Abstract
The GenBank/EMBL/DDBJ accession number for the fsr–gelE region sequence of E. faecium QS32 is FJ858146.
Enterococcus faecalis is the predominant species in human/animal-associated environments, and therefore the most studied species of this genus (Tannock & Cook, 2002). E. faecalis, together with Enterococcus faecium, are also the most frequently found species in dairy food products, with varying prevalence as a function of the country and cheese type (Aarestrup et al., 2002). E. faecalis is also responsible for up to 80 % of enterococcal-associated nosocomial infections, followed by E. faecium. One explanation for the over-representation of E. faecalis among clinical isolates may relate to its natural abundance, or to the presence of virulence factors (Hancock & Gilmore, 2000). Although harmless in healthy individuals, enterococcal clinical isolates become pathogenic in patients in intensive care units and in hospitalized patients with impaired immune systems. This has been associated with a variety of virulence factors carried by E. faecalis (Ogier & Serror, 2008).
One of the most studied E. faecalis virulence factors is gelatinase, a metalloprotease able to degrade several substrates, such as gelatin, casein, haemoglobin and other bioactive peptides, including E. faecalis sex pheromones (Kayaoglu & Orstavik, 2004). Gelatinase is encoded by gelE, which is in an operon with sprE, which encodes a serine protease (Qin et al., 2000). Its phenotypic expression requires the regulatory system encoded by the fsrABC operon (Lopes et al., 2006; Qin et al., 2000), recently renamed fsrABDC (Nakayama et al., 2006). fsr is homologous to the agr quorum sensing system of Staphylococcus aureus, and has also been implicated in virulence of E. faecalis independently of gelE (Bourgogne et al., 2006). The role of gelE and fsr loci in E. faecalis virulence has been demonstrated in different mammalian infection models (Mohamed & Murray, 2006), in the Caenorhabditis elegans nematode model (Sifri et al., 2002) and in the Arabidopsis thaliana plant model (Jha et al., 2005). More recently, gelatinase has been implicated in evasion of the immune system of the insect Galleria mellonella, suggesting that gelatinase may participate in virulence in this system (Park et al., 2007). Screening of both gelE and fsr in E. faecalis isolates from different origins revealed their dissemination in fermented milk products (Lepage et al., 2006; Lopes et al., 2006; Semedo et al., 2003). However, the roles for Fsr and gelatinase in the potential virulence of E. faecalis food strains have never been established. In the present study, two gelatinase-positive E. faecalis dairy strains carrying gelE and the complete fsr operon, and their respective isogenic gelE and fsrB deletion mutants, were tested for virulence in the G. mellonella infection model.
Bacterial strains, plasmids, and culture conditions.Bacterial strains used in this study are listed in Table 1. Enterococci were grown in M17 broth supplemented with 0.5 % (w/v) glucose (M17BGlu) or M17 agar supplemented with 0.5 % glucose (M17AGlu) at 37 °C without aeration. Escherichia coli strains were grown aerobically in Luria–Bertani broth or on LB agar at 37 °C. Antibiotics were used at the following concentrations: erythromycin, 30 µg ml–1 for Enterococcus faecalis and 150 µg ml–1 for Escherichia coli; ampicillin, 80 µg ml–1.
Table 1. Strains used in this study
General DNA techniques.
General molecular biology techniques were performed by standard methods (Sambrook et al., 1989). Restriction enzymes, polymerases and T4 DNA ligase were used according to manufacturers' instructions. PCR amplification was performed using a Biometra or Eppendorf thermocycler. When necessary, PCR products and DNA restriction fragments were purified with QIAquick purification kits (Qiagen) or Montage Life Science kits (Millipore). Plasmids were purified using the QIAprep Spin Miniprep kit (Qiagen). Electrotransformation of Escherichia coli and Enterococcus faecalis was carried out as described by Dower et al. (1988) and Dunny et al. (1991), using a Gene Pulser apparatus (Bio-Rad). Plasmid inserts were sequenced at Baseclear.
Construction of in-frame gelE and fsrB deletion mutants of strains QA29B and LSE4a.
Markerless gelE and fsrB deletion mutants of E. faecalis were constructed essentially as described by Brinster et al. (2007). Briefly, 5' and 3' flanking regions of fsrB and gelE were amplified from chromosomal DNA of each strain by PCR with primers OEF-232, OEF-233, OEF-234 and OEF-235, and OEF-236, OEF-237, OEF-238 and OEF-239, respectively (Table 2). The two cognate PCR fragments were fused by PCR using the external primers OEF232 and OEF235, and OEF-236 and OEF-239 for fsrB and gelE, respectively, and the resulting product was cloned into pGEM-T (Promega). The inserted PCR fragment was removed from its cloning vector by restriction enzymes and subsequently cloned into pG+host9 plasmid (Maguin et al., 1996), which was then electroporated into E. faecalis. The fsrB and gelE single- and double-crossover mutants were selected as described by Brinster et al. (2007). Successful targeted mutations of fsrB and gelE in strains LSE4a and QA29B were first identified by PCR screening and were confirmed by Southern blot analysis.
Table 2. Primers used in this study
Gelatinase activity assay.
The phenotypic assay of gelatinase activity was performed as described by Lopes et al. (2006). Briefly, Enterococcus strains were grown on agar plates containing 3 % (w/v) gelatin (Oxoid) and flooded with a saturated solution of ammonium sulfate (Merck). A transparent halo around cells indicated gelatinase activity.
G. mellonella mortality assay.
G. mellonella eggs were hatched at 25 °C, and the larvae were reared on bee's wax and pollen (Naturalim) until the last instars which were used for the infection experiments. Enterococcus strains were grown in M17BGlu and collected by centrifugation 1 h after they had reached stationary phase. Bacterial cells were washed with 0.9 % saline solution and stored as a dry, frozen pellet at –80 °C. Before inoculation, the frozen bacterial pellet was suspended in 1 ml saline solution and serial dilutions were plated on M17AGlu plates in order to determine the bacterial count of the pellet.
Groups of 20–30 G. mellonella larvae, starved for 24 h and weighing about 200 mg, were injected at the base of the last proleg with 10 µl each bacterial inoculum (∼2x108 cells ml–1) using a microinjector (KDS 100; KD Scientific) with a 1 ml syringe and 0.45x12 mm needles (Terumo). A control group of larvae received saline solution only. The size of the inoculum was confirmed by determining the number of c.f.u. on M17AGlu. Five infected larvae were kept per Petri dish, without food, at 37 °C and survival was monitored every 24 h for 2–5 days, depending on the experiment. Experiments were repeated at least three times. The mortality rate was compared using a two-tailed unpaired t-test at the 95 % confidence interval. Survival curves were constructed by the Kaplan–Meier method and compared by log-rank analysis (GraphPad Prism, version 4.0; GraphPad Software). P-values of <0.05 were considered statistically significant.
Sequence analysis of the fsr–gelE region of E. faecium QSE32.
PCR amplification of overlapping fragments of the fsr–gelE region was carried out using Expand High Fidelity DNA polymerase (Roche) and the primers indicated in Table 2. Sequencing was performed at Baseclear. The sequence was analysed using Vector NTI 10.3.0 (Invitrogen) and the final DNA sequence, and the deduced protein sequence were analysed using Vector NTI 10.3.0 (Invitrogen) and BLAST from the NCBI website ().
Increasing interest in using G. mellonella as a surrogate model to study virulence of various micro-organisms led us to compare virulence of enterococcal strains. We determined whether strains of enterococci other than E. faecalis GM could kill G. mellonella (Park et al., 2007). Mortality rates at 48 h post-infection are presented in Fig. 1. All E. faecalis strains tested (LN68, V583, LSE4a, OG1RF and QA29B) were able to kill between 60 and 98 % of G. mellonella larvae with inocula of about 2x106 c.f.u. The most virulent E. faecalis strains for G. mellonella were the clinical strain V583 and milk isolates LN68 and LSE4a. Maximum killing by LN68 was reached within 24 h (data not shown), indicating that this isolate was more rapidly lethal for G. mellonella. The clinical strain OG1RF and the cheese isolate QA29B were significantly less virulent with ∼72 and ∼60 % killing after 48 h, respectively. Interestingly, E. durans strain QN1 and E. faecium strain QSE32 did not kill G. mellonella larvae significantly. These data show that E. faecalis strains from both clinical and food origin have the ability to kill G. mellonella larvae, whereas E. durans and E. faecium species seem to be avirulent or of low virulence for G. mellonella under the conditions used.
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Gelatinase and Fsr affect E. faecalis virulence in G. mellonella
We asked whether the G. mellonella model could discriminate between the roles of gelE and fsrB in virulence, using OG1RF and isogenic strains TX5264 and TX5266 (kindly provided by B. Murray) deleted for gelE and fsrB, respectively. Larvae were infected with ∼2x106 c.f.u. OG1RF, TX5264 and TX5266. As shown in Fig. 2, the three strains have similar killing rates of G. mellonella at 48 h. However, killing was significantly delayed for larvae injected with the ΔgelE strain (TX5264); 50 % of the larvae were dead after 24 h, compared to 80 % of those injected with OG1RF or TX5266 (P<0.05). This result indicates that gelE, but not fsrB, contributes to OG1RF virulence in the G. mellonella infection model.
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To study the role of gelE and fsrB in the potential virulence of E. faecalis food isolates, we collected strains QA29B and LSE4a in two distant areas of cheese production in Portugal (Semedo et al., 2003; Lopes et al., 2006). According to PFGE and MLST analysis, these two isolates are not genetically related (data not shown). Besides the fsrABDC and gelE-sprE operons, both strains carried the virulence genes agg, esp and efaAfs, none of which was haemolytic (results not shown). We successfully generated independent in-frame gelE and fsrB deletion mutants by allelic exchange in E. faecalis QA29B and LSE4a, as described by B. Murray and co-workers (Qin et al., 2001; Sifri et al., 2002). As expected, the resulting ΔfsrB and ΔgelE deletion mutants failed to produce detectable gelatinase in a standard plate assay (data not shown). Next, their virulence was examined using the G. mellonella infection model. As for OG1RF, inactivation of the gelE gene in the two strains significantly decreased killing of G. mellonella larvae; 65 and 32 % of larvae infected with the gelE mutants were dead after 48 h, compared to 88 and 60 % of those infected with LSE4a and QA29B, respectively (P<0.05). Moreover, inactivation of fsrB in LSE4a and QA29B clearly reduced virulence compared to the isogenic WT strains (Fig. 3). After 48 h post-infection, the QA29B ΔfsrB mutant killed 15 % of the G. mellonella larvae, versus 60 % killing by the isogenic WT strain. Thus, in contrast to results in OG1RF, fsrB appears to be implicated in infection by certain E. faecalis isolates.
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Sequence analysis of the E. faecium QSE32 fsr–gelE region
To check whether the low virulence of the E. faecium QS32 strain in G. mellonella could result from the absence of a functional Fsr system, we sequenced the entire fsr–gelE region of this strain. The gene organization was identical to E. faecalis V583, and the sequence shared 98 % identity at both the nucleotide and amino acid sequence level with gelE of E. faecalis V583 (data not shown). Therefore, we concluded that the Fsr system of E. faecium QS32 is most probably functional and that gelE conferred gelatinase activity. The low virulence of E. faecium QS32 seems to be independent of the functionality of the fsr–gelE region. Simple invertebrates, the nematode C. elegans and insects Drosophila melanogaster and G. mellonella have recently attracted interest as models for screening of virulence factors of pathogenic microbes, including E. faecalis, or for elucidating their effects in the host (Maadani et al., 2007; Park et al., 2007; Schneider et al., 2007). Although adaptive immunity is unique to vertebrates, the innate immune response seems to be well conserved between vertebrates and invertebrates. In contrast to the nematode, which lacks the cellular immune response involved in phagocytosis of bacteria (Fares & Greenwald, 2001), insects have both cellular and humoral responses, making them attractive models to study bacteria–host interactions (Vallet-Gely et al., 2008). Although the G. mellonella infection model has been efficiently used to identify virulence factors in Gram-negative bacteria and in fungi (Schell et al., 2008; Mylonakis et al., 2005; Brennan et al., 2002; Choi et al., 2002; Cotter et al., 2000, Xu et al., 1991), this model has been mainly used to characterize virulence factors in Bacillus cereus and Bacillus thuringiensis. These bacteria are virulent to G. mellonella by oral infection (Salamitou et al., 2000; Fedhila et al., 2006) and by injection into the haemocoel (Bouillaut et al., 2005). A recent study demonstrated killing of G. mellonella larvae by at least one E. faecalis strain isolated from larval cadavers (Park et al., 2007). In the present study, we used the G. mellonella model to compare virulence of several Enterococcus strains from food and clinical origins. Using this simple infection model, we demonstrated the efficacy of the G. mellonella model, and establish for the first time that gelatinase and Fsr virulence factors of E. faecalis contribute to the virulence of food isolates.
E. durans and E. faecium strains were relatively avirulent or of low virulence, killing only 0–3 % of larvae, whereas E. faecalis strains had killing rates of 60–98 %. Although we used different strains of E. faecalis and E. faecium in this study, our results with G. mellonella correlate well with the C. elegans model, for which E. faecium strains were less virulent than E. faecalis strains (Garsin et al., 2001; A. Alberti, personal communication).
Attenuated virulence of gelE-deficient E. faecalis strains in the G. mellonella infection model is consistent with results in mammal and nematode models (Engelbert et al., 2004; Sifri et al., 2002; Qin et al., 2000). This result correlates with the recent finding that gelatinase degrades cecropin, an antimicrobial peptide important for the immune system in the haemolymph of larvae of G. mellonella (Park et al., 2007).
Since inactivation of gelE does not abolish E. faecalis virulence, other factors might be involved in G. mellonella virulence. Although SprE protease is still expressed in gelE-inactivated strains, it does not seem to have insecticidal activity (Park et al., 2007). Based on the high genome diversity between E. faecalis isolates (Bourgogne et al., 2008; Lepage et al., 2006), it is likely that the strains used in this study carry other factors involved in virulence in G. mellonella or differentially express them. Interestingly, the OG1RF genome encodes a putative extracellular protease (OG1RF_0194), which remains to be characterized (Bourgogne et al., 2008).
The major regulatory system of gelatinase, the fsr operon, is among the most probable candidates to be involved in virulence in G. mellonella. While no significant difference in virulence was observed between larvae inoculated with the OG1RF and the fsrB isogenic mutant, we found that fsrB contributes to virulence in G. mellonella in the two food isolates studied. The result with OG1RFΔfsrB is in agreement with a previous study in which fsrB inactivation in OG1RF did not modify virulence in a rat endocarditis model (Singh et al., 2005). The authors proposed that residual production of gelatinase was sufficient to allow induction of endocarditis. In this case, however, residual gelatinase production would have had to be strongly induced in vivo, especially as virulence was evaluated at just 24 h post-infection. As proposed above, fsrB may directly or indirectly regulate other factors important for virulence in OG1RF. Since inactivation of fsrB in LSE4a and QA29B food isolates appears to be more relevant for virulence, we propose that OG1RF has additional factors that contribute to its pathogenic potential, as suggested by its intrinsic virulence in a mouse peritonitis model (Bourgogne et al., 2008), or that these food isolates lack factors important for virulence in such a model. Taken together, present results and previous work suggest that the genetic background of the strain may be as important as the infection or host model used to assess virulence. Singh et al. (2005) used an endocarditis model to demonstrate that a gelE sprE double mutant significantly decreased the evolution of endocarditis compared to infection by WT OG1RF; the fsrB mutant was not significantly attenuated compared to wild-type OG1RF. Using different models, i.e. rabbit endophthalmitis, mouse peritonitis, C. elegans and G. mellonella (this study), the fsrB mutant strain of OG1RF was more attenuated than the double protease mutant (Engelbert et al., 2004; Sifri et al., 2002). An important conclusion of this work is that the role of virulence genes studied in one model cannot always be extrapolated to other models.
Transfer of virulence or antibiotic resistance genes has been observed in vitro (Eaton & Gasson, 2001) and in vivo (Mater et al., 2005; Lester et al., 2006). Although our finding does not preclude that enterococcal food isolates will be virulent in other infection or animal models or pathogenic for humans, it indicates the in vivo activity of the gelatinase and Fsr present in food isolates, and calls for surveillance in monitoring virulence traits in food strains which represent a potential reservoir for virulence genes.
In summary, we used the G. mellonella infection model as a surrogate virulence model to compare virulence of three Enterococcus species, and E. faecalis isolates and isogenic deletion mutants. Our data show that fsrB and to a lesser extent gelE significantly contribute to the virulence of E. faecalis food isolates. This simple animal model may provide insights for risk assessment of food isolates.
The authors thank B. Murray for kindly providing us with strains TX5264 and TX5266, Christophe Buisson for his help in setting up the G. mellonella virulence assays at UBLO, and A. Alberti for permitting citation of unpublished data. F. G. is grateful to Fundação para a Ciência e Tecnologia for grant SFRH/BD/18757/2004. The work of the Portuguese group was funded by Fundação para a Ciência e a Tecnologia (FCT) through Project grant PDC/CVT/67270/2006, co-financed through FEDER. The work of the French group was funded by the Institut National de la Recherche Agronomique. This work was supported by the bilateral cooperation project Portugal/France (GRICES/EGIDE, Pessoa program, 2006/07).Edited by: T. J. Mitchell
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Received 19 May 2009; accepted 19 August 2009.
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