attachement & colonization –Toxins Exotoxins –Membrane active toxins –Protein synthesis inhibitors –Cell signaling inhibitors –Superantigens –proteases Endotoxins –Immune avoidance factors

9. Role of protein secretory pathways in virulence PS Type II (retractable) –Subunits in inner, outer and periplasmic space –G subunit polymerize/depolymerize –Extends/retracts past outer membrane through complex D –like a piston pushes out the secreted proteins to periplasmic space –Ex. Cholera toxin PS Type II mechanism resemble pili type IV used for twitching motility

10. Type III protein secretory system many G- bacteria, live in close association with their hosts secrete regulatory proteins via injectisome directly into host cells –to modulate host cell activities –evolutionary resemblance to flagellum increase virulence potential –Avoids receptor use –Avoids dilution of secreted proteins outside pathogen Ken Miller talks about PSIII and flagellum

11. Salmonella SPI-1 and SPI-2 are type III secretory systems 12 pathogenicity islands in S. typhi SPI-1, a type III secretory system Injects 13 different toxins (effector proteins) Subvert signaling, remodel cytoskeleton Induce membrane ruffles, take S.typhi SPI-2: alter vesicle trafficking –Prevent phogosome-lysosome fusion –Pathogen avoids innate immunity

12. Injectisome: a type III secretory virulence factor

13. General SecA dependent secretory system Toxin secretion by type IV secretory system Resemble conjugation apparatus of gram negative bacteria Bordetella pertussis toxin secreted through general SecA pathway to periplasm Type IV collects toxin in periplamic space Exports across outer membrane

14. Adhesins: Microbial Attachment Human body expels invaders –Mucosa, dead skin constantly expelled –Liquid expelled from bladder –Coughing, cilia in lungs –Expulsion of intestinal contents Adhesins: surface proteins, glycolipids, glycoproteins –assist in attachment and colonization of host tissues Pili (fimbriae) Hollow fibrils with tips to bind host cells

15. Adhesins: Pili type I e.g. Pyelonephritis pili of uropathogenic E.coli attachment to P-blood group antigen upper uninary tract infection Pili assemble on outer membrane First, general SecA dependent secretion to periplasm PapG,E,F & major subunit Pilin A PapD chaperon sorting/delivery to PapC Secretion and pilus formation PapG recognizes the digalactoside on P-blood group antigen of host kidney cells

16. Adhesins: Pili type IV Found on P. aeruginosa, V. cholera, pathogenic E. coli & N. meningitidis Mediates attachment and twitching motility Resemble type II secretory system Pil A is major structural pilin PilC,Y1 tip attachment proteins Assembly: PilA preprotein signal sequence removed by PilD PilQ mediates export across outer membrane PilF/T mediates energy dependent assembly/disassembly of pilus

17. Type IV pili: bacterial attachment and motility

18. Exotoxins soluble, heat-labile, proteins usually released into the surroundings as bacterial pathogen grows most exotoxin producers are gram-positive often travel from site of infection to other tissues or cells where they exert their effects

19. More About Exotoxins Some toxin genes born on plasmids or prophages the most lethal substances known highly immunogenic can stimulate production of neutralizing antibodies (antitoxins) can be chemically inactivated to form immunogenic toxoids –e.g., tetanus toxoid

20. Membrane-disrupting exotoxins Alpha toxin of S. aureus Forms 7-membered oligomeric beta-barrel Cause cytoplasmic leakage Phospholipase of Clostridium perfringens removes charged head group of phospholipids in host-cell plasma membranes –membrane destabilized, cell lyses and dies –Also called α-toxin or lecithinase

21. AB type Exotoxins Composed of two subunits A“A” subunit – responsible for toxic effect –ADP-ribosyltion of target proteins eg. diphtheria toxin –Cleave 28S rRNA, eg. Shiga toxin B“B” subunit – binds to target cell, delivers A subunit Diphtheria exotoxin B subunit mediates receptor binding Endocytosis and fusion membrane vesicles eg. ER or endosomes B recycles back to membrane “A” escapes and enters cytoplasm In the cytoplasm A catalyses ADP- ribosylation of EF2, halts translation Cell death ensues Diphtheria toxin targets EF2 disrupts translation

22. Anthrax toxin composed of, –Protective antigen (B subunit): delivers EF and LF (A subunits) –Edema factor raises cAMP levels Causes fluid secretion, tissue swelling –Lethal factor cleaves protein kinases Blocks immune system from attacking Anthrax toxin: a deadly protease Bacillus anthracis

23. Animation: anthrax toxin mode of action

24. Superantigens Are bacterial and viral proteins that can activate T-cells in the absence of a real bacterial antigen mediate the binding of MHC-II and T-cell receptors (almost 30% of T- cell population) eg. Staphylococcal enterotoxin B (SEB) Massive activation results in producing lots of cytokines Results in tissue damage and shock and multi-organ failure

25. Animation: Superantigens

26. Endotoxins lipopolysaccharide in gram-negative cell wall can be toxic to specific hosts –called endotoxin because it is bound to bacterium and released when organism lyses and some is also released during multiplication –toxic component is the lipid portion, lipid A heat stable toxic (nanogram amounts) weakly immunogenic generally similar, despite source

27. Immune avoidance mechanisms Once inside host cell, how to avoid death? –Cell ingests pathogens in phagosome Some pathogens use hemolysin to break out –Shigella dysenteriae, Listeria monocytogenes –Phagosome fuses with acidic lysosome Some pathogens secrete proteins to prevent fusion –Salmonella, Chlamydia, Mycobacterium, Legionella Some pathogens mature in acidic environment –Coxiella burnetii—Q fever

28. Survival inside phagocytic cells escape from phagosome before fusion with lysosome –microbes use actin-based motility to move within and spread between mammalian host cells Burkholderia pseudomallei forming actin tails and protrude through membrane and extend infection to nearby cells Surviving within the Host

29. Outside host cell, how to avoid death? –Complement, antibodies bind pathogen Some pathogens secrete thick capsule –Streptococcus pneumoniae, Neisseria meningitidis Some pathogens make proteins to bind antibodies –Staphylococcus aureus cell wall Protein A »Binds Fc fragment »Antibodies attach “upside down” »Prevents opsonization Some pathogens cause apoptosis of phagocytes