1 E0364 Inserm Université Lille II (Faculté de Médecine Henri Warembourg) Institut Pasteur de Lille, Lille, France
2 U629 Inserm Institut Pasteur de Lille, Lille, France
3 Division of Biophysics, Research Center Borstel, Borstel, Germany
4 Unidad de Investigacion, Hospital Son Dureta and Institut Universitari d'Investigació en Ciències de la Salut (IUNICS), Palma Mallorca, Spain
Correspondence
M. Marceau
michael.marceau{at}ibl.fr
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ABSTRACT |
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These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession numbers for the Y. pseudotuberculosis pmrF, phoPphoQ and pmrApmrB operons are AF336802, AF333125 and AY259243 respectively.
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INTRODUCTION |
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It has been reported that the Gram-negative enteropathogen Yersinia pseudotuberculosis (a member of the Enterobacteriaceae related to Salmonella) is highly resistant to polymyxin and -helical peptides (Bengoechea et al., 1998a
, b
). In Yersinia pestis (a clone that recently emerged from Y. pseudotuberculosis: Achtman et al., 1999
), it has been shown that this resistance is controlled by PhoPPhoQ (Oyston et al., 2000
) and probably depends on the production of 4-aminoarabinose (Rebeil et al., 2004
). In the present work, we confirm the crucial role of a Salmonella-like pmrF operon in the control of resistance of Y. pseudotuberculosis to certain classes of antimicrobial peptides. However, this operon may play a role in cellular processes other than those allowing survival in mammals, and its two-component-system-mediated expression differs from that of the corresponding Salmonella chromosomal locus.
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METHODS |
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DNA sequencing and sequence analysis.
DNA was sequenced by the dideoxy chain-termination method using the ABI PRISM dichloRhodamine Dye Terminator Sequencing kit with AmpliTaq DNA polymerase FS (Perkin Elmer) according to the manufacturer's instructions. Extension products were analysed with the Applied Biosystems model ABI 377XL automated DNA sequencer (Perkin Elmer). The nucleotide sequences obtained were analysed with Perkin-Elmer software (Sequence Navigator). The nucleotide sequence data for the Y. pseudotuberculosis pmrF, phoPphoQ and pmrApmrB operons have been deposited in the GenBank nucleotide sequence database under accession numbers AF336802, AF333125 and AY259243 respectively.
Synthetic oligonucleotides and PCR.
Oligonucleotide primers (Table 2) were custom-synthesized (Sigma and Genset) for PCR generation of DNA fragments used for cloning or probing. PCR amplification was performed as described elsewhere (Sebbane et al., 2001
) with AmpliTaq Gold polymerase (Perkin Elmer Applied Biosystems). Digoxigenin-labelled PCR products were generated with the PCR DIG Labelling Mix (Roche Diagnostics) and purified on Dye-ex Spin columns or Qiaquick PCR purification kit (Qiagen).
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Lipid A extraction and MALDI-TOF mass spectrometry analysis.
Lipid A was isolated as previously described (Zhou et al., 1999). Briefly, an overnight culture grown at 37 °C on LB was centrifuged and the cell pellet was then washed once with PBS. Cells were resuspended in 0·8 ml PBS, and a single-phase Bligh/Dyer mixture was made by addition of 2 ml methanol and 1 ml chloroform. After 60 min at room temperature, the insoluble material was collected by centrifugation for 20 min. The pellet was washed once with 5 ml of a fresh, single-phase Bligh/Dyer mixture consisting of chloroform/methanol/water (1 : 2 : 0·8, by vol.) and was then dispersed in 1·8 ml 12·5 mM sodium acetate (pH 4·5) containing 1 % SDS; the mixture was boiled for 30 min in order to cleave the glycosidic bond between lipid A and KDO. Lipid A was recovered by converting the hydrolysed material into a two-phase Bligh/Dyer mixture by addition of 2 ml chloroform and 2 ml methanol. After low-speed centrifugation, the lower phase was collected and washed twice with 4 ml of the upper phase derived from a fresh, neutral, two-phase chloroform/methanol/water (2 : 2 : 1·8, by vol.) Bligh/Dyer mixture. The washed lower phase was dried under nitrogen.
MALDI-TOF mass spectroscopy analyses of lipid A were performed with the Bruker-Reflex III two-stage reflection time-of-flight mass analyser (Bruker Daltonics) in a linear TOF configuration with an acceleration voltage of 20 kV. Details of the methods used are given by Lindner (2000). Mass scale calibration was performed externally with similar compounds of known chemical structure.
Purification of His-tagged recombinant PhoP proteins under native conditions.
The recombinant E. coli strain harbouring pQE60PhoH6.2 was grown in 1 litre of LB medium supplemented with ampicillin (100 µg ml1) and kanamycin (25 µg ml1). When the OD600 reached 0·8, expression of the gene encoding the recombinant protein was induced with 1 mM IPTG for 0·5 h. The cells were harvested by centrifugation and then resuspended in 5 ml of lysis buffer (300 mM NaCl, 50 mM Na2HPO4, 10 mM imidazole, pH 8) per g fresh weight. Cells were lysed using a French press with a pressure of 1000 p.s.i. (approx 7 MPa). The lysate was clarified by centrifugation at 10 000 g for 20 min. The supernatant was filtered on a 0·45 µm filter before being loaded onto a 1 ml HiTrap Affinity column (Amersham) pre-equilibrated with lysis buffer. The column was washed first with 10 ml lysis buffer and then with 5 ml lysis buffer containing 50 mM imidazole. Elution was performed with 10 ml lysis buffer containing 250 mM imidazole. The collected fractions were analysed by SDS-PAGE, using a 12·5 % polyacrylamide gel and Coomassie blue staining. Samples were dialysed overnight against PBS/10 % (v/v) glycerol, dispensed into aliquots and stored at 20 °C.
Electrophoretic mobility shift assays.
The binding of purified PhoP(His)6 protein to PCR products from gene promoter regions was assessed by electrophoretic mobility shift assays, as previously described (Himpens et al., 2000).
Antimicrobial peptide activity assays.
Polymyxin B and cecropin B were purchased from Sigma; defensin A was purified from Phormia terranovae (a kind gift from P. Bullet). The formulation of the incubation medium was identical to that of RPMI 1640 (Invitrogen, ref. 21870) except for Ca(NO3)2 and MgSO4, which were added separately at appropriate concentrations. The bactericidal effects of antimicrobial peptides were determined using Y. pseudotuberculosis cells obtained from cultures in LB broth (which naturally contains iron) at 28 °C for 1618 h (OD620 0·4) followed by an additional 3 h incubation in LB broth at the appropriate temperature and in the presence of 300 µM of the iron chelators deferoxamine mesylate or 2,2'-dipyridyl (Sigma) when necessary. Bactericidal assays were performed as follows. First, stock solutions of cecropin B, polymyxin B and defensin A (0·1 mg ml1 in sterile distilled water) were diluted in the incubation medium at the appropriate concentrations. Then 100 µl volumes of these working solutions of defined peptide concentration were placed in each well of a 96-well microtitre plate. Ten microlitres of Y. pseudotuberculosis cell suspension was added to each well (final bacterial concentration 5x106 c.f.u. ml1). As a control, bacterial suspensions were concomitantly added to wells containing 100 µl incubation medium lacking added antimicrobial peptides. Microtitre plates were shaken gently for 30 s and then incubated at 37 °C for 2 h. To assess viability of Y. pseudotuberculosis, 50 µl of mixture from each well was serially diluted in sterile distilled water. Diluates were plated on LB and incubated at 28 °C for 4872 h. Bacterial survival was defined as the ratio of the number of viable bacteria after 2 h contact with peptides to the number of viable bacteria in the absence of peptides. Micrococcus luteus (highly sensitive to defensins) and S. enterica serovar Typhimurium strain LT2 were used as internal controls for defensin A and iron chelator (deferoxamine mesylate or 2,2'-dipyridyl) activity respectively.
Experimental infection in a mouse model.
Six-week-old female outbred OF1 mice (Iffa Credo) were challenged either by the intravenous (i.v.) route (0·3 ml bacterial suspension in sterile PBS) or by the intragastric (i.g.) route (0·2 ml bacterial suspension in sterile distilled water, using a gastric tube). Mice were starved for 18 h prior to gastric inoculation. Bacterial inocula were prepared from overnight cultures in LB at 28 °C. The cultures were centrifuged and the bacterial pellets were washed once and resuspended in distilled water or PBS. Animals were kept in positive-pressure cabinets during experimentation, and mortality was monitored daily for 21 days after challenge. For each experimental infection, the presence of the virulence plasmid pYV was confirmed by PCR on bacterial thermolysates using primers YOPH1 and YOPH2, which are internal to yopH, a gene located on pYV (Carnoy et al., 2000). Infected animals were monitored for 3 weeks, and the 50 % lethal dose (LD50) was calculated from groups of 5 (i.v. model) or 10 (i.g. model) mice according to the method of Reed & Muench (1938)
.
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RESULTS |
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The pmrF operon is not required for full virulence of Y. pseudotuberculosis in a mouse model
Antimicrobial peptides are major effectors of innate immunity in mammals, and so we investigated whether the pmrF operon contributes to the progression of host infection by Y. pseudotuberculosis. The virulence of the pmrF mutant was assessed in the OF-1 mouse. The Y. pseudotuberculosis pmrF mutant was found to be as virulent as the parental strain when administered intravenously (LD50<102). Its LD50 also did not differ significantly from that of the wild-type in an oral model of infection, and the kinetics of animal death were similar, regardless of the infecting strain again, in contrast to the situation in Salmonella (Gunn et al., 2000).
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DISCUSSION |
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In S. typhimurium, pmrF transcription is mediated by both the PhoPPhoQ and PmrAPmrB two-component regulatory systems but is only under the control of PhoPPhoQ in Y. pseudotuberculosis. In line with this result, PhoP was found to participate directly in the transcriptional activation of this operon in the latter species. This situation is similar to what was observed in Ps. aeruginosa (McPhee et al., 2003), although both two-component systems are suspected to control pmrF expression in this latter organism. It is noteworthy that no putative Salmonella-, Ps. aeruginosa- or E. coli-like phoP promoter-binding direct repeats were found upstream of either the pmrF or phoPQ operons. A lack of conserved motifs in these promoting regions has also been observed in Ph. luminescens (Derzelle et al., 2004
).
Y. pseudotuberculosis PmrA and PmrB exhibit relatively low levels of identity (56 and 51 %, respectively) with the respective homologous proteins in Salmonella, thus raising the possibility that the two Y. pseudotuberculosis and Salmonella PmrAPmrB systems may not in fact be homologues. Interestingly, this situation is very similar to that recently observed in Er. carotovora, where the PmrA and PmrB subunits (Hyytiainen et al., 2003) share only 59 % and 56 % protein identity with their respective Salmonella counterparts and 65 % and 58 % with those of Y. pseudotuberculosis. Just as in Salmonella, the Erwinia PmrB protein is likely to be able to sense iron as an input signal directly, as illustrated by the presence of conserved iron-receptor-like EXXE motifs. However, in Er. carotovora, the PmrAPmrB system functions differently and, moreover, has been shown to regulate virulence functions specific to plant pathogens, in addition to antimicrobial peptide resistance (Hyytiäinen et al., 2003
). These observations strongly suggest that this two-component system has diverged differently in these three species, contrasting with core systems like OmpREnvZ, which remain highly conserved (i.e. >90 % identical). The CtrACtrB two component system has recently been shown to regulate distinct pathways (and thus different genes) in Brucella abortus and Caulobacter crescentus, despite the high degree of homology (>80 % identity at the amino acid level) for the corresponding regulator subunits in these two species (Bellefontaine et al., 2002
). PmrAPmrB is thus a second example showing that a ubiquitous two-component regulatory system may control different regulons in different bacterial species.
What, then, might be the role of PmrAPmrB? In Yersinia, this system is probably involved in regulation of genes other than those contributing to lipid A substitution with 4-aminoarabinose. In contrast to Salmonella, E. coli and Er. carotovora, the Yersinia pmrA and pmrB genes are not associated with pmrC to form the pmrCAB operon in fact, pmrC is probably absent from the Yersinia genome (Chain et al., 2004). Instead, as judged by the presence of a very short intergenic region (5 nucleotides), pmrA and pmrB are putatively co-transcribed with dacB, which encodes a product 79 % identical to an E. coli penicillin-binding DD-carboxypeptidase/DD-endopeptidase (Korat et al., 1991
). This latter finding suggests that PmrAPmrB could be involved in peptidoglycan homeostasis in Yersinia cells. Fully annotated genome sequences of pathogenic yersiniae are either available now (for Y. pestis and Y. pseudotuberculosis) or will be released soon (for Y. enterocolitica): transcriptome analysis of pmrA and pmrB mutants and their parental counterparts should clarify this point.
As shown in Fig. 3(b), pmrF transcription responds to pH changes, and our results indicate that this regulation was PmrAPmrB-independent. As already mentioned by other workers, the PhoPPhoQ system is unlikely to participate in this process either (Garcia Vescovi et al., 1996
). Accordingly, a (twofold) pH-induced transcriptional activation was still observed in a phoP-null background (Fig. 3
), suggesting that an additional regulator may control pmrF expression. To identify other potential activators or repressors, Tn5 transposon mutagenesis was carried out in Y. pseudotuberculosis. The regulation mutants isolated (other than phoP and phoQ mutants) were all found to have a transposon copy inserted within the miaA gene. The miaA product is a tRNA N6-isopentenyladenosine synthetase involved in the first step of the transformation of the adenosine located at position 37 (ms2i6A37) of some tRNAs into 2-methyl-N6-isopentenyladenosine. Recently, this tRNA modification was found to be essential for optimal expression of virulence factors in the bacterial pathogens Agrobacterium tumefaciens and Shigella flexneri (Durand et al., 1997
; Gray et al., 1992
). In the latter organism, it has been reported that a miaA mutant displayed a tenfold reduction in transcription of the virF regulon due to a post-transcriptional downregulation of VirF activator expression (Durand et al., 2000
). miaA inactivation in Y. pseudotuberculosis was associated with a slight inhibition of bacterial growth (as in Sh. flexneri). Transcription of the pmrF operon in two independent mutants (which differed only in the orientation of Tn5 within miaA) was significantly reduced, although it was not as low as that obtained in Y. pseudotuberculosis phoP mutants (data not shown). The exact mechanism of this unexpected additional control of pmrF operon transcription is unknown. However, the dramatic decrease in phoP mRNA levels in these insertion mutants (unpublished data) suggests that MiaA might modulate expression of the pmrF operon via regulation of PhoP either by controlling the production of a regulator that mediates phoPQ transcription or by interfering with autoregulation of the PhoPPhoQ system. These hypotheses are currently undergoing further investigation. However, to the best of our knowledge, MiaA activity has yet to be reported as being pH-dependent: therefore, we assume that MiaA per se is unlikely to account for the pH-dependence of pmrF transcription, and that other pmrF-regulating elements exist. Failure to detect putative regulatory elements by transposon mutagenesis could be due to the deleterious effects of inactivation on the bacterial cell or, alternatively, to a very low frequency of Tn5 intragenic insertion.
pmrF inactivation attenuates Salmonella virulence when bacteria are administered by an oral route (Gunn et al., 2000). In contrast, we found that arabinosylation of LPS had no detectable effect on the oral pathogenicity of Y. pseudotuberculosis in a mouse model. This discrepancy might be partly explained by the distinctly different lifestyles of the two enteropathogens in the host. Following oral ingestion, Salmonella cells enter the intestinal mucosa and then are taken up by (and survive within) resident phagocytes. Bacterial survival and replication in these cells depend, at least in part, on resistance (through lipid A modification of LPS) to killing by antimicrobial peptides (Gunn et al., 2000
). Like Salmonella, Y. pseudotuberculosis is an entero-invasive pathogen but after having crossed the epithelial barrier, it resists attack by mucosal macrophages via a plasmid-encoded type III secretion process (Cornelis, 2002
), thus blocking phagocytosis.
Growth of Y. pseudotuberculosis at temperatures below 37 °C results in increased resistance to polymyxin B, consistent with previous Yersinia studies showing that temperature downshifts are associated with higher levels of incorporation of 4-aminoarabinose into lipid A (Bhagya Lakshmi et al., 1989; Kawahara et al., 2002
). Nevertheless, we found that transcription of the Y. pseudotuberculosis pmrF operon was abolished at low temperatures (M. Marceau and others, unpublished results). Upregulation of lipid A substitution must therefore occur at a post-transcriptional level by an as yet unknown, cold-induced regulatory mechanism. The existence of such a mechanism (designed to counterbalance reduced transcription levels of the pmrF operon) strongly suggests that lipid A modifications may also play a crucial role outside the mammalian host.
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ACKNOWLEDGEMENTS |
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Received 23 June 2004;
revised 24 August 2004;
accepted 26 August 2004.
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