A Genomic Virulence Reference Map of Enterococcus faecalis Reveals an Important Contribution of Phage03-Like Elements in Nosocomial Genetic Lineages to Pathogenicity in a Caenorhabditis elegans Infection Model
ABSTRACT
In the present study, the commensal and pathogenic host-microbe interaction of Enterococcus faecalis was explored using a Caenorhabditis elegans model system. The virulence of 28 E. faecalis isolates representing 24 multilocus sequence types (MLSTs), including human commensal and clinical isolates as well as isolates from animals and of insect origin, was investigated using C. elegans strain glp-4 (bn2ts); sek-1 (km4). This revealed that 6 E. faecalis isolates behaved in a commensal manner with no nematocidal effect, while the remaining strains showed a time to 50% lethality ranging from 47 to 120 h. Principal component analysis showed that the difference in nematocidal activity explained 94% of the variance in the data. Assessment of known virulence traits revealed that gelatinase and cytolysin production accounted for 40.8% and 36.5% of the observed pathogenicity, respectively. However, coproduction of gelatinase and cytolysin did not increase virulence additively, accounting for 50.6% of the pathogenicity and therefore indicating a significant (26.7%) saturation effect. We employed a comparative genomic analysis approach using the 28 isolates comprising a collection of 82,356 annotated coding sequences (CDS) to identify 2,325 patterns of presence or absence among the investigated strains. Univariate statistical analysis of variance (ANOVA) established that individual patterns positively correlated (n = 61) with virulence. The patterns were investigated to identify potential new virulence traits, among which we found five patterns consisting of the phage03-like gene clusters. Strains harboring phage03 showed, on average, 17% higher killing of C. elegans (P = 4.4e−6). The phage03 gene cluster was also present in gelatinase-and-cytolysin-negative strain E. faecalis JH2-2. Deletion of this phage element from the JH2-2 clinical strain rendered the mutant apathogenic in C. elegans, and a similar mutant of the nosocomial V583 isolate showed significantly attenuated virulence. Bioinformatics investigation indicated that, unlike other E. faecalis virulence traits, phage03-like elements were found at a higher frequency among nosocomial isolates. In conclusion, our report provides a valuable virulence map that explains enhancement in E. faecalis virulence and contributes to a deeper comprehension of the genetic mechanism leading to the transition from commensalism to a pathogenic lifestyle.
INTRODUCTION
Enterococcus faecalis is a Gram-positive commensal bacterium of the mammalian gastrointestinal (GI) tract (1) and a leading cause of nosocomial infections worldwide (2). In humans, the normal abundance of E. faecalis in the intestinal lumen ranges between 105 and 108 CFU/g of feces without causing any obvious deleterious effects on the host (3, 4). However, if either perturbations of the host/commensal balance that weaken the host immune system or environmental factors such as use of antibiotics that inadvertently facilitate outgrowth of resistant E. faecalis occur, life-threatening infections might arise. Moreover, the intrinsic robustness enables E. faecalis to withstand multiple stresses (5, 6) and provides a fitness advantage with respect to host adaptation and colonization in environments such as the hospital setting (7–9). The aptitude of this bacterium for acquisition and transfer of mobile genetic elements (plasmids, transposons, and prophages) has facilitated the spread of virulence traits and antibiotic resistance genes among isolates (10–14).
Notwithstanding several studies undertaken in the last few decades having identified putative virulence determinants that may augment the ability of E. faecalis to cause disease (reviewed in reference 15), the incidence of many pathogenicity factors has been reported to be independent from the E. faecalis isolation source. The availability of 5 complete and more than 300 draft E. faecalis genome sequences (http://www.ncbi.nlm.nih.gov/genome/808) has provided the opportunity to examine the genomic diversity among strains and identify specific traits that may contribute to virulence (16–18). However, none of these experimental investigations have performed comparative estimations of the abilities of different E. faecalis isolates to cause infection employing a live model system with the purpose of correlating E. faecalis pathogenicity to the whole-genome content.
Several studies have established the soil nematode Caenorhabditis elegans as a suitable surrogate animal model for in vivo study of host-microbe interactions with E. faecalis. Through comparison of the levels of longevity of nematodes infected with isogenic deletion mutants versus parental strains and screening of transposon insertion mutant libraries, several gene categories required for enterococcal pathogenicity in C. elegans have been identified, including virulence factors such as cytolysin (Cyl) (19) and gelatinase (Gel) and serine protease (20) and factors playing a role in cell metabolism and physiology (21, 22). Notably, these reports have demonstrated that several virulence factors required for infections of C. elegans and mammals coincide (21, 23).
In the present study, we investigated the pathogenicity potential of a collection of 28 E. faecalis strains in the C. elegans infection model and the correlation between the virulence phenotype and their whole-genome content. Consistent with previous reports, we found that cytolysin (Cyl) and gelatinase (Gel) are the major factors involved in C. elegans killing by E. faecalis. Further, using a comparative pangenomic analysis combined with statistical regression model testing, a virulence reference map that extends beyond the production of cytolysin and gelatinase was established. This allowed us to identify an important role of a phage03-like element in pathogenicity among nosocomial isolates.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
Bacterial strains used in this study are listed in Table S1 in the supplemental material. Genome-sequenced strains were selected to represent the genetic diversity of E. faecalis and included human, animal, and insect isolates. The human isolates comprised clinical isolates from urinary tract, liver, and blood infections, adult and baby commensal isolates from the gastrointestinal tract, and the probiotic strain Symbioflor 1. The animal-associated strains were of porcine, canine, phocine, or insect origin. The collection consisted of 24 different sequence types (ST) from 17 known clonal complexes (CC), including the most prevalent nosocomial clonal lineages. The years of isolation of the selected strains spanned 1926 to 2005, and the strains originated in most parts of the world, including the United States, New Zealand, Europe, Japan, and Solomon Islands (16).
E. faecalis strains were cultured in brain heart infusion (BHI) medium (Oxoid Ltd., United Kingdom) or Todd-Hewitt broth (THB; Oxoid Ltd., United Kingdom) at 37°C. Escherichia coli strains were grown in Luria Bertani (LB) medium at 37°C. When required for selective growth, chloramphenicol (Cm) was added at 10 μg/ml for E. coli and 15 μg/ml for E. faecalis. For detection of β-galactosidase activity, X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) was used at 80 μg/ml for E. coli and 120 μg/ml for E. faecalis.
Cytolysin assay.
Hemolytic activity was assayed by culturing E. faecalis on blood agar plates supplemented with 5% (vol/vol) defibrinated horse blood, 1% (wt/vol) glucose, and 0.03% (wt/vol) l-arginine (Sigma-Aldrich) (24). Overnight cultures of the strains to be tested were diluted 1:100, spotted onto fresh plates, and incubated at 37°C or 25°C for 24 h under anaerobic conditions. Colonies able to lyse erythrocytes were surrounded by a transparent halo.
Gelatinase assay.
The production of gelatinase in E. faecalis strains was assessed by the use of TH agar plates containing 3% gelatin (25). Overnight cultures were diluted 1:100 and spotted onto fresh plates. After incubation at 37°C or 25°C overnight, plates were placed at 4°C for 5 h. The presence of a clear halo around the colonies indicated hydrolysis of gelatin.
Nematode strains and growth conditions.
The host infection assay was carried out using the C. elegans mutant glp-4 (bn2ts); sek-1 (km4) strain, which is unable to produce offspring at the restrictive temperature of 25°C, thus allowing a prolonged experiment of 7 days without progeny interference (26). After ingestion, E. faecalis is able to survive the mechanical digestion and establish a long-lasting, deadly infection in the intestine of C. elegans (19). The temperature-sensitive sterile C. elegans glp-4 (bn2ts); sek-1 (km4) mutant strain was obtained from the Caenorhabditis Genetic Center, Minneapolis, MN, and propagated at the permissive temperature of 15°C in nematode growth medium (NGM) seeded with E. coli OP50 as a food source (27). After 5 days, gravid worms were harvested by rinsing the plates with M9 buffer (27), transferred to 15-ml tubes, and collected by centrifugation for 2 min at 280 × g at room temperature (RT). The supernatant was discarded, and sodium hypochlorite solution (Sigma-Aldrich) was added to release the eggs. Eggs were washed twice with M9 buffer and incubated to hatch at 25°C overnight. Newborn worms were transferred to NGM plates seeded with E. coli OP50 for 48 to 50 h to obtain synchronized sterile young adult worms to be used in the killing assay.
C. elegans-E. faecalis pathogenicity assay.
The C. elegans mortality assay was performed as described previously (26) with some minor modifications. Briefly, a tube containing 2 ml of BHI medium was inoculated at 1:100 with the overnight culture of the appropriate E. faecalis strain and grown at 37°C for 4 to 5 h to reach an absorbance at 620 nm of 0.4 to 0.5. A 10-μl volume of the culture was spotted onto the center of a BHI plate. The plates were incubated overnight at 37°C and allowed to equilibrate for at least 1 h at RT before transferal of the worms. Young adult nematodes grown at 25°C were washed from plates containing E. coli OP50 using sterile M9 buffer. Approximately 200 to 300 worms were transferred to a BHI plate seeded with the E. faecalis strain to be tested. Polymyxin B (5 μg/ml) was added to the medium to selectively prevent E. coli growth. The plates were incubated at 25°C for 8 h. Nematodes were then carefully washed three times with 2 ml of sterile M9 buffer in order to minimize the carryover of bacterial cells. Approximately 30 nematodes were transferred to each agar plate; all experiments were performed as technical replicates. Plates were incubated at 25°C with 80% to 85% relative humidity. Worms were scored daily with a dissecting microscope for viability. The bacterial isolates were ranked from the most to the least virulent based on the time required to kill 50% of the host population (LT50). Strains with no nematocidal effect are referred to as apathogenic.
Plasmid construction and generation of E. faecalis mutants.
To achieve the construction of phage03 deletion mutants, the phage03-like integration site was PCR amplified using E. faecalis OG1RF chromosomal DNA as the template. The generation of an amber point mutation at the fsrB codon corresponding to Leu-65 was achieved by using the genomic DNA of E. faecalis OU510 (28) as the template for PCR amplification of a 2.0-kb fragment. The two fragments of 2.2 kb and 2.0 kb were independently digested with restriction enzymes EcoRI and PstI and ligated into similarly digested pLT06, giving pSL01 and pSL02, respectively.
pSL01 and pSL02 were electroporated into E. coli EC1000, which supplies RepA in trans, for replication of the plasmids at the permissive temperature of 30°C (29). Transformants were screened for the presence of the insertion using primers OriF/KS05seqR (see Table S2 in the supplemental material). To exclude mutations occurring during cloning, the fragments were sequenced using the same primers. The plasmids were purified using E.Z.N.A. plasmid minikit I (Omega Bio-tek). Vector pSL01 was electroporated in both E. faecalis JH2-2 and E. faecalis V583, while pSL02 was introduced only to V583, using standard procedures (30). Gene replacement was achieved by double-crossover homologous recombination following the procedure described by Thurlow et al. (31). To select for the second recombination event, the integration mutants were passaged for 2 days in THB with no selection at 30°C. The chromosomal DNA of the white colonies arose on M9YEG plates supplemented with 10 mM p-chlorophenylalanine, and X-Gal was screened by PCR with primers int1-F/int2-R (see Table S2).
The phage03 isogenic deletion mutants were named E. faecalis SL100 (JH2-2 mutant) and SL110 (V583 mutant), while the frame-shifted fsrB V583 mutant was named SL111 (see Table S1 in the supplemental material). Gene replacement in these strains was confirmed by PCR and sequencing using primers flanking the targeted region (see Table S2).
Monitoring of growth in liquid laboratory medium.
Overnight cultures of E. faecalis strains were diluted 100-fold in fresh GM17 medium and grown to an optical density at 620 nm (OD620) of 0.2. Cells were back-diluted 100-fold in GM17, and a total volume of 300 μl of bacterial inoculum in fresh medium was added to a 96-well plate (Nunc; Thermo Fisher Scientific, Denmark). Cultures were incubated at 37°C under static conditions, and absorbance at 620 nm was measured at 15-min intervals for 11 h with a SPECTROstar Nano microplate reader (BMG Labtech, USA). The experiments were performed as independent triplicates.
Sequence analysis.
At the time that this investigation began, the genome sequences of 27 of the 28 wild-type E. faecalis isolates included in this study were publicly available (see Table S1 in the supplemental material) whereas a 3.26-Mbp high-quality draft assembly of the E. faecalis MMH594 genome was generated by our group (see Text S1 in the supplemental material). For each genome, the RefSeq annotated proteins were downloaded. A sequence comparison was conducted where each annotated protein sequence was subjected to BLAST searches against all annotated proteins. The best hit for every query sequence in each genome was extracted, and a relative score was computed. This score was the obtained BLAST score divided by the maximum attainable BLAST score for the same query sequence; i.e., a relative score of 1.0 means that the query was found with 100% identity in the genome. A threshold of 0.75 was used to identify a sequence as Present (>0.75) or Absent (<0.75) in each genome. Thus, each annotated sequence has a Present/Absent pattern (0's and 1's) over the 28 genomes. Many sequences share the same Present/Absent pattern, and in the downstream analysis we considered only distinct patterns, with a lookup table assigning each sequence to its distinct pattern. Reciprocal or opposite patterns (0 replaced with 1 and vice versa) were considered reflections of the same distinct pattern. Patterns without variation across the 28 genomes (Present in all strains) were discarded since they lack discriminating power.
On the basis of the BLAST all-versus-all alignments, we identified a set of 1,006 core gene families. In cases where a gene family contained paralogs, only the ortholog (best-matching sequence) from each genome was considered. Each gene family was aligned with MUSCLE (32). The protein sequence alignments were back-translated to DNA alignments, and then sequences were concatenated. The raw distance (p-distance) between genomes was computed from this alignment, and a neighbor-joining tree (Bionj in the R-package ape) was constructed (33).
For construction of a tree based on the Present/Absent proteins, each genome was represented as a vector of 82,356 1's or 0's corresponding to whether each sequence in the pan-genome was present (>75% similar) or absent in the genome. Based on these vectors, the Manhattan distance values of comparisons of all pairs of genomes were computed, and the dendrogram tree was constructed by hierarchical clustering using average linkages.
PCA.
The experimental data were also illustrated with a principal component analysis (PCA). A matrix with one column for each sample time (48, 72, 96, 120, 144, and 168 h) and one row for each experimental unit (strain, batch, and plate) was filled with percent survival data; i.e., row i contains the time series of survival percentages for experimental unit i over the 6 sample times. This matrix was used as the input for a standard PCA, and each experimental unit was plotted in the two first principal directions (see Fig. 2).
Analysis of variance.
The percentage of nematode survival at 72 h after exposure to E. faecalis was used in an analysis of variance (ANOVA) to search for associations with various categorical variables. A maximum value of 100 means that all nematodes survived, while the minimum value of 0 means that all died. The experiments were conducted in different batches and time series, and a potential bias in the results from any batch was described as a random effect in an ANOVA model.
The first fixed effects included in the model were the effects of gelatinase (Gel) and cytolysin (Cyl) as well as their interaction:where αi (i = 1 or 2) and βj (j = 1 or 2) are the fixed effects of Gel and Cyl, respectively, i.e., α takes value 0 or 1 depending on whether survival y is observed for a Gel-negative (Gel−) or Gel-positive (Gel+) strain, and the definitions of the terms are similar for β, Cyl−, and Cyl+. The term bh (h = 1,…, 8) is the random effect of the batch, and eijhk (k = 1, 2, or 3) (representing triplicate plates where the nematodes were kept for each treatment) is the residual term.
(1)
A model similar to that reported in equation 1 was also used for other fixed effects in addition to those seen with Gel and Cyl, e.g., for investigating the effect of the presence or absence of a set of genes, as described in the sequence analysis subsection above:where γg (g = 1 or 2) is 0 or 1, depending on whether the genes considered are absent or present in the strain from which the corresponding response y is observed. Different patterns of presence or absence were tested against survival in this model, one by one; i.e., the g fixed effect was reestimated for each distinct pattern.
(2)
Finally, we also combined model 1 and model 2, yielding the following equation:where the effect of a gene being present or absent competes with the Gel and Cyl effects in explaining the observed variations in survival.
(3)
In all cases, the fixed effects were estimated and the null hypothesis of no effect was tested against the two-sided alternative in each case. When we searched for associated Present/Absent patterns, we conducted many hypothesis tests and, for this reason, we computed adjusted P values to correct for multiple testing, using the Benjamini-Hochberg approach of controlling the false-discovery rate (36).
Nucleotide sequence accession number.
The draft assembly of the E. faecalis MMH594 genome generated by our group has been submitted to GenBank under accession number AOPW00000000.
RESULTS
C. elegans infection by E. faecalis.
To test the hypothesis that the degree of E. faecalis virulence in the C. elegans model correlates with the bacterial gene content, age-synchronized cohorts of C. elegans were challenged with the individual 28 E. faecalis isolates and the LT50 (the time at which 50% nematode lethality is detected) was used to rank the strains from the most to the least virulent.
E. faecalis MMH594 was the most aggressive isolate in killing the nematodes, showing an LT50 of 48 ± 3 h (Fig. 1). In contrast, ATCC 4200, EF62, T2, E1825, CH188, and Symbioflor 1 were apathogenic, as more than 85% of the C. elegans nematodes were alive at the end of the experiment, a percentage similar to that observed for E. coli OP50. Infection with E. faecalis V583, Merz96, D6, OG1RF, T3, E1022, HH22, Fly1, or TX0104 had a strong impact on the longevity of the nematodes, halving the C. elegans population in 68 ± 6 h, whereas E. faecalis JH-1, ARO1/DG, T8, DS5, E1Sol, HIP11704, T11, T1, and X98 exhibited intermediate killing. The slowest nematocidal activity (LT50 >99 h) was observed for E. faecalis E1052, E1877, and JH2-2.
FIG 1
A principal component analysis (PCA) revealed that the data for all sample times were explained by two variables, where the first principal component (PC1) accounted for 94% and the second principal component (PC2) for 4% of the variability (Fig. 2). Strains with high negative values corresponding to the first component were apathogenic in the C. elegans model, whereas strains associated with high positive values were the most virulent (Fig. 2).
FIG 2
To examine whether the virulence phenotype in C. elegans was related to clonal lineages, the phylogenetic relationship between the E. faecalis strains was analyzed by constructing a core genome-based tree (see Fig. S1 in the supplemental material). However, and not surprisingly for an highly recombinogenic organism such as E. faecalis, only limited correlation between pathogenicity and origin could be observed, thus indicating that the clonal lineage could not be predictive of the ability of a strain to be virulent in this model.
Role of gelatinase and cytolysin pathotypes in C. elegans killing by E. faecalis.
Cytolysin and gelatinase are two established enterococcal virulence factors known to play a significant role in C. elegans killing by E. faecalis (19, 20). Among the E. faecalis strains included in this study, we confirmed that 10/28 were cytolytic on blood agar plates and that 15/28 showed a positive gelatinase phenotype, at both 37°C and 25°C. Notably, E. faecalis MMH594, Merz96, JH-1, and T1 were producers of both cytolysin and gelatinase, while E. faecalis Symbioflor 1, ATCC 4200, EF62, T2, E1825, and CH188 showed neither cytolysin nor gelatinase activity. As expected, despite the presence of the gelE gene, some strains (ATCC 4200, CH188, HIP11704, T2, T8, X98, JH2-2, E1052, and E1825) failed to produce gelatinase due to partial deletions of the fsr operon, which activates gelatinase expression (37). Strains ATCC 4200 and T2 were unable to produce cytolysin, although their genomes harbor an intact but nonfunctional cyl operon and its associated regulatory genes.
Figure 3 depicts the 28 E. faecalis strains divided in 4 subsets based on the Gel and Cyl phenotypes. The bars indicate nematode survival at 72 h. Comparing the fractions of live nematodes in each subset, it was evident that gelatinase and cytolysin are major factors of C. elegans killing. However, the substantial variation in survival within each subset indicated that Gel and Cyl are not the only determinants of E. faecalis virulence. Interestingly, all the gelatinase and cytolysin nonproducers were avirulent in C. elegans, with the exception of E. faecalis JH2-2. Observation of the pathotypes showed that the strains with a gelatinase- or cytolysin-positive phenotype were of both clinical and commensal origin. This indicates that nonclinical cytolysin- or gelatinase-producing bacteria can also be potentially infectious in this model.
FIG 3
Construction of a genomic reference map of E. faecalis pathogenicity in C. elegans.
Aiming to elucidate the genetic basis of the nematocidal activity of the individual isolates and the observed virulence variation, a bimodal mathematical and bioinformatics analysis approach was employed. A total of 79,108 annotated protein sequences for 27 sequenced strains were downloaded from the RefSeq database in addition to 3,248 proteins translated from the identified coding sequences (CDS) in the MMH594 genome. After all-against-all BLAST analysis performed as described in Materials and Methods, every sequence was given a Present/Absent pattern over the 28 genomes using a cutoff value of a 0.75 relative BLAST score. This revealed that 48,821 of the unique annotated protein sequences (encoded by the corresponding variants of core genes) were present in all strains. The remaining sequences were organized into 2,325 protein patterns, into which proteins showing identical Present/Absent patterns across all genomes were grouped.
A Present/Absent protein pattern-based tree was constructed, and it resolved the examined strains into three lineages (Fig. 4). Highly pathogenic isolates in the C. elegans model (MMH594, V583, HH22, and TX0104), all belonging to the nosocomial clonal complex 2 (CC2) group, occupy a separate lineage (lineage A) in relation to the other strains employed in the study. Lineage C, although it includes commensal isolates EF62 and E1052, consists of clinical isolates (HIP11704, CH188, JH2-2, T8, ATCC 4200, and T2) exhibiting phenotypes ranging from moderately virulent to apathogenic. Lineage B consisted of two subgroups. In subgroup I, the majority of the strains are highly or moderately lethal nematode killers, with the exception of strain E1877, which is a strain of low pathogenicity. The two isolates Symbioflor1 and commensal E1825 form subgroup II in lineage B and have no nematocidal effect. Taken together, the data show a significant correlation between Present/Absent protein profiles and the E. faecalis phenotype of virulence toward C. elegans.
FIG 4
Next, to evaluate the contribution of cytolysin and gelatinase to E. faecalis pathogenicity, each of the 28 strains was coded by its Gel and Cyl phenotype (positive or negative), and an analysis of variance using a mixed-effect model was conducted as described for equation 1. This analysis revealed a strong (P < 0.0001) negative impact of Gel and Cyl that accounted for 40.8% and 36.5%, respectively, of the observed variations in survival of the nematodes. The interaction was not synergistic but showed that a Gel+ Cyl+ phenotype is (significantly) 26.7% less virulent than the sum of their separate contributions, i.e., that there is saturation or an antagonistic interaction between these two virulence traits.
Next, we fitted each of the 2,325 distinct Present/Absent patterns to worm survival, as described in equation 2, resulting in the identification of 414 patterns statistically associated with C. elegans killing (see Table S3 in the supplemental material). In addition, we performed a similar ANOVA considering other factors. Strains were grouped by capsular polysaccharide locus serotype, human or nonhuman origin, clinical or commensal origin, and gastrointestinal or urinary tract origin, but none of these showed a significant association with nematode survival. Similarly, the year of isolation did not reveal any significant impact on virulence. Clinical isolates from blood produced 27% higher killing (P < 0.001) of the worms than those from other sources.
Having observed that gelatinase and cytolysin have a strong and significant negative impact on nematode survival, the ANOVA was then extended as described in equation 3 to include both the two pathogenicity traits and the effect of Present/Absent patterns. This resulted in 376 patterns positively correlated with virulence (see Table S4 in the supplemental material); i.e., these patterns are capable of explaining significant additional variations in survival even when Gel and Cyl are also included in the model.
After a process of binning and manual curation, 61 candidate patterns remained that included 168 protein-coding sequences with a potential role during nematode infection (see Table S5 in the supplemental material). On the basis of these results, we found that 86% (52/61) of the patterns included annotated CDS identified in E. faecalis V583 and, importantly, that a total of 25 were associated with previously described mobile genetic elements (MGEs). These included genes that cover only parts of the MGEs (phage01 [n = 5], phage04 [n = 11], phage05 [n = 10], phage06 [n = 6], phage07 [n = 1], E. faecalis pathogenicity island [PAI] [n = 3], efaC1 [n = 10], and the vanB vancomycin resistance region [n = 1]), with the exception of phage03, which was almost completely represented (n = 54). In addition to the MGEs, 9 patterns included a putative phage element corresponding to the gene cluster ef3217 to ef3227 and genes for surface structural proteins previously described to be enriched in isolates of high-risk clonal complex 2 (17).
Functional genomic analysis of pathogenesis in noncytolytic and gelatinase-negative strain JH2-2 identifies involvement of phage03 in virulence.
As shown in Fig. 3, JH2-2 was the only cytolysin- and gelatinase-negative isolate that exhibited significant nematocidal activity. It was therefore of interest to identify genes that promoted E. faecalis JH2-2 host fitness and pathogenicity in C. elegans. This strain is a plasmid-free derivative of the JH2 clinical isolate (38), and its virulence, relative to that of the JH2 isolate, in the intravenous mouse infection model has been shown in a previous report (39). However, the role of genetic traits involved in the development of disease has not been further elucidated.
To identify potential virulence factor-coding genes, we performed an in silico subtractive analysis of the genome of E. faecalis JH2-2 (GCA_000148445.1) against the genomes of strains showing an avirulent phenotype in the nematode model, namely, E. faecalis 62, E. faecalis CH188, E. faecalis ATCC 4200, E. faecalis T2, and E. faecalis E1825. The genome of E. faecalis V583 was used as the reference. Moreover, to rule out possible intrinsic errors in gene annotations and predictions present in the publicly available E. faecalis draft genomes, the search output was manually examined using BLAST. Besides, we employed Mauve 3.2.1 software to perform global alignment of the same genomes (34). These combined approaches identified 135 genes present in JH2-2 but absent from the apathogenic strains (see Table S6 in the supplemental material). A large fraction of these genes encode MGEs with high sequence homology to that identified in the E. faecalis V583 genome (40).
Among the gene clusters identified by comparative genomics analysis, the phage03-like element in JH2-2 was among the patterns with the lowest t values in the ANOVA, suggesting a potential involvement in virulence. This candidate pathogenicity trait was therefore selected for further functional studies.
We designed experiments to assess whether the presence of the intact phage03-like element affects E. faecalis JH2-2 pathogenicity in C. elegans. Markerless in-frame deletion of the phage03-like region from E. faecalis JH2-2 was achieved by allelic replacement using the pSL100 vector (see Table S2 in the supplemental material). We compared the levels of growth in GM17 broth of the phage03-like deletion mutant, named SL100, and of the E. faecalis JH2-2 wild-type strain. As shown in Fig. 5a, the growth yield of SL100 was significantly higher than that of E. faecalis JH2-2, with increased cell density in the stationary phase of growth. This suggests that the reduced E. faecalis JH2-2 growth may have been due to the metabolic burden imposed by the presence of the phage03-like DNA. Subsequently, we evaluated the contribution of the phage03-like element to nematocidal activity. SL100 was found to be severely attenuated in nematode killing compared to E. faecalis JH2-2 at each sampling point (P < 0.05, unpaired t test), and the results showed 76.5% increased C. elegans survival at the end of the experiment (Fig. 5b). Similarly, infection of Galleria mellonella larvae with approximately 5 × 106 CFU/ml of SL100 led to significantly (P = 0.02, unpaired t test) increased survival compared to the JH2-2 wild-type strain results (see Fig. S2 in the supplemental material). Removal of phage03 from JH2-2 resulted in a 30% reduction in killing of the larvae between 12 and 20 h postinfection. The reduced life span of the nematodes and wax moth larvae infected with JH2-2 compared to SL100 indicates that the phage03-like element contributes to increased E. faecalis virulence, revealing a role of the phage in the development of infection.
FIG 5
Phage03-like elements are widely distributed among nosocomial isolates.
Enterococcal phages contribute to bacterial evolution by facilitating the spread of virulence and antimicrobial resistance determinants via transduction (41). Paulsen and coworkers have shown that approximately 25% of the total genome of E. faecalis V583 consists of MGEs and seven integrated prophage regions (42). However, only a few studies have investigated how prophage-carried genes can engender virulence in E. faecalis (41, 43, 44).
The sequence of the phage03-like element in E. faecalis JH2-2 (HMPREF9496_01004 to HMPREF9496_01061 and HMPREF9496_01491 to HMPREF9496_01507) has a length of approximately 48 kb and closely resembles the region spanning ef1417 to ef1489 in the genome of E. faecalis V583. This region contains 71 coding sequences with 81% similarity to the region in E. faecalis V583 and an average G+C content of 35% (see Table S7 in the supplemental material). A phage03-like element is also found in the E. faecalis MMH594 and HH22 CC2 strains and in the E. faecalis Fly1 animal isolate, among the strains used in this study. ANOVA showed that the phage03-containing strains displayed on average 17% higher killing toward C. elegans (P = 4.4e−6).
Among the 71 genes, only 16 have an assigned function based on homology to genes encoding annotated proteins whereas the rest share no similarity to those encoding known proteins. The phage03 element is flanked by a 76-bp inverted repeat, indicating the presence of attL and attR attachment sites (45). No phage particles could be induced by mitomycin C treatment (data not shown). The inability to detect phage particles is in accordance with previous observations that found only low production of the phage particles following mitomycin C treatment (41).
Comparative analysis of the phage integration regions in the five E. faecalis strains revealed different integration sites, suggesting independent acquisitions of phage DNA by different E. faecalis strains. The phage03 element sequences in E. faecalis MMH594, V583, HH22, JH2-2, and Fly1 reveal that, consistent with other temperate bacteriophages found in lactic acid bacteria, the genes are organized to form functional modules for lysogeny, DNA replication, packaging, head and tail assembly, and host lysis and that they are regulated in a coordinated manner (Fig. 6) (46). The sequences share high similarity, with the exception of the regions containing genes encoding proteins with a predicted function in virion DNA replication and host lysis.
FIG 6
In addition to E. faecalis JH2-2, MMH594, V583, HH22, JH2-2, and Fly1, BLAST searches against NCBI available genomes showed the presence of a phage03-like element in a total of 93 enterococcal isolates, of which 73 were of confirmed nosocomial origin (see Table S8 in the supplemental material).
Deconstruction of E. faecalis V583 virulence.
Aiming to corroborate the significance of phage03 elements for the pathogenicity of E. faecalis in C. elegans, we analyzed a series of isogenic mutants of E. faecalis V583 (Fig. 7). The selected strain belongs to the high-risk clonal complex 2 group of strains that are prevalent in nosocomial infections (47).
FIG 7
As a first step, we tested the hypothesis that plasmids contribute to virulence in V583. Infection of C. elegans was performed by employing the E. faecalis V19 plasmid-cured derivative (48), and it revealed that extrachromosomal DNA found in V583 does not contribute to pathogenicity in this model (P = 0.09, unpaired t test). Next, we evaluated the impact of the gelatinase production in the killing of nematodes by employing the V583ΔgelE mutant. As depicted in Fig. 7, deletion of a gelatinase gene reduced virulence at 72 h postinfection by 40% (P < 0.05, unpaired t test), which is consistent with previous results (20). Then, to assess the contribution from the Fsr master regulatory system, a frame-shifted fsrB mutant, named SL111, was constructed by allelic replacement. This strain produced 56% less killing than the parent strain (P < 0.05, unpaired t test). We therefore hypothesized that the remaining virulence potential could be partially attributed to the phage03 element. An isogenic in-frame phage03 deletion mutant of E. faecalis V583 named SL110 was constructed by employing the pSL100 vector system (see Table S2 in the supplemental material) as described above for the analogous element in E. faecalis JH2-2. Infecting C. elegans with E. faecalis SL110, we observed a 17% reduction in the killing compared to the wild-type strain results (Fig. 7). SL110 was still capable of efficiently killing the nematode, since it harbored a functional fsrABDC operon and the gelE gene; however, the deletion of the phage03 element led to decreased killing ability by SL110 after 48 and 72 postinfection (P = 0.0003, unpaired t test). Taken together with the observation in JH2-2, these results provide evidence that the phage03 element acts as a virulence factor toward C. elegans.
DISCUSSION
The emergence of E. faecalis strains as prominent nosocomial pathogens and the occurrence of enterococcal infections not treatable with most available antibiotics emphasize the need for understanding the complex mechanism by which this organism causes life-threatening diseases (49). The availability of large amounts of whole-genome sequencing data has facilitated in silico comparative analyses of the gene content of enterococcal strains representing a wide spectrum of biotypes, clonal lineages, and isolation sources. As result, these investigations have contributed to the determination of the size and composition of the E. faecalis core genome (40, 50), the genetic background for antibiotic resistance (51), and the identification of potential virulence factors that can influence the bacterial lifestyle (16). However, there is still a lack of understanding of how the genetic variation between commensal and nosocomial strains could explain the alleged behavior associated with these.
In the present study, we combined statistical and comparative genomic approaches, followed by a functional genomic study, with a model host infection system to examine the correlation between E. faecalis whole-genome content and its impact in the host-microbe interaction. We showed that infection of C. elegans by 28 E. faecalis isolates resulted in a wide range of virulence phenotypes, with six strains behaving in a commensal manner, with no nematocidal effect, and 22 strains displaying a virulence phenotype of from high to low levels of pathogenicity. Principal component analysis showed that the first and second principal components account for 98% of the variance in the virulence data, thus indicating a significant relationship between E. faecalis pathogenicity toward C. elegans and gene content. We therefore employed a comparative approach to analyze the genomes of the 28 E. faecalis strains to identify variations in the genetic makeup that could explain commensalism or opportunistic lifestyle in the nematode. A tree based on Present/Absent gene patterns identified by the comparative genomic analysis resolved the 28 strains into three lineages and showed a significant correlation between Present/Absent profiles and the phenotype of E. faecalis virulence toward C. elegans (Fig. 4). Moreover, the tree highlighted a deep division between isolates belonging to CC2 and the other strains employed in the study.
Statistical ANOVA established a strong contribution of cytolysin and gelatinase to nematode killing which accounted for 40.8% and 36.5%, respectively, of the observed variations in survival of the nematodes. However, the presence of the two factors explained only 50.6% of the variation. Speculatively, it is conceivable that the lack of synergism between gelatinase and cytolysin is caused by saturation or antagonistic interactions between the two factors when coproduced by a strain.
In agreement with previous studies, evaluation by ANOVA of the E. faecalis isolation source for the association with increased virulence toward C. elegans indicated that the isolate origin had no impact on virulence in the nematodes (52). The fact that non-clinically isolated strains also had the potential to be infectious (Fig. 3) is consistent with the ambivalent nature of E. faecalis in relation to its host, with the ability of apparently harmless strains to disrupt the commensal relationship with the host and cause severe diseases.
In addition, in line with a previous investigation that reported a low correlation between the presence of capsule among E. faecalis clinical isolates and the ability to establish infection (53), our data showed that the distributions of cell wall polysaccharides are equal at all levels of E. faecalis virulence, and no significant difference in C. elegans pathogenicity was observed.
Beside other reports that have clearly demonstrated that gelatinase and cytolysin are two prominent E. faecalis virulence factors exerting a crucial role in nematode death (19, 20), our study also identified a strong positive impact of these two factors on nematode killing. The mathematical model, computed to include the a priori knowledge of the cytolysin and gelatinase genotype and to fit to the virulence at 72 h, established a virulence reference map of statistically significant traits that can be predictive of E. faecalis virulence (see Table S5 in the supplemental material). Among the resulting patterns, a predominant (41%) fraction is comprised of MGEs. The presence of a multiplicity of mobile elements has been previously described for E. faecalis V583 and is considered a major driving force of genome evolution (42, 44, 50). In addition, a study from our group reported the enrichment of structures of MGEs among strains belonging to the high-risk nosocomial clonal complex 2 (CC2) group (17). The prevalence of MGEs among factors contributing to E. faecalis pathogenicity supports the notion that highly virulent isolates benefit from the introduction of fitness and virulence factors that confer increased adaption under conditions of stress and infection.
Patterns 1408 and 1523, including CDS EF1827 to EF1830 and EF1833, emerged in our work as contributing to virulence in nematodes. The associated genes reside in a 23.9-kb region corresponding to the 5′-end fragment of fsrC (ef1820) and the 3′-end segment of ef1841 of E. faecalis V583 whose deletion has been reported to cause the lack of a positive gelatinase phenotype among strains carrying the gelE gene (54). Interestingly, the genes encode sugar transport system components related to the glpK pathway and are involved in glycerol catabolism in E. faecalis (55). As described in other bacterial species, growth in the presence of glycerol as the carbon source leads to the activation of a transcription activator also modulating the expression of virulence genes (56).
The list of candidate genes that might contribute to enterococcal pathogenicity includes variable cell surface structure components, such as EF2164 (pattern 666), EF0967 (pattern 609), and EF3250 and EF3251 (pattern 1577), previously identified as enriched among isolates of the high-risk clonal complex 2 (17). Surface-exposed proteins play an important role in pathogenicity, as they are often involved in colonization of host tissues and evasion of host defenses (57, 58). Studies in C. elegans have ruled out the role of a number of surface structures, such as the AS aggregation substance, the Esp enterococcal surface protein, the Ace adhesin to collagen, and biofilm-associated pilus protein A, in mediating nematode killing (21, 59), suggesting that their host target may be absent. However, Creti and colleagues demonstrated that deletion of the ef3314 gene, encoding a surface protein structurally similar to Esp, affects the pathogenicity potential of the resulting E. faecalis mutant in C. elegans, thus demonstrating that some surface factors contribute to pathogenesis in the worm (22). Further functional studies are therefore required to elucidate the significance of the identified traits in enterococcal virulence.
Among the non-V583 annotated loci, pattern 1275 included two proteins holding a CaaX amino terminus and a DJ-1/PfpI domain, respectively. These domains are common to proteases; therefore, a potential role of these genes in virulence is plausible (60).
Polylysogeny is a common feature in the E. faecalis genome and is considered to influence the relationship of E. faecalis with its host, as these elements may confer properties advantageous for the dissemination of the bacterial strain (17, 41). Duerkop and colleagues demonstrated that temperate phages of E. faecalis ST6 can modulate the assemblies of bacterial communities in the mammalian intestinal tract (61). A more recent study showed that five (phage01, phage03, phage04, phage05, and phage07) of the seven V583 prophages are capable of excision from the bacterial chromosome and that four of them produced infective virions and are therefore possible mediators of horizontal gene transfer (44). Bacteriophage-encoded proteins such as platelet binding proteins were established as factors that provide mechanisms to invade host tissues and damage host cells, enriching the plethora of E. faecalis virulence traits (43, 44).
Our comparative genomic approach pointed toward a significant enrichment of a phage03-like element in nosocomial and clinical isolates. We demonstrated that phage03 deletion resulted in a decline of E. faecalis JH2-2 infectivity both in C. elegans and in G. mellonella larvae. In agreement with previous reports that have described the absence of phage03 in the genome of E. faecalis food isolates (45) and its enrichment among clonal lineages associated with nosocomial diseases (17, 40), we detected the presence of phage03 in the genome of CC2 strains E. faecalis V583, MMH594, and HH22. BLAST searches against the available E. faecalis genomes showed that this phage03-like element was also found with a higher incidence in nosocomial isolates whereas it is rarely found in other strains of different origins (see Table S8 in the supplemental material). We therefore examined the role of the phage in the pathogenicity of nosocomial isolates by assessing the virulence of an isogenic phage03 mutant of E. faecalis V583. The mutant displayed attenuation in nematode killing as well as in the G. mellonella model compared to the parent strain, providing additional evidence that phage03 plays a role in E. faecalis virulence. To our knowledge, this is the first study to have demonstrated that a phage element endows clinical and nosocomial isolates with factors promoting increased pathogenicity in vivo during infection in a model organism.
Though the mechanism by which phage03 impacts E. faecalis virulence remains to be elucidated, the ability of this element to form infective virions indicates that its genes are expressed and that it can act as an efficient vehicle for horizontal gene transfer, spreading traits contributing to boosting strain fitness (44). Excision of the phage has been associated with treatment with fluoroquinolones (44), supporting the idea that application of antibiotics in the nosocomial environment may facilitate the creation of conditions favorable for the emergence of highly pathogenic clonal lineages.
Analysis of the phage03 sequence revealed the presence of proteins that could play a role in bacterial virulence. ef1420 encodes a protein of unknown function previously reported to significantly decrease E. faecalis infectivity in the G. mellonella model (62). This protein was predicted to contain a lipobox motif and is expected to be a surface-exposed element (45, 63). Lipoproteins represent approximately 25% of the proteins predicted to be associated with E. faecalis cell envelope, and they are reported to be involved in substrate binding and in delivery to ABC transporters, acquisition of sugars, protein folding, antibiotic resistance, and cell envelope stability (64, 65). In addition, lipoproteins play a significant role in virulence, as they mediate adhesion to the extracellular matrix (ECM), initiation of the inflammatory process followed by activation of the immune system, induction of phagosome escape, and translocation of virulence factors (63).
Besides EF1420, two proteins (EF1460 and EF1474) contain a LysM domain in the C terminus thought to contribute to the anchoring to peptidoglycan (66) whereas EF1426 is a VirE domain-containing protein conceivably associated with virulence by exerting a role in the type IV secretion pathway in other organisms (67). It is noteworthy that many phage03 proteins have an unknown function but nevertheless might play a role in and contribute to E. faecalis pathogenicity.
In conclusion, using a statistical and comparative genomic approach combined with an in vivo assessment of E. faecalis virulence during infection in C. elegans, our results establish a set of nonorthologous gene patterns that may be predictive of E. faecalis pathogenicity. Our report highlights the importance of MGEs as potential contributors to enterococcal pathogenicity and demonstrates that phage03, a prevalent element among nosocomial clonal lineages, is responsible for enhanced virulence. Future functional investigations are required to validate any involvement of the identified patterns in E. faecalis virulence and to gain a deeper understanding of the genetic mechanism underlying enterococcal pathogenesis.
ACKNOWLEDGMENTS
We are grateful to Hilde Nilsen (University of Oslo) for valuable support and guidance in setting up the nematode survival experiments. We thank the Norwegian Sequencing Centre for providing the MMH594 genome sequencing service.
This work was supported by the project number 191452 from the Research Council of Norway.
Supplemental Material
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Published In
Infection and Immunity
Volume 83 • Number 5 • May 2015
Pages: 2156 - 2167
Editor: A. Camilli
PubMed: 25776747
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© 2015 American Society for Microbiology.
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Received: 16 October 2014
Returned for modification: 23 November 2014
Accepted: 7 March 2015
Published online: 15 April 2015
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A Genomic Virulence Reference Map of Enterococcus faecalis Reveals an Important Contribution of Phage03-Like Elements in Nosocomial Genetic Lineages to Pathogenicity in a Caenorhabditis elegans Infection Model. Infect Immun
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