Target gene selection, selection of a plasmid-free derivative of E. faecalis V583 and production of a library of targeted mutants

Analysis of the E. faecalis V583 genome led to the selection of 223 putative virulence gene candidates encoding i) surface exposed proteins such as cell wall-anchored proteins, lipoproteins and transporters and proteins involved in surface polysaccharides or lipopolysaccharides synthesis; ii) putative transcriptional regulators; iii) proteins of unknown function identified as specific to E. faecalis; iv) enzymes involved in carbohydrate metabolism; v) proteins involved in stress resistance; or vi) proteins similar to known virulence factors, such as hyaluronidase, internalin-like protein, and proteases. A percentage of 85 and 15 of the selected genes are part of the core genome or located on mobile genetic elements, respectively, including 17 genes that are present in the pathogenicity island [23].

To avoid problems related to the presence of replicating plasmids we selected a plasmid-cured derivative of strain V583, as described in the Materials and Methods section, named VE14089. This strain has not undergone major DNA rearrangements, as indicated by DNA microarray analysis, pulsed field gel electrophoresis, Southern blotting, long-range PCR and DNA sequencing analyses, with one exception: a 20.5-kb region of pTEF1 (from efa0063 up to efa0006) is integrated between chromosomal genes ef3209 and ef3210, and is flanked by two copies of IS1216. As expected, the cured strain was sensitive to erythromycin and gentamicin and related macrolides and aminoglycosides resulting from plasmid loss, but remains as resistant as the wild-type strain V583 to other antibiotics tested. We compared both strains for several phenotypes. The cured strain demonstrated a growth defect in comparison to the parental strain which was more pronounced under aerobic than anaerobic conditions. However, the cured strain had an only minor reduced ability to cause lethality in the Galleria mellonella virulence model (Fig. S1), indicating that strain VE14089 remained relevant for E. faecalis genetic studies on virulence.

The system for generating targeted chromosomal insertions by homologous recombination is based on the conditional replication of two plasmids [26]. Plasmid pVE14218 (RepA−) is a cloning vector with a tetracycline marker used to clone internal fragments of each gene to be inactivated. Plasmid pG+host3 is a thermosensitive helper plasmid that provides the RepA replication protein to pVE14218 at permissive temperature. The pVE14218 derivative plasmids were integrated in the targeted loci as described in Materials and Methods. Thus, from a total of 223 gene disruptions attempted on candidates, 177 isogenic V583-derived mutants were successfully obtained, which targeted genes in 14 of the 20 gene role categories as defined by the JCVI CMR database (Table S1). Unsuccessful mutagenesis could be explained either by an essential role of the targeted genes in the conditions used and/or their poor recombinogenic potential. One-third of the mutants target functions involved in cell envelope and in transport. The whole mutant library was then submitted to several test conditions. The target genes were assigned as affecting four categories of phenotypes related to host adaptation, namely: (i) stress and antibiotic resistance, (ii) resistance to opsonophagocytosis, (iii) adherence to Caco-2 epithelial cells and (iv) virulence in a Galleria mellonella insect model.

Mutant growth behavior, fitness in stressing environment and resistance to antibiotics

Growth behaviour and fitness under oxidative stress conditions have been tested for the entire E. faecalis mutant library using microtiter plate assays. The analyses evidenced more or less important growth defects under non-stress condition in 10 of the mutants (Table S1). The strongest effects were observed in strains with mutations in genes encoding putative glycosyltransferases (EF2167, EF2170 and EF2196). The majority of the strains (125) showed no significant differences in sensitivity to exposure to H₂O₂ compared to the parental strain. The 52 mutants that were more sensitive to H₂O₂ were grouped in three categories with respect to the degree of their sensitivities (slightly, highly and extremely sensitive) to the stressing agent (Table S2). The most sensitive mutants were affected in genes encoding transcriptional regulators (EF0073 and EF1599) belonging to the Cro/CI family, EF0107 present in the Arginine deiminase operon [27] and EF1525, one of the three Fur-paralogs, proteins implicated in envelope metabolism (EF1172, EF1746, EF2170, EF2180, EF2196, EF2198), a paralog of the virulence factor Gls24 (EF0604 encoded by the PAI), an ABC transporter (EF1408) and the antioxidant activity and virulence factor MsrB (EF3164) [28]. Of note, no mutant more resistant to H₂O₂ than the parental strain was identified.

Antibiotic resistance of the VE14089 strain and all its isogenic mutants was determined for 34 antibiotics that, together, cover all known mechanisms of antibiotic action (Table S3). More than 88% of the mutants showed no modification of their antibiotic resistance, including 7 mutants in genes annotated as having a role in drug resistance (ef0420, ef0785, ef1042, ef1814, ef1943, ef2068 and ef2772; see Table S3). Of the 21 mutants displaying a modified antibiotic resistance profile, increased sensitivity to ceftriaxone was observed in a mutant with an interrupted npr-gene (ef1211). This gene encodes the well characterized NADH peroxidase of E. faecalis and its main cellular role is to protect cells against internal metabolically-derived oxidative stress [29]. Two mutants putatively affected in the cell envelope were more sensitive to fusidic acid (ef1746 and ef2181); this might relate to changes in cell permeability. Five mutants showed increased resistance to amoxicillin and 2 to chloramphenicol; the targeted genes are predicted to have roles in signal transduction (n = 3), in cell envelope (n = 2), in cellular processes (n = 1) and in protein fate (n = 1). Among the low number of mutants that showed altered antibiotic sensitivity, we noted a significant enrichment of genes affected in the categories “cell envelope” and “signal transduction” compared to the representation of these two categories in the entire mutant collection (Table 1).

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Table 1. Percentage of mutants in each JCVI role category.

https://doi.org/10.1371/journal.pone.0029023.t001

Resistance to opsonophagocytosis

Opsonic killing of bacteria constitutes a reliable in vitro surrogate of protective immunity in vivo and occurs in the presence of specific antibodies, complement, and phagocytes [30]. Different structures on the bacterial surface can be the target of opsonic antibodies and the loss of such structures will render the mutant more resistant to opsonic killing. Specific mechanisms of the bacteria to withstand the protective immune response of the host can be observed in the same assay by identifying mutants that become hyper-susceptible to opsonic killing when one of these factors is eliminated [31].

Sera raised against E. faecalis V583 were tested for their opsonic killing activity against the various mutant strains. From 177 mutants tested, only 3 were more resistant to opsonophagocytosis while 11 were more sensitive (1 with complement alone and 10 without antiserum, see Table S4). Among these, 10 (9 sensitive and 1 resistant mutant) carried mutations in genes predicted to have a role in the cell envelope. In contrast to the VE14089 strain, most of these mutants were efficiently killed by a combination of polymorphonuclear neutrophils (PMNs) with complement, while the additional effect of antibody opsonization was negligible. Two mutants targeted in genes probably involved in wall-teichoic acid synthesis (ef1172, ef1173) were susceptible to complement and PMNs. Three mutants displayed significantly higher resistance to opsonic killing compared to the wild-type. Two were mutated in adjacent transcriptional regulator genes ef0600–ef0601, which are part of the pathogenicity island of strain V583 enriched in CC2 complex [32] and both encode regulators of the TetR family of transcriptional regulators. One strain mutated in gene ef2490 (cpsF) was also more resistant to opsonic killing. CpsF is responsible for the sero-specificity of serotype C to which VE14089 as a derivative of V583 belongs [33]. It was shown previously that removal of CpsF changes this strain to sero-group D thereby abrogating the activity of anti-serotype C antibodies [34]. Disruption of capsule genes ef2491 (cpsE) and ef2492 (cpsD) did not increase susceptibility to killing by complement and PMNs. This result may be explained by the fact that LTA unmasked by capsular polysaccharide also protects E. faecalis from opsonic killing [33], [34], [35].

Adhesion to Caco-2 epithelial cells

Non-invasive bacteria that colonize the gastrointestinal tract, such as entero-hemorrhagic Escherichia coli (EHEC), adhere tightly to epithelial cells and promote pedestal formation [36]. Although there is no consensus about the capacity of enterococci to invade epithelial cells, adherence to such cells is likely to contribute to enterococcal pathogenesis. To evaluate this ability in the 177 E. faecalis mutants, we used a 96-well microtiterplate screening model for adherence of E. faecalis to cells of the human colon carcinoma Caco-2 epithelial cell-line. This cell-line has already proven to be a valuable model for testing adherence of E. faecalis to epithelial cells [37], [38].

Eighty-four percent of the mutants were not modified with respect to their adhesion to the intestinal epithelial cells. Four mutants displayed a decreased ability to adhere (Table S5); the interrupted genes have predicted roles in signal transduction (PTS system; ef0553), in cell envelope (ef0252) and in cellular processes (ef1076). In one of the adhesion-defective mutants, a hypothetical protein (EF0876) was affected that carries a domain found in the streptococcal regulator Mga, which is known to regulate production of adhesins in group A streptococci [39]. Interestingly, 24 mutants showed increased adhesion to the epithelial cells, 9 of which carried mutations in genes with a predicted role in the cell envelope, such as polysaccharide biosynthesis or degradation.

Virulence in the Galleria mellonella insect model

Increasing interest in using G. mellonella as a surrogate model to study virulence of various microorganisms led us to compare the virulence of the 177 mutant strains with that of the parental strain. One third of the mutants (n = 57) were modified in virulence in this model: 49 showed decreased virulence and 8 were more virulent (Fig. S2, Table S6). Most of the mutations of both mutant categories are in genes potentially involved in cell envelope metabolism, regulatory functions and transport. Eight genes encode proteins of unknown function, including 3 of which the products are predicted to locate at the cell envelope (ef1420, ef2250 and ef2276). A slight enrichment in the category “Regulatory Function” is observed (Table 1). The diversity of the regulator classes affecting E. faecalis virulence indicates that this process relies on a large number of functions in this model. Bacterial virulence in insects such as the Lepidoptera G. mellonella lies in the ability to resist the innate immune response, which includes the action of lysozyme, antimicrobial peptides and reactive oxygen and nitrogen species such as hydrogen peroxide and nitric oxide [40]. Among the attenuated mutants, some carry mutations in genes well known to participate in resistance to antimicrobial peptides of E. faecalis or other bacteria. These include genes involved in the dealanylation of LTA (ef2746 and ef2748), in the biosynthesis of teichoic acids (ef1172 and ef1175), in membrane phospholipid synthesis (ef1027, an mprF-like gene) or potential efflux pumps (ef0785, ef1760, ef1814 and ef2068).

Combined phenotypic analysis of the E. faecalis mutant library

All the data collected for each of the mutants constructed in this study were entered in an easy-access phenotypic database (http://genome.jouy.inra.fr/phenotype/). From the 177 mutant strains, more than 1/3 showed no detectable phenotype and 36% (n = 64), 19% (n = 34) and 6% (n = 11) were tested positive for one, two and more than two phenotypes, respectively. A Venn diagram was constructed according to the intersection of predicted genes in the 5 phenotyping tests, with the exception of the increased adhesion phenotype (Fig. 1). In this analysis, a handful of phenotype combinations were observed, the most frequent being the combination of resistant to H₂O₂ and virulent in G. mellonella. Noticeably, the majority (13/14) of the mutants affected in the opsonophagocytosis assay displayed at least another phenotype, with sensitivity to H₂O₂ and virulent in G. mellonella being the most prevalent. Furthermore, half of the mutants with modified antibiotic resistance are affected in virulence. One mutant exhibited four phenotypes (ef1746) while 8 mutants displayed three phenotypes (ef1027, ef1172, ef1705, ef2170, ef2196, ef2198, ef2167, ef2992). The affected genes mostly encode proteins with predicted roles in cell envelope metabolism, in polysaccharide synthesis or degradation, or in transport or substrate binding.

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Figure 1. Venn diagram representation of the results of the five phenotypic tests performed on the mutants obtained in this study.

Only genes that are common to at least two phenotypes are listed. The increased adhesion phenotype was not included due to difficulties in constructing the Venn diagram with these data. Stars indicate mutants with increased adhesion phenotype. ^§ The 10 mutants combining phenotypes for H₂O₂ stress and virulence are not shown due to lack of space in the figure. They were inactivated in ef0073, ef0465, ef0814, ef1212, ef1420, ef1493, ef1741, ef1760, ef1798 or ef2442.

https://doi.org/10.1371/journal.pone.0029023.g001

Bacterial strains, plasmids and culture conditions

Enterococcus faecalis V583 is a vancomycin resistant strain [25] harboring three plasmids. Nucleotide sequences of the chromosome and plasmids are available at http://www.tigr.org. For technical reasons we conducted the study with a plasmid cured strain. Plasmid curing was carried out by a procedure combining three conditions known to lead to plasmid loss: the use of novobiocin [77] and growth at 42°C in a medium lacking its buffering component [78]. Strain V583 was cultured for 96 hours under these conditions and the procedure was repeated several times. The successive strains obtained were: V583 EryS (carrying pTEF2, and pTEF1 free of the EryR cassette), V32 (containing pTEF1 free of the EryR cassette), and V19, renamed in this study VE14089 (free of replicating plasmids). A transcriptional microarray was performed on strain VE14089 using the Affymetrix GeneChip, SLARE1 [79], the details of transcriptome experiments and analysis are described as additional information in Text S1. Microarray data have been deposited in the ArrayExpress public database at http://www.ebi.ac.uk/ arrayexpress with the series accession number E-MEXP-3068. The microarray analysis revealed that genes efA0063 to efA0006 from plasmid pTEF1 were still present in this strain but PCR analysis at the borders of this region demonstrated that a circular form of the plasmid was not present. To identify the site of chromosomal integration, pulsed-field gel electrophoresis of NotI, SfiI, or SmaI digested VE14089 genomic DNA and subsequent Southern hybridization was performed as described previously [80]. An intragenic probe for efA0078 was amplified using the primers, efA78F 5′-GGACTTGATCCATTGAAGAC-3′, and efA78R 5′-GGTAAAACCTGACTATTGCC-3′, and radiolabeled by incorporation of [α-³²P]dCTP-labelled deoxynucleotides (Perkin Elmer) using Ready-To-Go DNA labelling beads (GE). Southern blot analysis revealed a chromosomally integrated plasmid-fragment on two possible SmaI restriction fragments of 105 kb or 110 kb. To determine the site of integration, long-range PCRs were performed in the 110-kb region, which allowed delimiting the insertion within an intergenic region between the genes ef3209 and ef3210. Finally, the precise integration site of the genes efA0063 to efA0006 was determined by sequencing.

E. coli was cultivated at 37°C in LB broth (Difco) in a glass tube with shaking. E. faecalis strains were grown in M17 broth (Difco) supplemented with 0.5% glucose (GM17), or on GM17 agar plates at an appropriate temperature. E. coli VE14188 (dam⁻dcm⁻ RepA⁺) was used to amplify plasmids to be introduced in E. faecalis. To maintain the conditionally replicating plasmid pVE14218 and derivative plasmids, tetracycline was used at 12.5 µg/mL for E. coli and at 10 µg/mL for E. faecalis. To maintain plasmid pGhost3 (RepA^Ts), chloramphenicol was used at 10 µg/mL.

Transformation of E. coli and of E. faecalis

E. coli VE14188 and E. faecalis were transformed by electroporation using a BioRad Gene Pulser apparatus according to the method of Dower et al. [81] and Holo and Nes [82], respectively. Briefly, 40 µL of electrocompetent cells of E. faecalis VE14412 were mixed with 2 µL of plasmid DNA (100 ng). After electric shock, 1 mL of SM17MC medium (GM17 with 0,5 M saccharose, 10 mM MgCl₂ and 10 mM CaCl₂, pH 6,8–7,0) were added and the cell suspension was incubated at 28°C for 2 h without shaking. One hundred µL of 10⁰ and 10⁻¹ dilutions were spread on GM17 agar plates with 10 µg/mL tetracycline and incubated at 28°C for 48 h.

Extraction of plasmid DNA from E. coli and of total DNA from E. faecalis

DNA of pVE14218 was purified using cesium chloride gradient centrifugation [83] and cognate derivatives plasmids were purified according to the protocol of the QIAprep Spin Miniprep Kit (QIAGEN).

Total DNA from E. faecalis was extracted using a modified protocol described by Brinster et al., 2007 [84]. Bacterial cells were pelleted by centrifugation at 4°C, 8,000 rpm for 3 min. The cell pellet was washed once in 1 ml of TES (50 mM Tris, pH 7.5; 20% sucrose, 5 mM EDTA), resuspended in 300 µL of TES+lysozyme (5 mg/mL) and incubated for 30 min at 37°C. Protoplasts were mixed with 300 µL of saline solution (150 mM NaCl; 10 mM EDTA, pH 8) and lysed by the addition of 20 µL of 20% SDS. One volume of phenol/chloroform/isoamyl alcohol 25∶24∶1 (PCI) was added and the suspension was homogenised. DNA was separated from cellular debris by centrifugation for 10 min at 14,000 rpm at 10°C. The PCI step was repeated once or twice on the aqueous phase containing soluble DNA. Subsequently, the DNA was precipitated by addition of one volume of isopropanol. After centrifugation at 14,000 rpm, 10°C for 10 min, the supernatant was discarded, the DNA pellet was washed in cold 70% ethanol, centrifuged for 5 min and then air-dried. DNA was finally resuspended in 50 µL of TE+RNAse (100 µg/mL), incubated at least 30 min at 37°C. and stored at −20°C.

Generation of integrants of E. faecalis by thermal shock

Single colonies of E. faecalis containing both pGhost3, which provides functional RepA^Ts, and the conditionally replicating pVE14218-derivative were cultivated in 50 ml of GM17 (without antibiotic) under static conditions for 2 h at 28°C. Then, the cultures were transferred to the non-permissive temperature for pGhost3 (42°C) for two hours to inhibit plasmid replication. Next, serial dilutions were plated on GM17 containing 10 µg/ml tetracycline (to select for plasmid integrants) and on GM17 (to evaluate the integration rate) and incubated for 24 h at 42°C. Thus, chromosomal integration of the specific pVE14218 derivative and loss of pGhost3 was selected for. Integration at the targeted locus was validated by Southern Blot analysis using the DIG DNA labeling and Detection kit (Roche) and/or PCR.

H₂O₂ stress resistance assay

Two ml of an overnight culture of E. faecalis was harvested, and cells were suspended in 10 ml of GM17 medium. A volume of 180 µl of the resuspended culture was added to each well of a microtiter plate to which 20 µl of 0 mM, 5 mM, 6 mM or 7 mM H₂O₂ was added (thus giving 1/10^th final concentration; plates were incubated for 2 h at 37°C without agitation. Twenty µl of culture from each well was then used to inoculate another microtiter plate containing in each well 180 µl of GM17. Plates were incubated at 37°C in a plate reader (BioRad Model 680), and growth was monitored for 24 h by for OD₆₀₀ every 30 min after plate agitation for 10 s.

Antibiotic resistance assay

Resistance to 34 antibiotics, covering different classes. Both intrinsic and acquired resistances were assessed, at first using disk susceptibility tests. Screens were done according to the recommendations of the disk providers. Subsequently, the minimal inhibitory concentration (MIC) was determined for each antibiotic using either the microdilution, agar dilution or E-test method and results were interpreted according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS). The MICs were determined for mutants showing relevant antibiotics phenotypes by the disk diffusion assays. MIC values of mutant that were at least 2-fold different from that of the parent strain VE14089 were considered significant.

Resistance to opsonophagocytosis

Rabbit sera raised against whole bacterial cells and known to contain opsonic antibodies against E. faecalis V583 were tested for their opsonic killing activity against E. faecalis reference strains as described previously [31]. Polymorphonuclear neutrophils (PMN) were freshly prepared from human blood collected from healthy adult volunteers and percent killing was calculated by comparing the colony counts in the inoculum (i.e., viable counts at time 0 – T0) to the colony counts after a 90-minute incubation at 37°C (T90), using the following formula: ((mean cfu at T0 – mean cfu at T90)/(mean cfu at T0))×100. The killing activity of sera in combination with complement and PMN was compared to control samples with human sera and complement but without PMN. While the VE14089 strain was killed only by a combination of PMNs, complement, and antibodies, we observed the following differences in some of the mutants: Killing by complement alone (Complement Sensitivity: Co-Sens), Killing by complement and PMNs but without antiserum (Serum Independent: Ser-Ind), and a general higher resistance to killing with all three components (Higher Resistance, High-Res).

Adhesion of E. faecalis to Caco-2 cells

The Caco-2 cell line from human colon adenocarcinoma was obtained from the cell bank of the Centro de Investigaciones Biologicas (CIB-CSIC, Madrid, Spain). Caco-2 cells were cultured at 37°C under 5% CO₂ in 24-well plastic microplates (TTP) in RPMI medium 1640 (Invitrogen) containing 10% Foetal Bovine Serum (Invitrogen), without antibiotics, until the cells were grown to ±95% confluence. The cell monolayers were washed with sterile Phosphate Buffered Saline without Ca²⁺ and Mg²⁺ (Biowhitaker). The RPMI medium was removed, and fresh RPMI medium was added before infection. The bacterial inoculum consisted of a suspension in RPMI without Foetal Bovine Serum of bacteria obtained from 1 ml (∼10⁹ bacteria) of an overnight culture in 5 ml BHI at 37°C, centrifuged during 5 min at 6000 rpm. Briefly, bacterial suspensions were added to ±95% confluent Caco-2 monolayers at a multiplicity of infection (MOI) of 50∶1. Infected cell monolayers were centrifuged for 5 min at 500 rpm at room temperature and incubated for 2 h at 37°C under 5% CO₂. They were then washed 3 times with PBS. A 300-µl volume of triton X-100 (Sigma) in 1% PBS was added per well. Homogenates were collected and serial dilutions were plated on BHI containing 10 µg/µl tetracycline for mutants and on BHI for controls (strains V583 and VE14089) and incubated for 24 h at 37°C. Colonies were counted to assess adherent/invasive colony forming units (CFU). Experiments were performed three times, in duplicate.

Galleria mellonella killing assay

E. faecalis mutants were grown in GM17 containing 1 µg/ml of tetracycline and collected by centrifugation 1 h after they had reached the stationary phase. The VE14089 strain was grown in GM17. Bacterial cells were washed twice with 0.9% saline solution and stored as a dry frozen pellet at −80°C. At least two days before inoculation, the frozen bacterial pellet was suspended in 1 ml of saline solution and serial dilutions were plated on GM17 agar plates in order to determine the bacterial count of the pellet.

Groups of 30 last instar G. mellonella larvae, weighing about 200 mg each, were injected at the base of the last proleg with 10 µl of a bacterial inoculum (∼4×10⁸ cells per ml) using a microinjector (KDS 100, KD Scientific Inc.) with a 1-ml syringe and 0.45- by 12-mm needles (Terumo). A control group of larvae received 10 µl of a saline solution only. The inoculum size was confirmed by determining the number of CFU on GM17 agar. The infected larvae were kept, five per Petri dish, at 37°C and survival was monitored every 24 h for 2 to 5 days depending on the experiment. Survival obtained with mutant strains might be overestimated if gene reversion occurs, although in view of the short time of the assays, we consider this an unlikely event. Survival curves were constructed by the Kaplan-Meier method and compared by log-rank analysis (GraphPad Prism, version 4.0; GraphPad Software, Inc.). Whereas P values of >0.05 were considered not significantly different from the VE14089 (nsd), P values of <0.05 were considered statistically significant with the following classification: 0.005<P<0.05 (decreased or increased), 0.0005<P<0.005 (decreased+ or increased+) and P<0.0005 (decreased++ or increased++).

Phenotypic data base construction

Information on the mutant construction (primers and plasmids used) and on the results of the screening was added in the database FAECALVIR provided by Migale (INRA, Jouy-en-Josas, France) and implemented by all the partners of the study (http://genome.jouy.inra.fr/phenotype/).

Ethics statement

The animal welfare committees of the University of Freiburg (Regierungspräsidium Freiburg Az 35/9185.81/G-07/15) approved animal experiments. Rabbit experiments were performed in compliance with the German animal protection law (TierSchG). The animals were housed and handled in accordance with good animal practice as defined by FELASA and the national animal welfare body GV-SOLAS.

Human PMNs were obtained from healthy volunteers in accordance with a protocol approved by University of Freiburg Institutional Review Board for Human Subjects (approval number 116/04). Written informed consent was obtained from all human volunteers.