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)
Shigella acid tolerant
Estimated 165 million cases/year > 600,000 deaths
99% in developing countries; 69% < 5 years old
Dysentery – in adults often self-limiting, but can be fatal in young children
Reiter’s syndrome – autoimmune disease associated with infection by a number of Gram-negative pathogens, including Shigella, Campylobacter, Salmonella - displays a strong association with a particular MHC haplotype – HLA-B27, suggesting a genetic predisposition
Complications
Haemolytic uraemic syndrome (HUS; see lecture EHEC)
Reiter’s syndrome: autoimmune reaction
- reactive arthritis, conjunctivitis, urethritis

3. Shigella dysenteriae Shigella flexneri Shigella boydii Shigella sonnei
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
Appears to be highly host-adapted – can cause disease in non-human primates, but not other animals
Some texts say only S. dysenteriae produce Shiga toxin, but not quite accurate, others can produce very small quantities in vitro – leaves open possibility that higher quantities might be produced in vivo.
Produces much more Shiga toxin
1000 – 10,000 fold less toxin,
in vitro – but in vivo??

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
No reason to suppose any significant differences in invasion mechanisms employed by other Shigella species (or EIEC) – though more limited, all studies on these entirely consistent with studies on S. flexneri
No suitable animal models – so studies limited to various cultured cells
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 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?
IcsB
Multiply in cytoplasm
IpaB and IpaC form ‘bulb’ at tip of ‘needle complex’ that acts as pore in host cell membrane and projects active domains of IpaB and C inside cell.
IpA injected into host cell – mutants in IpA greatly reduce, but do not abolish invasion completely.
IpaB and/or IpaC suspected to also mediate escape from phagosome – deduced from abilities of the proteins to lyse membranes at low pH – cannot be tested directly ‘cos mutants also defective is earlier entry step
IcsA – OM protein that provides actin nucleation site – so despite being very different type of organism than Listeria (Gram-neg v Gram-pos), motility within cell achieved by essentially same mechanism, albeit with diff surface proteins
Mutants defective in IcsB can polymerise actin on surface and move about cell, but can’t escape 2nd vacuole – so presumably IcsB lyses this
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 initially via M cells, in vivo
Multiplication in enterocytes
production of IL-8
Cell damage
LPS release
Recruits
PMNs
The inflammatory response will disrupt tight junctions and open paracellular route for further Shigella invasion – exacerbating severity
Inflammation
Shigella induce apoptosis in macrophages
& release of proinflammmatory cytokine IL-1b
Multiplication in host cells
helps Shigella evade PMNs

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
- cell detaches from neighbours
- loses contact with extracellular matrix
- rounds up
also called ICE
= Interleukin 1b converting enzyme)

11. Salmonella : Nontyphoidal and thyphoid fever
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 ( million organisms)

17. 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.).

18. 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

19. 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+

20. 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

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