Yiping He - Center for Food Safety Engineering
Yiping He - Center for Food Safety Engineering
Yiping He - Center for Food Safety Engineering
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Microarray analysis of quorum-sensing<br />
quorum sensing<br />
regulated gene expression in<br />
Campylobacter jejuni<br />
<strong>Yiping</strong> <strong>He</strong><br />
Microbial Biophysics and Residue Chemistry<br />
Research Unit<br />
Chin Chin-Yi Yi Chen<br />
Microbial <strong>Food</strong> <strong>Safety</strong> Research Unit<br />
Microaerobic workstation<br />
Optimal growth conditions <strong>for</strong> C. jejuni:<br />
5% O 2, 10%CO 2, 85% N 2, 82% relative humidity, 37 o C or 42 o C<br />
LuxS/AI-2 dependent quorum sensing<br />
• Quorum-sensing (QS) is a bacterial cell-to-cell<br />
communication process in response to high cell density<br />
• QS regulates gene expression by producing and<br />
releasing autoinducers (AIs)<br />
• Three major types of autoinducers: AI-1, AI-2, peptides<br />
• AI-2 is the product of the LuxS enzyme, which is widely<br />
conserved in bacteria<br />
• LuxS/AI-2 dependent QS controls a variety of cellular<br />
activities including the production of virulence factors<br />
and toxins, biofilm <strong>for</strong>mation, and swarming motility<br />
Campylobacter jejuni<br />
• A major cause of bacterial gastroenteritis and human diarrhea<br />
• A Gram-negative, microaerophilic, motile, spiral-shaped bacterium<br />
Some of the virulence factors in C. jejuni<br />
� Motility<br />
• An important factor <strong>for</strong> C. jejuni colonization in chicken<br />
• Alterations in flagellar gene expression affect its pathogenesity<br />
� Oxidative stress defense:<br />
• Highly sensitive to the O 2 metabolites/oxidative stress<br />
• Lacks the oxidative stress response factors: SoxR and OxyR<br />
• Utilizes: superoxide stress defense system (SodB)<br />
peroxide stress defense system (KatA, AhpC and Tpx)<br />
� Chemotaxis<br />
� Adhesion and invision<br />
� Toxin production<br />
� Iron acquisition<br />
� <strong>He</strong>at-shock response<br />
AI-2 biosynthesis and methyl cycle in E. coli<br />
MetF<br />
CH3-THF<br />
5, 10-methylene THF<br />
CH3-THPG<br />
???<br />
AI-2<br />
MetH, MetE<br />
THF, THPG<br />
DPD<br />
Homocysteine<br />
LuxS<br />
SRH<br />
Adenine<br />
Lsr<br />
LsrK P P<br />
P<br />
P<br />
LsrFG<br />
ATP ADP<br />
Methionine<br />
SahH<br />
Pfs<br />
Degraded AI-2<br />
2 μm<br />
SAM synthetase<br />
(MetK)<br />
SAM<br />
Methyltransferases<br />
SAH (toxic)<br />
11/27/2007<br />
1
Amino acid sequence alignment of LuxS<br />
E.coli MPLLDSFTVDHTRMEAPAVRVAKTMNTPHGDAITVFDLRFCVPNKEVMPERGIHTLEHLF 59<br />
S.typhimurium MPLLDSFAVDHTRMQAPAVRVAKTMNTPHGDAITVFDLRFCIPNKEVMPEKGIHTLEHLF 59<br />
V.harveyi MPLLDSFTVDHTRMNAPAVRVAKTMQTPKGDTITVFDLRFTAPNKDILSEKGIHTLEHLY 59<br />
H.influenzae MPLLDSFKVDHTKMNAPAVRIAKTMLTPKGDNITVFDLRFCIPNKEILSPKGIHTLEHLF 59<br />
C.jejuni MPLLDSFKVDHTKMPAPAVRLAKVMKTPKGDDISVFDLRFCIPNKDIMSEKGTHTLEHLF 60<br />
******* ****:* *****:**.* **:** *:****** ***:::. :* ******:<br />
EE.coli coli AGFMRNHLNGNGVEIIDISPMGCRTGFYMSLIGTPDEQRVADAWKAAMEDVLKVQDQNQI 119<br />
S.typhimurium AGFMRDHLNGNGVEIIDISPMGCRTGFYMSLIGTPDEQRVADAWKAAMADVLKVQDQNQI 119<br />
V.harveyi AGFMRNHLNGDSVEIIDISPMGCRTGFYMSLIGTPSEQQVADAWIAAMEDVLKVENQNKI 119<br />
H.influenzae AGFMRDHLNGDSIEIIDISPMGCRTGFYMSLIGTPNEQKVSEAWLASMQDVLGVQDQASI 119<br />
C.jejuni AGFMRDHLNSNSVEIIDISPMGCRTGFYMSLIGTPDEKSIAKAWEAAMKDVLSVSDQSKI 120<br />
*****:***.:.:**********************.*: ::.** *:* *** *.:* .*<br />
E.coli PELNVYQCGTYQMHSLQEAQDIARSILERDVRINSNEELALPKEKLQELHI- 170<br />
S.typhimurium PELNVYQCGTYQMHSLSEAQDIARHILERDVRVNSNKELALPKEKLQELHI- 170<br />
V.harveyi PELNEYQCGTAAMHSLDEAKQIAKNILEVGVAVNKNDELALPESMLRELRID 171<br />
H.influenzae PELNIYQCGSYTEHSLEDAHEIAKNVIARGIGVNKNEDLSLDNSLLK----- 166<br />
C.jejuni PELNIYQCGTCAMHSLDEAKQIAQKVLNLGISIINNKELKLENA-------- 164<br />
**** ****: ***.:*::**: :: .: : .*.:* * :<br />
LuxS-dependent AI-2 production<br />
Relativve<br />
light units<br />
1.0E+08<br />
1.0E+07<br />
1.0E+06<br />
1.0E+05<br />
1.0E+04<br />
1.0E+03<br />
1.0E+02<br />
1.0E+01<br />
0 2 4 6 8 10 12<br />
Assay Time (Hours)<br />
∆luxS<br />
WT<br />
MH broth<br />
• Wild-type C. jejuni produces functional AI-2<br />
• There is no AI-2 production in the luxS mutant<br />
• LuxS is a key enzyme in the AI-2 biosynthesis pathway<br />
Reduced motility in the ΔluxS mutant<br />
Halo diameter 35±1.22 mm 28±1.58 mm<br />
Construction of ΔluxS mutant in C. jejuni<br />
gatB<br />
luxS<br />
cj1199<br />
Primer 1 Primer 2 Primer 3 Primer 4<br />
Cm R<br />
Electroporate into C. jejuni<br />
Select <strong>for</strong> chloramphenicol-resistant clones<br />
First amplification<br />
Second amplification<br />
Restriction digest<br />
Ligation with Cm R cassette<br />
Verify mutant by PCR &<br />
sequencing<br />
Growth curves of the wt and ΔluxS mutant<br />
of C. jejuni<br />
Viable cell couunt<br />
(CFU/ml)<br />
1.0E+10<br />
1.0E+09<br />
1.0E+08<br />
1.0E+07<br />
1.0E+06<br />
1.0E+05<br />
doubling time:<br />
WT, 42°C<br />
∆ luxS, 42°C<br />
WT, 37°C<br />
∆ luxS, 37°C<br />
0 4 8 12 16 20 24 28 32<br />
Time (Hours)<br />
wild-type ΔluxS mutant<br />
37 o C 77 min 89 min<br />
42 o C 53 min 67 min<br />
Resistance of the wt and ΔluxS mutant<br />
to CHP and H 2O 2<br />
Diameter of growth innhibition<br />
zone (mm)<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
3% 1% 0.33%<br />
CHP<br />
CHP: cumene hydroperoxide<br />
H 2O 2: hydrogen peroxide<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
wt<br />
∆luxS<br />
3% 1% 0.33%<br />
H 2O2<br />
11/27/2007<br />
2
Expression microarray<br />
Oligo array<br />
Wild-type ΔluxS mutant<br />
Scanned microarray image<br />
In both H 2O 2 treated & untreated conditions:<br />
• Triplicates of cell samples and chips were used.<br />
• In each chip, there are triplicate sets of spotted oligos.<br />
• EEach h set t of f oligos li represents t >1600 ORF ORFs of f CC. jjejuni. j i<br />
• 9 replicate data was normalized and analyzed.<br />
• The up/down-regulated genes were selected by 1.5 fold<br />
cut-off combining with P value < 0.05.<br />
Differentially expressed genes in the ΔluxS<br />
Efflux<br />
ssystem stem<br />
ABC<br />
transporter<br />
PstABS<br />
MetF<br />
CH3-THF<br />
5, 10-methylene THF<br />
CH3-THPG<br />
AI-2<br />
MetE<br />
THF, THPG<br />
DPD<br />
Homocysteine<br />
LuxS<br />
SRH<br />
Adenine<br />
Methionine<br />
Pfs<br />
Oxidative stress<br />
response genes<br />
SAM synthetase<br />
(MetK)<br />
SAM<br />
Methyltransferases<br />
SAH (toxic)<br />
+H 2O 2<br />
Flagellar<br />
genes<br />
Expression microarray<br />
Oligo array<br />
H 2O 2 treated wild-type H 2O 2 treated ΔluxS mutant<br />
Selected genes regulated by luxS with/without H2O2 treatment<br />
Locus/Genes Gene product Fold change<br />
(wt/∆luxS)<br />
+ H2O2 - H2O2<br />
Cj1198 (luxS) S-ribosylhomocysteinase 331.6 195.5<br />
Cj1199 putative iron/ascorbate-dependent oxidoreductase 7.4 11.1<br />
Cj1200 putative periplasmic protein 2.2 3.2<br />
Cj1201(metE) homocysteine methyltransferase 1.66 0.95<br />
Cj1202(metF) 5,10-methylenetetrahydrofolate reductase 1.8 1.36<br />
Cj1096c(metK) S-adenosylmethionine synthetase 1.5 0.98<br />
Cj0613(pstS) possible periplasmic phosphate binding protein 2.1 2.9<br />
Cj0615(pstA) putative phosphate transport system permease protein 1.26 1.6<br />
Cj0616(pstB) phosphate ABC transporter, ATP-binding protein 1.9 2.1<br />
Cj0727 ABC transporter, periplasmic substrate-binding protein 2.1 1.57<br />
Cj0728 putative periplasmic protein 18 1.8 148 1.48<br />
Cj0301c(modB) molybdenum ABC transporter 2.0 1.12<br />
Cj1220(groES) co-chaperonin 1.69 1.27<br />
Cj0334(ahpC) alkyl hydroperoxide reductase 1.56 1.03<br />
Cj0779(tpx) probable thiol peroxidase 1.61 1.08<br />
Cj0117(pfs) S-adenosylhomocysteine nucleosidase 0.66 0.68<br />
Cj0697(flgG2) putative flagellar basal-body rod protein 0.46 0.84<br />
Cj0698(flgG) flagellar basal-body rod protein 0.51 0.70<br />
Cj0040 flagellar assembly protein 0.48 0.81<br />
Cj0041 hypothetical protein 0.52 0.70<br />
Cj0042(flgD) putative flagellar hook assembly protein 0.49 0.63<br />
Cj0043(flgE) flagellar hook protein 0.58 0.64<br />
Cj1031 putative outer membrane component of efflux system 0.76 0.74<br />
Cj1032 putative membrane fusion component of efflux system 0.57 0.87<br />
Cj1033 putative integral membrane component of efflux system 0.64 0.99<br />
Cj1034c possible dnaJ-like protein 0.61 0.71<br />
Real time RT-PCR verification of the<br />
microarray results<br />
Log2 of wt/ΔluuxS<br />
4<br />
2<br />
0<br />
-2<br />
cj1199<br />
cj1200<br />
metE<br />
metF<br />
pstB<br />
pstS<br />
cj0727<br />
cj0728<br />
aphC<br />
flgG2<br />
real-time PCR<br />
microarray<br />
flgG<br />
LuxS regulated genes<br />
cj0040<br />
cj0041<br />
cj1032<br />
cj1034<br />
11/27/2007<br />
3
Summary<br />
• In C. jejuni, AI-2 production is dependent on LuxS.<br />
• The ∆luxS mutant exhibits slightly slower growth, lower<br />
motility, and higher sensitivity to oxidative stress than<br />
those of the wt strain.<br />
• Microarray y analysis y revealed that AI-2/LuxS regulates g<br />
the expression of genes involved in AI-2 biosynthesis and<br />
methyl cycle, ABC transporter, membrane efflux system,<br />
stress responses, and cell motility.<br />
• Expression of the oxidative stress genes (ahpC and tpx)<br />
was lower in the ΔluxS mutant than that in the wt when<br />
the cells were treated with H2O2, supporting the<br />
observed higher sensitivity of the ∆luxS mutant to<br />
oxidative stress.<br />
Acknowledgements<br />
Bacterial Epidemiology &<br />
Antimicrobial Resistance<br />
ARS ARS-USDA, USDA, Athens, GA<br />
Jonathan G G. Frye, Frye Ph Ph.D. D<br />
Microarray Facility<br />
Fox Chase Cancer <strong>Center</strong><br />
Yuesheng Li, Ph.D.<br />
Microbial <strong>Food</strong> <strong>Safety</strong><br />
Terence Strobaugh<br />
Pina Fratamico, Ph.D.<br />
John Luchansky, Ph.D.<br />
Ph.D.<br />
Microbial Biophysics &<br />
Residue Chemistry<br />
Peter Irwin, Ph.D.<br />
George Paoli, Ph.D.<br />
Shu Shu-I I Tu, Ph. D.<br />
11/27/2007<br />
4