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SBM 2044: Lecture 11

SBM 2044: Lecture 11. AIMS:. Introduce invasive Shigella sp Outline v ir genes & mechanisms involved in invasion of host cells by Shigella Outline other virulence mechanisms - apoptosis in macrophages. Shigellosis. Dysentery (bloody stools + pus + mucus; scant volume).

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SBM 2044: Lecture 11

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  1. SBM 2044: Lecture 11 AIMS: • Introduce invasive Shigella sp • Outline vir genes & mechanisms involved in invasion • of host cells by Shigella • Outline other virulence mechanisms • - apoptosis in macrophages

  2. Shigellosis • Dysentery (bloody stools + pus + mucus; scant volume) • invasion & multiplication in colonic epithelial cells • Transmitted by faecal-oral route • Direct transmission common - low I.D.50 (< 100 cells) • Shigellaacid tolerant • Estimated165 million cases/year > 600,000 deaths • 99% in developing countries; 69% < 5 years old • Complications • Haemolytic uraemic syndrome (HUS; see lecture EHEC) • Reiter’s syndrome: autoimmune reaction - reactive arthritis, conjunctivitis, urethritis

  3. Produces much more Shiga toxin 1000 – 10,000 fold less toxin, in vitro – but in vivo?? Shigella sp. • Gram-negative, non-spore- forming rods • Facultative aerobes • Non-motile • Natural habitat: Humans? • Environment? - faeces, contaminated water, etc) • Four species cause shigellosis - symptoms can range from • mild watery diarrhoea to severe dysentery Shigella dysenteriae Shigella flexneri Shigella boydii Shigella sonnei

  4. Shigella sp. – virulence mechanisms • Production of Shiga toxin • Toxin structure & action – see lecture EHEC • Role in shigellosis - unclear • Animal models: monkeys only • S. dysenteriae Stx mutant still caused dysentery, but less severe & less haemorrhaging • HUS – see lecture 10 E.coli • Invasion & multiplication in colonic epithelial cells • Studied mainly in S. flexneri & tissue cultured cells • Overall similar to Listeria, but details differ

  5. Shigella & EIEC invasion mechanisms • 33 genes required for invasion, including: • ipaA-D: ‘ invasion plasmid antigens’ so-called because proteins • originally detected by patients antisera • ipgA-F: identified by mutagenesis & sequencing, (‘invasion • plasmid genes’) • icsA & icsB: intracellular spread (identified by mutagenesis) • 20 genes encoding a Type III secretion system • - mxi: membrane excretion of Ipa • - spa: surface presentation of inv antigens

  6. Shigella or EIEC: Overview of invasion of host cells Activation of Type III secretion system depolymerisation of actin filaments • Adhesion • IpaA injected into cytoplasm • IpaB + IpaC – needle ‘bulb’ pore, with cytoplasmic active domains • IpaC required for actin polymerisation • IpaD – regulates secretion? a5b1 integrin IpaA/B/C/D Spread Ipa B/C? IcsB Multiply in cytoplasm IcsA

  7. Invasion overview: Entry Distinct from the ‘zipper-phagocytosis’ uptake seen with Listeria & Yersinia – reflects different signalling mechanisms Shigella sp. & Salmonella sp. initiated via Type III secretion Extensive actin polymerisation + membrane perturbations (ruffles) - not confined to immediate vicinity of adhering bacteria ‘signals’ ‘triggered’ phagocytosis

  8. Shigella & EIEC polarized CaCo2 cells via baso-lateral • surfaces only. • Shigella may invade initiallyvia M cells, in vivo Multiplication in enterocytes production of IL-8 Recruits PMNs Cell damage LPS release Inflammation Multiplication in host cells helps Shigella evade PMNs Shigellainduce apoptosis in macrophages & release of proinflammmatory cytokine IL-1b

  9. Recall: Necrosis v apoptosis • Programmed • Suicide pathway activated • Cell shrinks • DNA & nucleus fragment • Membrane changes - phagocytosis • Cell engulfed ‘cleanly’ • Accidential • Injury - cell swells • Osmotic lysis • Contents released into tissues • Triggers inflammation

  10. Apoptosis mediated by proteases called caspases • Family (>12) of cysteine proteases that cleave targets • adjacent to aspartic acid. • Most specific for other procaspases – activate cascade, leading to cleavage of nuclear lamins, DNase inhibitor, • & cytoskeleton components • Some cleave > 1 substrate protein - multiple roles in cell • Caspase-I - cleaves inactive IL-1b precursor to produce active IL-1b, a proinflammatory cytokine = Interleukin 1bconverting enzyme) also called ICE

  11. SBM 2044 Lecture 10 Salmonella : Nontyphoidal and thyphoid fever

  12. Salmonella • Gram negative rods • Motile with peritrichous flagella

  13. Salmonella that infects humans • Salmonella Typhi • Salmonella Choleraesuis • Salmonella Paratyphi A • Salmonella Paratyphi B • Enter host via the oral route, usually with contaminated food or drink.

  14. 4 clinical syndromes, plus the carrier state, are associated with the genus Salmonella • gastroenteritis: nausea, vomiting and diarrhoea; caused mainly by S. enterica • focal infection of vascular endothelium; caused by serovars Choleraesuis and Typhimurium • infections of particular organ systems; osteomyelitis in patients with sickle cell disease; commonly by S. typhimurium • typhoid fever; caused by serovars S. typhi and S. paratyphi A and B.

  15. Salmonella • Vast no. of serological varieties (serovars) • Antigens are distinguishable among serovars: somatic (O), flagellar (H), and capsular (K) • Acid sensitive (hypochlorhydria, achlorhydria) – express > 40 proteins for pathogenesis • A large inoculum is needed to produce a disease (10-100 million organisms)

  16. Electron photomicrograph demonstrating invasion of guinea pig ileal epithelial cells by Salmonella typhimurium. Arrows point to invading Salmonella organisms. (Courtesy Akio Takeuchi, Walter Reed Army Institute of Research, Washington, D.C.).

  17. Entry: • Salmonella enter M cells and the apical membrane of epithelial cells • Ruffling of the plasma membrane – cytoskeletal rearrangement, and uptake of organisms within phagocytic vesicles (BME) • Type III secretion system is encoded by Salmonella pathogenicity island 1 (SPI1) • Macrophages might help, leading to dissemination • Into deeper tissue beyond the intestine by intestinal dendritic cells

  18. Damage • Host-epithelial cells interaction activates the inflammatory response and damage to intestinal mucosa • Mitogen-activated protein kinase (MAPK)  receptor on the cell surface  phospholipase A2, release arachidonic acid, produce PG+leukotrienes   Ca2+

  19. How do Salmonella survive in macrophages? • Regulation by two-component regulator or signal system, two genes: PhoP and PhoQ • PhoP and PhoQ act to modify the bacterial lipopolysaccharide  hence resistance to innate kiling by host immune system i.e. cationic peptides

  20. Salmonella • http://youtube.com/watch?v=VFGb3RKm-4o • Treatment: • Not antibiotics • Antimicrobial therapy for systemic nontyphoidal Salmonella infection • Typhoid vaccine • Salmonella carriers  jail?

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