Transcript No Slide Title

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
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)
Shigella acid tolerant
 Estimated 165 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
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
Produces much more Shiga toxin
1000 – 10,000 fold less toxin,
in vitro – but in vivo??
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
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
Shigella or EIEC: Overview of invasion of host cells
 Adhesion
Activation of Type III secretion system
• IpaA injected into cytoplasm
depolymerisation of actin filaments
• 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?
Multiply in cytoplasm
IcsA
IcsB
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
‘signals’
Extensive actin polymerisation + membrane
perturbations (ruffles) - not confined to
immediate vicinity of adhering bacteria
‘triggered’ phagocytosis
 Shigella & EIEC polarized CaCo2 cells via baso-lateral
surfaces only.
• Shigella may invade initially via M cells, in vivo
Multiplication in enterocytes
production of IL-8
Cell damage
LPS release
Recruits
PMNs
Inflammation
Shigella induce apoptosis in macrophages
& release of proinflammmatory cytokine IL-1b
Multiplication in host cells
helps Shigella evade PMNs
Recall: Necrosis v apoptosis
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Accidential
Injury - cell swells
Osmotic lysis
Contents released into tissues
Triggers inflammation
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Programmed
Suicide pathway activated
Cell shrinks
DNA & nucleus fragment
Membrane changes - phagocytosis
Cell engulfed ‘cleanly’
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
also called ICE = Interleukin 1b converting enzyme)
SBM 2044 Lecture 10
Salmonella :
Nontyphoidal and thyphoid fever
Salmonella
• Gram negative rods
• Motile with peritrichous
flagella
Salmonella that infects humans
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Salmonella Typhi
Salmonella Choleraesuis
Salmonella Paratyphi A
Salmonella Paratyphi B
Enter host via the oral route, usually with
contaminated food or drink.
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.
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)
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.).
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
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+
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
• Salmonella
• http://youtube.com/watch?v=VFGb3RKm-4o
• Treatment:
– Not antibiotics
– Antimicrobial therapy for systemic nontyphoidal
Salmonella infection
– Typhoid vaccine
– Salmonella carriers  jail?