Yiping He - Center for Food Safety Engineering

Microarray analysis of quorum-sensing
quorum sensing
regulated gene expression in
Campylobacter jejuni
Yiping He
Microbial Biophysics and Residue Chemistry
Research Unit
Chin Chin-Yi Yi Chen
Microbial Food Safety Research Unit
Microaerobic workstation
Optimal growth conditions for C. jejuni:
5% O 2, 10%CO 2, 85% N 2, 82% relative humidity, 37 o C or 42 o C
LuxS/AI-2 dependent quorum sensing
• Quorum-sensing (QS) is a bacterial cell-to-cell
communication process in response to high cell density
• QS regulates gene expression by producing and
releasing autoinducers (AIs)
• Three major types of autoinducers: AI-1, AI-2, peptides
• AI-2 is the product of the LuxS enzyme, which is widely
conserved in bacteria
• LuxS/AI-2 dependent QS controls a variety of cellular
activities including the production of virulence factors
and toxins, biofilm formation, and swarming motility
Campylobacter jejuni
• A major cause of bacterial gastroenteritis and human diarrhea
• A Gram-negative, microaerophilic, motile, spiral-shaped bacterium
Some of the virulence factors in C. jejuni
� Motility
• An important factor for C. jejuni colonization in chicken
• Alterations in flagellar gene expression affect its pathogenesity
� Oxidative stress defense:
• Highly sensitive to the O 2 metabolites/oxidative stress
• Lacks the oxidative stress response factors: SoxR and OxyR
• Utilizes: superoxide stress defense system (SodB)
peroxide stress defense system (KatA, AhpC and Tpx)
� Chemotaxis
� Adhesion and invision
� Toxin production
� Iron acquisition
� Heat-shock response
AI-2 biosynthesis and methyl cycle in E. coli
MetF
CH3-THF
5, 10-methylene THF
CH3-THPG
???
AI-2
MetH, MetE
THF, THPG
DPD
Homocysteine
LuxS
SRH
Adenine
Lsr
LsrK P P
P
P
LsrFG
ATP ADP
Methionine
SahH
Pfs
Degraded AI-2
2 μm
SAM synthetase
(MetK)
SAM
Methyltransferases
SAH (toxic)
11/27/2007
1

Amino acid sequence alignment of LuxS
E.coli MPLLDSFTVDHTRMEAPAVRVAKTMNTPHGDAITVFDLRFCVPNKEVMPERGIHTLEHLF 59
S.typhimurium MPLLDSFAVDHTRMQAPAVRVAKTMNTPHGDAITVFDLRFCIPNKEVMPEKGIHTLEHLF 59
V.harveyi MPLLDSFTVDHTRMNAPAVRVAKTMQTPKGDTITVFDLRFTAPNKDILSEKGIHTLEHLY 59
H.influenzae MPLLDSFKVDHTKMNAPAVRIAKTMLTPKGDNITVFDLRFCIPNKEILSPKGIHTLEHLF 59
C.jejuni MPLLDSFKVDHTKMPAPAVRLAKVMKTPKGDDISVFDLRFCIPNKDIMSEKGTHTLEHLF 60
******* ****:* *****:**.* **:** *:****** ***:::. :* ******:
EE.coli coli AGFMRNHLNGNGVEIIDISPMGCRTGFYMSLIGTPDEQRVADAWKAAMEDVLKVQDQNQI 119
S.typhimurium AGFMRDHLNGNGVEIIDISPMGCRTGFYMSLIGTPDEQRVADAWKAAMADVLKVQDQNQI 119
V.harveyi AGFMRNHLNGDSVEIIDISPMGCRTGFYMSLIGTPSEQQVADAWIAAMEDVLKVENQNKI 119
H.influenzae AGFMRDHLNGDSIEIIDISPMGCRTGFYMSLIGTPNEQKVSEAWLASMQDVLGVQDQASI 119
C.jejuni AGFMRDHLNSNSVEIIDISPMGCRTGFYMSLIGTPDEKSIAKAWEAAMKDVLSVSDQSKI 120
*****:***.:.:**********************.*: ::.** *:* *** *.:* .*
E.coli PELNVYQCGTYQMHSLQEAQDIARSILERDVRINSNEELALPKEKLQELHI- 170
S.typhimurium PELNVYQCGTYQMHSLSEAQDIARHILERDVRVNSNKELALPKEKLQELHI- 170
V.harveyi PELNEYQCGTAAMHSLDEAKQIAKNILEVGVAVNKNDELALPESMLRELRID 171
H.influenzae PELNIYQCGSYTEHSLEDAHEIAKNVIARGIGVNKNEDLSLDNSLLK----- 166
C.jejuni PELNIYQCGTCAMHSLDEAKQIAQKVLNLGISIINNKELKLENA-------- 164
**** ****: ***.:*::**: :: .: : .*.:* * :
LuxS-dependent AI-2 production
Relativve
light units
1.0E+08
1.0E+07
1.0E+06
1.0E+05
1.0E+04
1.0E+03
1.0E+02
1.0E+01
0 2 4 6 8 10 12
Assay Time (Hours)
∆luxS
WT
MH broth
• Wild-type C. jejuni produces functional AI-2
• There is no AI-2 production in the luxS mutant
• LuxS is a key enzyme in the AI-2 biosynthesis pathway
Reduced motility in the ΔluxS mutant
Halo diameter 35±1.22 mm 28±1.58 mm
Construction of ΔluxS mutant in C. jejuni
gatB
luxS
cj1199
Primer 1 Primer 2 Primer 3 Primer 4
Cm R
Electroporate into C. jejuni
Select for chloramphenicol-resistant clones
First amplification
Second amplification
Restriction digest
Ligation with Cm R cassette
Verify mutant by PCR &
sequencing
Growth curves of the wt and ΔluxS mutant
of C. jejuni
Viable cell couunt
(CFU/ml)
1.0E+10
1.0E+09
1.0E+08
1.0E+07
1.0E+06
1.0E+05
doubling time:
WT, 42°C
∆ luxS, 42°C
WT, 37°C
∆ luxS, 37°C
0 4 8 12 16 20 24 28 32
Time (Hours)
wild-type ΔluxS mutant
37 o C 77 min 89 min
42 o C 53 min 67 min
Resistance of the wt and ΔluxS mutant
to CHP and H 2O 2
Diameter of growth innhibition
zone (mm)
50
40
30
20
10
0
3% 1% 0.33%
CHP
CHP: cumene hydroperoxide
H 2O 2: hydrogen peroxide
50
40
30
20
10
0
wt
∆luxS
3% 1% 0.33%
H 2O2
11/27/2007
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Expression microarray
Oligo array
Wild-type ΔluxS mutant
Scanned microarray image
In both H 2O 2 treated & untreated conditions:
• Triplicates of cell samples and chips were used.
• In each chip, there are triplicate sets of spotted oligos.
• EEach h set t of f oligos li represents t >1600 ORF ORFs of f CC. jjejuni. j i
• 9 replicate data was normalized and analyzed.
• The up/down-regulated genes were selected by 1.5 fold
cut-off combining with P value < 0.05.
Differentially expressed genes in the ΔluxS
Efflux
ssystem stem
ABC
transporter
PstABS
MetF
CH3-THF
5, 10-methylene THF
CH3-THPG
AI-2
MetE
THF, THPG
DPD
Homocysteine
LuxS
SRH
Adenine
Methionine
Pfs
Oxidative stress
response genes
SAM synthetase
(MetK)
SAM
Methyltransferases
SAH (toxic)
+H 2O 2
Flagellar
genes
Expression microarray
Oligo array
H 2O 2 treated wild-type H 2O 2 treated ΔluxS mutant
Selected genes regulated by luxS with/without H2O2 treatment
Locus/Genes Gene product Fold change
(wt/∆luxS)
+ H2O2 - H2O2
Cj1198 (luxS) S-ribosylhomocysteinase 331.6 195.5
Cj1199 putative iron/ascorbate-dependent oxidoreductase 7.4 11.1
Cj1200 putative periplasmic protein 2.2 3.2
Cj1201(metE) homocysteine methyltransferase 1.66 0.95
Cj1202(metF) 5,10-methylenetetrahydrofolate reductase 1.8 1.36
Cj1096c(metK) S-adenosylmethionine synthetase 1.5 0.98
Cj0613(pstS) possible periplasmic phosphate binding protein 2.1 2.9
Cj0615(pstA) putative phosphate transport system permease protein 1.26 1.6
Cj0616(pstB) phosphate ABC transporter, ATP-binding protein 1.9 2.1
Cj0727 ABC transporter, periplasmic substrate-binding protein 2.1 1.57
Cj0728 putative periplasmic protein 18 1.8 148 1.48
Cj0301c(modB) molybdenum ABC transporter 2.0 1.12
Cj1220(groES) co-chaperonin 1.69 1.27
Cj0334(ahpC) alkyl hydroperoxide reductase 1.56 1.03
Cj0779(tpx) probable thiol peroxidase 1.61 1.08
Cj0117(pfs) S-adenosylhomocysteine nucleosidase 0.66 0.68
Cj0697(flgG2) putative flagellar basal-body rod protein 0.46 0.84
Cj0698(flgG) flagellar basal-body rod protein 0.51 0.70
Cj0040 flagellar assembly protein 0.48 0.81
Cj0041 hypothetical protein 0.52 0.70
Cj0042(flgD) putative flagellar hook assembly protein 0.49 0.63
Cj0043(flgE) flagellar hook protein 0.58 0.64
Cj1031 putative outer membrane component of efflux system 0.76 0.74
Cj1032 putative membrane fusion component of efflux system 0.57 0.87
Cj1033 putative integral membrane component of efflux system 0.64 0.99
Cj1034c possible dnaJ-like protein 0.61 0.71
Real time RT-PCR verification of the
microarray results
Log2 of wt/ΔluuxS
4
2
0
-2
cj1199
cj1200
metE
metF
pstB
pstS
cj0727
cj0728
aphC
flgG2
real-time PCR
microarray
flgG
LuxS regulated genes
cj0040
cj0041
cj1032
cj1034
11/27/2007
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Summary
• In C. jejuni, AI-2 production is dependent on LuxS.
• The ∆luxS mutant exhibits slightly slower growth, lower
motility, and higher sensitivity to oxidative stress than
those of the wt strain.
• Microarray y analysis y revealed that AI-2/LuxS regulates g
the expression of genes involved in AI-2 biosynthesis and
methyl cycle, ABC transporter, membrane efflux system,
stress responses, and cell motility.
• Expression of the oxidative stress genes (ahpC and tpx)
was lower in the ΔluxS mutant than that in the wt when
the cells were treated with H2O2, supporting the
observed higher sensitivity of the ∆luxS mutant to
oxidative stress.
Acknowledgements
Bacterial Epidemiology &
Antimicrobial Resistance
ARS ARS-USDA, USDA, Athens, GA
Jonathan G G. Frye, Frye Ph Ph.D. D
Microarray Facility
Fox Chase Cancer Center
Yuesheng Li, Ph.D.
Microbial Food Safety
Terence Strobaugh
Pina Fratamico, Ph.D.
John Luchansky, Ph.D.
Ph.D.
Microbial Biophysics &
Residue Chemistry
Peter Irwin, Ph.D.
George Paoli, Ph.D.
Shu Shu-I I Tu, Ph. D.
11/27/2007
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