1 Laboratoire de Microbiologie de l'Environnement, EA 956 soutenue par l'INRA, IRBA, Université de Caen, 14032 Caen Cedex, France
2 Institute of Microbiology, Catholic University of Sacred Heart, L. go F. Vito 1, 00168, Rome, Italy
Correspondence
Jean-Christophe Giard
jean-christophe.giard{at}unicaen.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the perR sequence of E. faecalis JH2-2 reported in this paper is DQ064645.
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INTRODUCTION |
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Enterococcus faecalis is a Gram-positive bacterium that is part of the commensal flora of humans and animals, but may also cause severe diseases and is one of the main causative agent of hospital-based infections (Gilmore et al., 2002). This hardy organism resists many kinds of environmental stresses and the emergence of multidrug-resistant strains has become a serious problem (Giard et al., 2003
; Jett et al., 1994
). Not only may E. faecalis encounter peroxide during the infection of tissue, it has the ability to generate superoxide and H2O2 when cultivated in an oxygenated medium (Huycke et al., 2002
). A survey of the E. faecalis genome sequence revealed that this bacterium possesses several genes encoding antioxidant enzymes, such as ahpCF, npr (encoding the NADH peroxidase), sodA (encoding the superoxide dismutase) and katA. Recently, we showed that a mutation in the ef2958 gene, named hypR, sensitized E. faecalis to H2O2 treatment and greatly affected the survival in murine peritoneal macrophages (Verneuil et al., 2004b
). This gene encodes a regulator of the LysR family and, in spite of structural and functional differences, was the most closely related gene to oxyR of E. coli (Verneuil et al., 2004a
). Transcriptional and DNA-shift analysis also revealed that expression of hypR and ahpCF was directly under HypR control, thus providing the first evidence of a new oxidative-stress regulon in E. faecalis (Verneuil et al., 2004b
).
In this report, we characterized the PerR-like regulator in E. faecalis. We then analysed the effect of a perR mutation on the transcription of genes with putative roles in the oxidative-stress response. PerR does not seem to be either a major regulator of oxidative-stress genes or involved in survival within macrophages, but, according to the results obtained in the mouse model, is a factor important for virulence.
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METHODS |
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H2O2 challenge conditions.
Wild-type and mutant cells (grown as described above) were harvested at an OD600 of 0·5 by centrifugation and resuspended in 0·9 % (w/v) NaCl with 20 mM H2O2. These cultures were placed into a 37 °C water bath and, at the desired time point, samples were taken for plate count. The number of c.f.u. was determined after 48 h incubation at 37 °C. The survival of the mutant and wild-type cells in the absence of peroxide stress has been determined and did not reveal any difference. Each point is the mean of at least three experiments with duplicate plating, and statistical comparison of means was performed by using Student's t-test. Survival at any given time point was determined as the ratio of the number of c.f.u. after treatment to the number of c.f.u. at the zero time point.
General molecular methods.
PCR was carried out in a reaction volume of 25 µl with 5 µg chromosomal DNA of E. faecalis JH2-2 by using PCR Master Mix (Eppendorf). The annealing temperature was 5 °C below the melting temperature of primers and PCR products were purified by using a QIAquick kit (Qiagen). Plasmids were purified by using a QIAprep Miniprep kit (Qiagen). For Southern blotting, membrane-bound restricted chromosome was hybridized with 32P-labelled PCR probe. Membranes were then exposed to a storage phosphor screen (Packard Instrument Company) for 5 h. Genomic DNA extraction and other standard techniques were carried out as described by Sambrook et al. (1989).
Construction of the perR (previously named ef1585) insertional mutant.
To construct an insertional mutant with a disruption in the E. faecalis perR gene, a 270 bp internal fragment (obtained by PCR amplification using the E. faecalis JH2-2 chromosome as template; see Table 1 for the sequences of primers used) was ligated into the suicide vector pUCB300 that had been digested with SmaI. The resulting plasmid obtained after transformation of E. coli XL1Blue was used to transform competent cells of E. faecalis JH2-2. Erythromycin-resistant colonies were selected on agar plates containing 150 µg erythromycin ml1. Integration was verified by PCR and Southern blot analysis. The stability of the insertion was determined by plate counts with and without antibiotic.
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Real-time quantitative PCR.
Specific primers to produce amplicons of equivalent length (100 bp) were designed by using the Primer3 software (available at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and are listed in Table 1. The amplification efficiencies were between 95 and 100 %. Total RNA (2 µg) was reverse-transcribed by using Omniscript enzyme (Qiagen) according to the manufacturer's recommendations. A 5 µl aliquot of the resulting cDNA-synthesis reaction mixture (dilution, 102) was used for subsequent PCR amplification with the appropriate forward and reverse primers (1 µM final concentration) and the QuantitTect SYBR Green PCR mix (Qiagen). As control, sham cDNA-synthesis reactions that lacked reverse transcriptase, followed by PCR amplification, were carried out to identify RNA preparations contaminated by residual genomic DNA. Quantification of 23S rRNA levels was used as an internal control, because its expression was not altered under oxidative-stress conditions. Amplification, detection (with automatic calculation of the threshold value) and real-time analysis were performed twice and in duplicate with two different RNA samples by using the Bio-Rad iCycler iQ detection system.
The value used for comparison of gene expression in various strains and environments was the number of PCR cycles required to reach the threshold cycle (CT) (between 17 and 26). To relate the CT value back to abundance of an mRNA species, CT was converted to n-fold difference by comparing mRNA abundance in the JH2-2 wild-type strain (harvested in the middle of the growing phase or after 30 min in presence of 2·4 mM H2O2) with that obtained with the perR-mutant strain. The n-fold difference was calculated by the formula n=2x when CT mutant<CT JH2-2, and by n=2x when CT mutant>CT JH2-2, with x=CT mutantCT JH2-2. A value >1 reflects a relative increase in mRNA abundance compared with the wild-type; a negative value reflects a relative decrease. Statistical comparison of means was performed by using Student's t-test.
Survival assays in mouse peritoneal macrophages.
Survival of E. faecalis in mouse peritoneal macrophages was tested by using an in vivo/in vitro infection model as described previously (Gentry-Weeks et al., 1999; Verneuil et al., 2004b
). Briefly, E. faecalis perR mutant and JH2-2 were grown aerobically at 37 °C in brain heart infusion (BHI) broth for 16 h. Then, the bacteria were pelleted and resuspended in an adequate volume of PBS for injection. Male BALB/c mice (10 weeks old) were infected with 107108 cells of each strain by intraperitoneal injection. After a 6 h infection period, peritoneal macrophages were collected by peritoneal wash (2·5 ml PBS), centrifuged and suspended in Dulbecco's modified Eagle's medium containing 10 mM HEPES, 2 mM glutamine, 10 % (v/v) bovine fetal serum and 1x non-essential amino acids, supplemented with vancomycin (10 µg ml1) and gentamicin (150 µg ml1). The cell suspension was dispensed into 24-well tissue-culture plates and incubated at 37 °C under 5 % CO2 for 2 h. After exposure to antibiotics (i.e. 8 h post-infection) to kill extracellular bacteria, the infected macrophages were washed and triplicate wells of macrophages were lysed with detergent. After dilution with BHI broth, the lysates were plated on BHI agar to quantify the viable intracellular bacteria. The same procedure was performed at 24, 48 and 72 h post-infection. To assess their viability, macrophages were detached from tissue-culture wells with cell scrapers, stained with trypan blue and viable macrophages were counted with a haemocytometer. All experiments were performed five times and results were subjected to statistical analysis by using Student's t-test.
Mouse peritonitis model.
Testing of the JH2-2 and perR-mutant strains was performed as described by Teng et al. (2002). Briefly, the strains were incubated in BHI broth overnight at 37 °C under constant agitation. The cells were harvested by centrifugation, washed twice with ice-cold 0·85 % saline solution and resuspended in the same solution to reach a density of approximately 1·5x1010 c.f.u. ml1. The inoculum size was confirmed by plating onto BHI agar. Dilutions (two- to 10-fold) of the bacterial suspension, prepared in chilled 0·85 % saline solution, were used as inocula, after 10-fold-diluting them in 25 % sterile rat-faecal extract (SRFE, from a single batch) (Pai et al., 2003
). Outbred ICR female mice, 46 weeks old (Harlan Italy S.r.l.), were used. Mice were injected intraperitoneally with 1 ml of each bacterial inoculum made in 25 % SRFE, then housed five per cage and fed ad libitum. Mice were monitored every 3 h and the number of surviving mice was recorded. The LD50 was determined as described by Reed & Muench (1938)
. Survival curves were obtained by the KaplanMeier method and compared by log-rank test using the GraphPad Prism software (GraphPad Software Inc.). Comparisons with P values <0·05 were considered to be significant. All strains were tested more than once.
Complementation of the perR mutant.
To complement the perR gene in trans, a PCR fragment containing perR and its promoter (see Table 1 for the sequences of primers used) was cloned into the pCU1 plasmid (CmR) (Augustin et al., 1992
). The resulted vector (pPER) was then transformed into the perR mutant. In order to compare the phenotypes of the complemented strain and the wild-type, plasmid pCU1 was also introduced into the JH2-2 and perR-mutant strains. Survival assays towards H2O2 and in the mouse peritonitis model were carried out as described above.
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RESULTS |
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Effect of the perR mutation on survival within macrophages and on virulence of E. faecalis
It has been demonstrated that E. faecalis was able to persist for an extended period in mouse peritoneal macrophages (Gentry-Weeks et al., 1999; Verneuil et al., 2004b
). In order to assess whether the perR gene affected the capability of E. faecalis to resist killing by macrophages, the intracellular survival of bacterial cells was monitored (Fig. 3
). No significant difference in the number of viable cells was observed between wild-type and mutant strains.
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DISCUSSION |
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The PerR and Fur regulons of B. subtilis have recently been updated by transcriptome and proteome analysis, which confirmed that PerR plays a crucial role in the control of expression of key enzymes involved in the oxidative-stress response, such as ahpCF (encoding the alkyl hydroperoxide reductase), katA (encoding catalase) or mrgA (encoding a Dps-like protein) (Mostertz et al., 2004). In B. subtilis, it was suspected that the resistance of the perR mutant reflected the absence of repression of these genes (Bsat et al., 1998
). This was obviously not the case for E. faecalis. Despite the identification of sequences nearly identical to the consensus sequence Per box in the promoter regions of ahpCF and dps in E. faecalis, none of them exhibited a conserved inverted-repeat structure (Fuangthong & Helmann, 2003
). Expression of the ahpCF operon in E. faecalis cultivated in the presence of H2O2 is known to be under the control of the recently discovered transcriptional regulator HypR, involved in survival within macrophages as well as resistance to H2O2 challenge (Verneuil et al., 2004b
). Therefore, our results seemed to exclude a dual regulation with PerR. In S. pyogenes, PerR seems able to bind specifically to a single site of the ahpC promoter, but transcriptional analyses revealed that ahpC regulation is independent of PerR (Brenot et al., 2005
). This latter result has also been observed in our study. Another important characteristic that links S. pyogenes and E. faecalis and distinguishes them from the B. subtilis model is the absence of the general stress sigma factor
B. Further investigation leading to the identification of members of the PerR regulon in E. faecalis and S. pyogenes will probably provide more insights about novel effectors of protection against H2O2.
In our study, we showed that the number of viable intracellular bacteria in infected murine macrophages was not reduced in the perR mutant over the 72 h infection period compared with the JH2-2 wild-type strain of E. faecalis. It was hypothesized that the oxidative-stress response leading to the production of enzymes that inactivate ROS generated by the oxidative burst may explain the ability of bacterial cells to survive inside macrophages (King et al., 2000; Verneuil et al., 2004b
). The ability of the perR mutant to survive in murine macrophages may reflect its capacity to cope with the oxidative challenge. Nevertheless, our data obtained in the mouse peritonitis model demonstrated that PerR is absolutely required for full virulence potential. PerR has been shown to be also required for virulence of S. aureus strain 8325-4 in a murine skin-abscess model of infection (Horsburgh et al., 2001
). In this bacterium, the ability of PerR to sense H2O2 and to regulate antioxidant defence and iron storage may be important for coordinating a survival response (Horsburgh et al., 2001
). Furthermore, a perR mutant of L. monocytogenes was significantly affected in virulence for mice (Rea et al., 2004
). Given these findings, together with the resistance against H2O2 challenge, it is likely that the virulence defect of the perR mutant is due to the alteration of a response other than oxidative stress, such as metallic-ion uptake or storage.
In conclusion, this report brings to light the fact that the B. subtilis model concerning the oxidative-stress response is different from that of E. faecalis and that, in the latter, the transcriptional regulator PerR is important for virulence in the mouse peritonitis model. Further work is being undertaken to characterize the precise role of the PerR regulator in the E. faecalis stress response.
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ACKNOWLEDGEMENTS |
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Received 6 July 2005;
revised 15 September 2005;
accepted 19 September 2005.
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