Salmonella 2015 - BSC Blackboard

Biology of Salmonella
Dr Sophie Helaine
Salmonella pathogenesis
Human diseases
Typhoid (S. enterica Typhi and S. Paratyphi): 17 million cases
and 600,000 deaths/yr - increasing multiple drug resistance
recurrence infection
Non-typhoidal (e.g S. enterica Typhimurium): 1.3 billion
cases and 3 million deaths/yr (major problem in sub-Sarahan
Africa)
Salmonella Typhi and

human typhoid
Typhoid prevalence, 2006
Salmonella Typhi and

human typhoid
Transmission (faecal-oral)
Chronic carrier state

Typhoid Mary
T0
48 h (10 days)
12 days
S. Typhi
Typhimurium
S.
Human diseases
Africa)
Murine model of Typhoid

fever
S. Typhimurium invasion of M cells
Fatal bacteraemia
Colonisation of Peyers
Patches, mesenteric lymph
nodes
Intracellular replication in spleen and liver

macrophages (up to 109 bacterial cells/orga
implies >30 rounds of cell division)
Total number of virulence genes
Estimates suggest approx.

of S. Typhimurium genome
quired for virulence
S. Typhimurium has 4600 ORFs => approx. virulence gene
Approx. genes identified to date
Most are present on Pathogenicity Islands/islets (> 60)
But, biochemical and physiological functions of many
remain unknown
Different virulence genes act at

different stages of S. Typhimurium
virulence
stage of
environmental
infection stimulus
response
genes
stomach
low pH
acid tolerance fur, atr
small
intestine
osmotic stress
anaerobiosis
motility
adhesion
flg, lpf and pef
epithelium
penetration
anaerobiosis
cell contact
invasion
bloodstream
complement
antimicrobial
peptides
macrophage
low pH, low Mg++
virulence
many (e.g.inv/spa)
serum resistance rfb, rfc, rck

peptide resistance sap, htr
complex
many
(pag, SPI-2, spv)
Salmonella an intracellular
pathogen
Salmonella-containing vacuoles in spleen cells
pathogen
Listeria
Shigella
Salmonella
Mycobacteria
Coxiella
Intracellular replication in macrophages

(16 h)
wild-type
Dhar and McKinney, Current Opinion in Microbiology 2007
Measuring replication by Fluorescence

Dilution
pDiGc
pDiGi
Helaine et al, PNAS 2010
Measuring replication by Fluorescence

Dilution
18 h
Bone-marrow derived
macrophages
Non-replicating bacteria > 50 %
Helaine et al, PNAS 2010
Bacterial persisters
Many bacterial species form persisters - highly tolerant to antibiotics
Persistent bacterial infections pose significant public-health problems
Typhoid fever in humans - 15% of treated patients suffer a relapse
A major breakthrough was the discovery that E. coli in vitro persisters are nonreplicating bacterial cells (Balaban et al, Science 2004)
Salmonella persisters induced by contact

Persisters are transiently tolerant to AB treatment
with host M
Survival of Salmonella to AB treatment
Interaction with host M, even transient, enhances the formation of

persister cells of Salmonella to 3 different classes of AB
Host induced Salmonella persisters are

Antibiotic tolerant bacteria
: non-lysed by exposure to beta-lactam
non-replicating
100
total intracellular
population
intracellular
persisters
% of Max
80
60
40
21 %
20
9%
0
0
10
10
10
GFP
10
10
Fluorescence
Host induced persisters are non-replicating
bacteria
arabinose-inducible
gfp
104
dsRed
10
+ arabinose
+arabinose
+ triton
103
DsRed
FL2-H
10
DsRed
FL2-H
IPTG-inducible
Non-replicating bacteria
Regrowth
102
101
+ cefotaxime
10
cefotaxime
0
O/N growth
10
10
100
+ IPTG
infect
101
102
FL1-H
103
GFP
Responsive non-replicating
after 24 h in macrophages
- IPTG
10
24 h
in macrophages
104
bacteria
10
10
10
FL1-H
Growth in rich medium
103
103
DsRed
DsRed
104
10
102
10
100
100
102
101
101
102
GFP
103
104
100
100
responding
Proportion of the non-replicating

bacteria resume growth
101
102
GFP
103
104
GFP
regrowing
4
10
Regrowing non-replicating bacteria

after 24 h in macrophages
+ cefotaxime 200 g/mL
10
Non-replicating bacteria
Regrowth
10
10
10
10
DsRed
DsRed
+arabinose
102
10
10
10
24 h
in macrophages
10
10
GFP
10
Responsive persisters after 24 h in IFN

activated C57 macrophages
10
102
10
10
10
10
10
GFP
10
10
Responsive and regrowing persisters in

C57 macrophages
+ cefotaxime 200 g/mL
Figure5
Non replicating bacteria reservoir for relapse of infection
Heterogeneity of intracellular population
Bacterial actors ? Host signals ?
Human diseases
Africa)
pathogen
Two T3SS
SPI-1 mediates invasion of host cells
and
intestinal secretory and inflammatory
responses
SPI-2 is required for bacterial replication
and immune modulation
pathogen
Two T3SS
Adhesion
Invasion
SPI-1 T3SS
Extracellular
SCV formation
SCV maturation
SPI-2 T3SS
Intracellular
Replication
Salmonella invasion of epithelial

cells occurs
via the
SPI-1
type
III secretion
effectors
induce
actin polymerisation,
membrane ruffling
and bacterial internalisation
system
Effector
pathogen
Two T3SS
Adhesion
Invasion
SPI-1 T3SS
Extracellular
SCV formation
SCV maturation
SPI-2 T3SS
Intracellular
Replication
effectors (SifA, SseJ, SseG, etc)
SPI-2 Type III

ecretion system
acidic pH triggers
secretion
translocon pore (SseC, SseD)

vacuolar membrane
H+
H+
translocon (SseB)
H+
H+
H+
H+
H+
secreton needle
bacterial outer
membrane
SsaC
SsaJ
bacterial inner
membrane
SsaV
SsaM
SpiC
SsaL
ssaV mutant
is null for
SPI-2 function
Intracellular replication defect of a SPI-2

null mutant in macrophages (16 h)
wild-type
SPI-2 mutant
Effectors of the SPI-2 T3SS
From Figueira and Holden, Microbiology, 2012
From Figueira and Holden, Microbiology, 2012
Figueira et al, manuscript in preparation
Human diseases
Africa)
typhoidal Salmonella in HIV-infected African a

NTS (S. Typhimurium and Enteritidis):
Predominantly cause self-limiting gastroenteritis
BUT
major cause of invasive disease in Africa, young
children and HIV-infected adults
Non specific fever (+/- diarrhea) Bacteremia
Case fatalities (22-45 %)
AB resistance
No vaccine
Salmonellae are the commonest cause

of bacterial bloodstream infection in
Africa
(Reddy EA, et al. Lancet Infect Dis 2010)
Okoro, et al. Nature Genetics, 2012
Log10 change in Salmonella (cfu/ml)
Host resistance to Salmonella relies on:

- cell-mediated immunity (macrophages, PMNs, NK
cells)
- complement killing (induced by Antibodies
OMPs)
HIV-uninfected sera
HIV-infected sera
Exposure to serum (min)
Lack of Ab?
MacLenan, et al. Science, 2010
HIV-uninfected sera
HIV-infected sera
Total anti- Salmonella IgG titer
Inhibitory factor: LPS IgG
Exposure to serum (min)
Diverting complement deposition away from OM

Impeding access to OM by cross linking O-antigens
Summary
Salmonella enterica different diseases, typhoid and nontyphoidal

Replicates in macrophages in membrane-bound vacuoles
Persists chronic infection, carriers/shedders
SPI-1 and SPI-2 Type III secretion systems are important
virulence components: SPI-1 for invasion and SPI-2 for
replication
More reading
Salmonella takes control: effector driven
manipulation of the host. McGhie et al., Curr
Opinion Microbiol 12: 117 (2009)
Salmonella interplay with host cells: Haraga et al.,
Nature Reviews Microbiol 6: 53 (2008)

Salmonella 2015 - BSC Blackboard

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Biology of Salmonella

Salmonella Typhi and

Typhoid prevalence, 2006

Salmonella Typhi and

Chronic carrier state

Murine model of Typhoid

S. Typhimurium invasion of M cells

Intracellular replication in spleen and liver

Total number of virulence genes

Estimates suggest approx.

Different virulence genes act at

acid tolerance fur, atr

low pH, low Mg++

serum resistance rfb, rfc, rck

Intracellular replication in macrophages

Dhar and McKinney, Current Opinion in Microbiology 2007

Measuring replication by Fluorescence

Helaine et al, PNAS 2010

Measuring replication by Fluorescence

Non-replicating bacteria > 50 %

Helaine et al, PNAS 2010

Salmonella persisters induced by contact

Interaction with host M, even transient, enhances the formation of

Host induced Salmonella persisters are

Growth in rich medium

Proportion of the non-replicating

Regrowing non-replicating bacteria

+ cefotaxime 200 g/mL

Responsive persisters after 24 h in IFN

Responsive and regrowing persisters in

+ cefotaxime 200 g/mL

Non replicating bacteria reservoir for relapse of infection

Heterogeneity of intracellular population

Bacterial actors ? Host signals ?

Salmonella invasion of epithelial

effectors (SifA, SseJ, SseG, etc)

SPI-2 Type III

translocon pore (SseC, SseD)

Intracellular replication defect of a SPI-2

Effectors of the SPI-2 T3SS

From Figueira and Holden, Microbiology, 2012

Effectors of the SPI-2 T3SS

From Figueira and Holden, Microbiology, 2012

Effectors of the SPI-2 T3SS

Figueira et al, manuscript in preparation

typhoidal Salmonella in HIV-infected African a

Salmonellae are the commonest cause

(Reddy EA, et al. Lancet Infect Dis 2010)

typhoidal Salmonella in HIV-infected African a

Okoro, et al. Nature Genetics, 2012

typhoidal Salmonella in HIV-infected African a

Log10 change in Salmonella (cfu/ml)

Host resistance to Salmonella relies on:

Exposure to serum (min)

MacLenan, et al. Science, 2010

Log10 change in Salmonella (cfu/ml)

typhoidal Salmonella in HIV-infected African a

Total anti- Salmonella IgG titer

MacLenan, et al. Science, 2010

typhoidal Salmonella in HIV-infected African a

Inhibitory factor: LPS IgG

typhoidal Salmonella in HIV-infected African a

Inhibitory factor: LPS IgG