Abstract
Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. Here, we identify genes that contribute to growth of E. faecium in human serum. We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, to completion and then compared its transcriptome during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. A high-throughput transposon sequencing (Tn-seq) approach was used to identify conditionally essential genes in E. faecium E745 during growth in serum. Genes involved in de novo nucleotide biosysnthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants also exhibited significantly attenuated virulence in an intravenous zebrafish infection model. We conclude that genes involved in carbohydrate and nucleotide metabolism of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism.
Introduction
Enterococci are commensals of the gastrointestinal tract of humans and animals, but some enterococcal species, particularly E. faecium and E. faecalis, are also common causes of hospital-acquired infections in immunocompromised patients (1). While E. faecalis has been recognized as an important nosocomial pathogen for over a century, E. faecium has emerged as a prominent cause of hospital-acquired infections over the last two decades (2). Since the 1980s, E. faecium acquired resistance to multiple antibiotics, including β-lactams, aminoglycosides and finally, to the glycopeptide vancomycin (3). Nosocomial infections are almost exclusively caused by a specific sub-population of E. faecium, termed clade A-1, which has emerged from a background of human commensal and animal E. faecium strains (4). Strains in clade A-1 carry genetic elements that are absent from animal or human commensal isolates and which contribute to gut colonization or pathogenicity (5–9).
E. faecium bloodstream infections frequently develop in patients undergoing immunosuppressive therapy, and can result from translocation of strains from the intestinal tract to the bloodstream (10). In addition, the use of intravenous catheters in hospitalized patients, is another risk factor for the introduction of E. faecium into the bloodstream (3, 11, 12). Currently, E. faecium causes approximately 40% of enterococcal bacteremias. Due to the accumulation of antibiotic resistance determinants in clade A-1 strains, E. faecium infections are more difficult to treat than infections caused by E. faecalis or other enterococci (13–15).
To cause bloodstream infections, E. faecium needs to be able to survive and multiply in blood, but the mechanisms by which it can do so, have not yet been studied. To thrive in the bloodstream, an opportunistic pathogen has to evade host immune mechanisms and to adjust its metabolism to an environment that is relatively poor in nutrients (16).
To identify genes that are conditionally essential in bacteria, high-throughput screening methods for transposon mutant libraries have been developed and optimized for many different bacterial species (17, 18). To perform high-throughput functional genomics in ampicillin-resistant, vancomycin-susceptible clinical E. faecium strains, we previously developed a microarray-based transposon mutagenesis screening method which was used to identify genes involved in the development of endocarditis (7), resistance to ampicillin (19), bile (20) and disinfectants (Guzman Prieto et al., submitted for publication). However, microarray-based methods for transposon mutant library screening are limited in their accuracy and can only be used in strains for which the microarray was designed. To address these limitations, several methods, including Tn-seq (21) and TraDIS (22), which are based on high-throughput sequencing of the junctions of the transposon insertion sites and genomic DNA, have been developed (23).
In this study, we set-up Tn-seq in the clinical E. faecium isolate E745 to identify genes that contribute to survival and growth in human serum. In addition, we determined the transcriptional response of E. faecium E745 in that same environment. Finally, we substantiated the role of several E. faecium genes that contribute to growth in serum and in virulence, in a high-throughput zebrafish infection model. Collectively, our findings show that metabolic adaptations are key to E. faecium growth in serum and contribute to virulence.
Materials and Methods
Bacterial strains, plasmids, and growth conditions
The vancomycin-resistant E. faecium strain E745 was used throughout this study. This strain was isolated from feces of a hospitalized patient, during a VRE outbreak in a Dutch hospital (24, 25). Unless otherwise mentioned, E. faecium was grown in brain heart infusion broth (BHI; Oxoid) at 37°C. The E. coli strains DH5α (Invitrogen) was grown in Luria-Bertani (LB) medium. When necessary, antibiotics were used at the following concentrations: chloramphenicol 4 µg ml−1 for E. faecium and 10 µg ml−1 for E. coli, and gentamicin 300 µg ml−1 for E. faecium and 25 µg ml−1 for E. coli. All antibiotics were obtained from Sigma-Aldrich. Growth was determined by measuring the optical density at 660 nm (OD660).
Genome sequencing, assembly and bioinformatic analysis
E. faecium E745 was sequenced using a combination of Illumina HiSeq 100 bp paired-end sequencing, long-read sequencing using the Pacific Biosciences RS II SMRT technology and the MinION system with R7 flowcell chemistry (Oxford Nanopore Technologies). Corrected PacBio reads were assembled using the Celera assembler (version 8.1) (26) and assembled contigs were then corrected by aligning Illumina reads using BWA (version 0.7.9a), with default parameters for index creation and BWA-MEM algorithm with -M option for the alignment (27). This approach resulted in 15 contigs, including one contig covering the entire 2.77 Mbp chromosome. After discarding contigs with low-coverage, the remaining contigs constituted 5 circular plasmid sequences and 5 non-overlapping contigs. These 5 contigs were aligned against the NCBI Genbank database and all were found to be part of the E. faecium plasmid pMG1 (28). Based on this alignment the presumed order of contigs was determined and confirmed by gap-spanning PCRs and sequencing of the products. A single gap between two contigs, could not be closed by PCR. Thus, we assembled Illumina reads together with MinION 2D reads using the SPAdes assembler (version 3.0) (29), which produced a contig that closed the gap, resulting in a complete assembly of this plasmid. Sequence coverage of chromosomal and plasmid sequences was determined with SAMtools (version 0.1.18) using short read alignments to the assembly, which were generated using BWA (version 0.7.9a). SAMtools was also used to identify possible base-calling and assembly errors, by aligning short reads to assembled contigs. A base was corrected using the consensus of aligned reads (30). The corrected sequences were annotated using Prokka (version 1.10) (31). The annotated genome of E. faecium E745 is available from NCBI Genbank database under accession numbers CP014529 – CP014535.
Transcriptome profiling
Approximately 3 x 107 cfu of E. faecium E745 were inoculated into 14 ml of BHI broth and heat-inactivated serum, and grown at 37°C until log phase. Cultures were centrifuged at room temperature (15 s; 21.380 g), and pellets were flash frozen in liquid N2 prior to RNA extraction, which was performed as described previously (19). The ScriptSeq Complete Kit (Bacteria) (Epicentre Biotechnologies, WI) was used for rRNA removal and strand-specific library construction. Briefly, rRNA was removed from 2.5 µg of total RNA. To generate strand specific RNA-seq data, approximately 100 ng of rRNA-depleted RNA was fragmented and reverse transcribed using random primers containing a 5′ tagging sequence, followed by 3′ end tagging with a terminal-tagging oligo to yield di-tagged, single-stranded cDNA. Following magnetic-bead based purification, the di-tagged cDNA was amplified by PCR (15 cycles) using ScriptSeq Index PCR Primers (Epicentre Biotechnologies, WI). Amplified RNA-seq libraries were purified using AMPure XP System (Beckman Coulter) and sequenced by a 100 bp paired end reads sequencing run using the Illumina HiSeq 2500 platform (University of Edinburgh, United Kingdom). Data analysis was performed using Rockhopper (32) using the default settings for strand specific analysis.
Confirmation of RNA-seq data by quantitative real-time RT-PCR (qRT-PCR)
Total RNA isolated as described previously was used to confirm the transcriptome analysis by qRT-PCR. cDNA was synthesized as described above and qRT-PCR on these cDNAs was performed using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Breda, The Netherlands) and a StepOnePlus instrument (Life Technologies). The expression of tufA was used as a housekeeping control. Ct values were calculated using the StepOne analysis software v2.2. Transcript levels, relative to tufA, of the assayed genes were calculated using REST 2009 V2.0.13 (Qiagen, Venlo, The Netherlands). This experiment was performed with three biological replicates.
Generation of mariner transposon mutant library in E. faecium
To create a transposon mutant library in E. faecium E745 suitable for Tn-seq, the mariner transposon cassette (carrying a gentamicin resistance gene) in the transposon delivery plasmid pZXL5 (19) was adapted as follows. The transposon from pZXL5 was amplified by PCR using the set of primers: pZXL5_MmeI_SacII_Fw and pZXL5_MmeI_SacII_Rv (primer sequences listed in Supplementary table 7). These primers introduced MmeI restriction sites in the inverted repeats on both sides of the transposon. The modified transposon delivery vector, termed pGPA1, was generated by the digestion of pZXL5 with SacII, followed by the insertion of the SacII-digested mariner transposon that contained MmeI restriction sites at its extreme ends. pGPA1 was electroporated into E. faecium E745 and the transposon mutant library was generated by selecting for gentamicin-resistant transposon mutants as described previously (19).
Tn-seq analysis of conditionally essential genes in E. faecium E745
The transposon mutant library created in E745 was prepared for Tn-seq analysis, similar to previously described procedures (33). To identify genes that are essential for the viability of E. faecium in BHI, we used ten experimental replicates of the mutant library. Aliquots (20 μl) of the transposon mutant library, containing approximately 107 cfu, were used to inoculate 20 ml BHI broth and grown overnight at 37°C. Subsequently, 1 ml aliquots of the cultures were spun down (15 s, 21.380 g) and used for the extraction of genomic DNA (Wizard genomic DNA purification kit, Promega Benelux). 2 μg of the extracted DNA was digested for 4 hr at 37°C using 10U MmeI (New England Biolabs) and immediately dephosphorylated with 1U of calf intestine alkaline phosphatase (Invitrogen) during 30 min at 50°C. DNA was isolated using phenol-chloroform extraction and subsequently precipitated using ethanol. The DNA pellets were then dissolved in 20 μl water. The samples were barcoded and prepared for Tn-seq sequencing as described previously (33). The sequence reads from all ten experimental replicates were mapped to the genome, and the mapped read-counts were then tallied for the analysis of the essentiality of the genes in the E. faecium E745 genome (further described below).
To identify genes that are required for growth in human serum, 20 μl aliquots of the frozen mutant library in E745 were inoculated in BHI broth and grown overnight as described above. Subsequently, bacterial cells were washed with physiological saline solution. Approximately 3x107 cfu were inoculated into 14 ml BHI broth, and approximately 3x106 cfu were inoculated into 14 ml human serum obtained from Sigma (Cat. No. H4522; Sterile filtered type-AB human serum) or heat-inactivated human serum (the same, after incubation for 30 min at 56°C). The different inoculum-sized were used in order for a similar number of divisions to occur during the experiment. Cells were incubated at 37°C for 24 hours without shaking and then further processed for Tn-seq (33). This experiment was performed in triplicate.
Tn-seq samples were sequenced (50 nt, single-end) on one lane of a Illumina Hiseq 2500 (Baseclear, Leiden, the Netherlands and Sequencing facility University Medical Center, Utrecht, The Netherlands), generating an average of 15 million high quality reads per sample.
Tn-seq data analysis
Raw Illumina sequence reads from Illumina sequencing were split, based on their barcode, using the Galaxy platform (34), and 16-nucleotide fragments of each read that corresponded to E745 sequences, were mapped to the E745 genome using Bowtie 2 (35). The results of the alignment were sorted and counted by IGV (36) using a 25-nucleotide window size and then summed over the gene. Read mapping to the final 10% of a gene were discarded as these insertions may not inactivate gene function. Read counts per gene were then normalized to the total number of reads that mapped to the genome in each replicate, by calculating the normalized read-count RPKM (Reads Per Kilobases per Million input reads) via the following formula: RPKM = (number of reads mapped to a gene x 106) / (total mapped input reads in the sample x gene length in kbp). Statistical analysis of the RPKM-values between the experimental conditions was performed using Cyber-T (37). Genes were determined to be significantly contributing to growth in human serum when the Benjamini-Hochberg corrected P-value was <0.05 and the difference in abundance of the transposon mutant during growth in BHI and serum was >2. To determine the essentiality of E.faecium genes during growth in the rich medium BHI, the normalized read-counts of the ten replicates in BHI were used as data input for the EL-ARTIST analysis, as described in the user manual of the ARTIST pipeline (38).
Isolation of mutants from the transposon mutant library pool
To recover a targeted transposon mutant from the complete mutant pool, a PCR-based screening strategy was developed (Supplementary figure 3). 40 µl of the transposon mutant library was inoculated into 40 ml of BHI broth with gentamicin and grown overnight at 37ºC with shaking (200 rpm). The overnight culture, containing approximately 109 cfu/ml, was then diluted to approximately 20 cfu/ml in 500 ml of BHI with gentamicin and kept on ice. Subsequently, 200 μl aliquots were transferred to wells of sterile 96 wells plates (n = 12, Corning Inc.). After overnight incubation at 37ºC without shaking, aliquots (15 μl) of each one of the 96 wells, were further pooled into a single new 96 well plate, as described in Supplementary figure 3.
PCRs were performed on the final plate in which the transposon mutants were pooled, to check for the presence of the Tn-mutants of interest, using the primer ftp_tn_both_ends_MmeI, which is complementary to the repeats flanking the transposon sequence, in combination with a gene-specific primer (Supplementary table 7). When a PCR was found to be positive in one of the wells of this plate, the location of the Tn-mutant was tracked backwards to the wells containing approximately 4 independent transposon mutants, by performing PCRs mapping the presence of the transposon mutant in each step. Cells from the final positive well were plated onto BHI with gentamicin and colony PCR was performed to identify the desired transposon mutant.
Growth of E. faecium E745 and individual mutants in human serum
Wild-type E745 and the mutant strains were grown overnight at 37°C in BHI broth. Subsequently, bacterial cells were washed with physiological saline and approximately 3 x 105 cfu were inoculated into 1.4 ml BHI broth or heat-inactivated serum. Cells were grown in 1.5 ml tubes (Eppendorf) in triplicate for each condition and incubated at 37°C for 24 hours without shaking. Bacterial growth was determined by assessing viable counts, for which the cultures were serially diluted using physiological saline solution and plated onto BHI agar followed by overnight incubation at 37°C.
Intravenous infection of zebrafish embryos
London wild-type (LWT) inbred zebrafish embryos, provided by the aquarium staff of The Bateson Center (University of Sheffield), were used for infection experiments. The parental E745 strain and its pyrK_2 and manY_2 transposon mutants were grown in BHI broth until they reached an optical density at 600 nm of approximately 0.5 and were then harvested by centrifugation (5,500 g, 10 min). Bacteria were microinjected into the circulation of dechorionated zebrafish embryos at 30 hours post fertilization, as previously described (39). Briefly, anesthetized embryos were embedded in 3% (w/v) methylcellulose and injected individually with approximately 1.2 x 104 cfu using microcapillary pipettes. For each strain, 29 to 32 infected embryos were observed for survival up to 90 hours post infection (hpi). This experiment was performed in triplicate.
Data availability
Sequence reads generated in this study have been made available at the European Nucleotide Archive under accession number PRJEB19025.
Results
The complete genome sequence of E. faecium E745
In this study, we implemented RNA-seq and Tn-seq analyses in E. faecium strain E745, an ampicillin-and vancomycin-resistant clinical isolate that was isolated from faeces of a hospitalized patient during an outbreak of VRE in the nephrology ward of a Dutch hospital in 2000 (24, 25). To allow the application of RNA-seq and Tn-seq in E. faecium E745, we first determined the complete genome sequence of this strain through a combination of short-read Illumina sequencing and long-read sequencing on the RSII Pacific Biosciences and Oxford NanoPore’s MinION systems. This resulted in a circular chromosomal sequence of 2,765,010 nt and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp (Supplementary table 1). Together, the chromosome and plasmids have 3,095 predicted coding sequences.
Transcriptome of E. faecium E745 during growth in rich medium and in human serum
The transcriptional profile of E745 was determined using RNA-seq during growth in rich medium (BHI) and in heat-inactivated human serum. A total of 99.9 million (15.6 - 17.6 million per sample) 100 bp paired-end reads were successfully aligned to the genome, allowing the quantification of rare transcripts (Fig. 1). A total of 3217 transcription units were identified, including 651 predicted multi-gene operons, of which the largest contains 22 genes (Fig. 1A and Supplementary table 2).
A comparative analysis of E745 during growth in BHI and in human serum, showed that 860 genes exhibited significantly (q<0.001 and a fold change in expression of >2 or <0.5 between cultures grown in BHI versus heat-inactivated serum) different expression between these conditions (Supplementary table 3). The large number (27.8% of genes on the E. faecium E745 genome) of differentially expressed genes, indicates that growth in human serum leads to a dramatic reprogramming of global E. faecium gene expression, involving both chromosomal and plasmid-located genes. Among the genes with the highest difference in expression between growth in serum and in rich medium, we identified a gene cluster with a role in purine biosynthesis (Fig. 1B). In addition, we found a 58.4 kbp prophage-like gene cluster that exhibited higher expression in E745 during growth in serum (Fig. 1C).
To confirm the RNA-seq analysis, we independently determined expression levels of eight genes during growth in serum versus growth in BHI by qPCR (Supplementary Fig. 1). RNA-seq and qPCR data were highly concordant (r2 = 0.98).
Construction and analysis of a transposon mutant library in E. faecium E745
A mariner-based transposon mutant library was generated in E. faecium E745 and Tn-seq (21) was performed on ten replicate transposon mutant libraries, that were grown overnight in BHI at 37°C, resulting in an average of 15 million Tn-seq reads for each library. To analyze the Tn-seq data, we divided the E745 genome in 25-nt windows. Of a total of 110,601 windows, 49,984 (45%) 25-nt windows contained one or more sequence reads. No positional bias was observed in the transposon insertion sites in the chromosome and plasmids of E.faecium E745 (Supplementary Fig. 2A). The genome-wide coverage of this transposon mutant library allowed the identification of genes that are conditionally essential in E.faecium. A total of 455 chromosomal genes were determined to be essential for growth of E.faecium E745 in BHI (Supplementary Table 4). An example of a gene identified as essential is shown in Supplementary Fig. 2B. Among the genes that were essential for growth of E.faecium E745 in BHI, 87% were present in all genome sequences of a set of 74 previously sequenced E. faecium strains (4) that represented the genetic and ecological diversity of the species. An additional 7% of the essential genes were present in 73 out of 74 E. faecium genomes (data not shown). The conserved presence of these essential genes among diverse E.faecium strains is in line with their crucial role in E. faecium viability. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum.
E. faecium E745 genes required for growth in human serum
In order to identify genes that contribute to growth of E. faecium E745 in human serum, we performed Tn-seq on cultures of the E. faecium E745 transposon mutant libraries upon growth in rich medium (BHI) and in human serum. The human serum was either used natively or heat-treated. The serum showed effective complement activity in both classical and alternative pathways, as determined by hemolytic assays (40), while this activity was abolished by heat-inactivation (data not shown). Minor differences were observed among conditionally essential genes between the experiments performed in native human serum or heat-inactivated serum (Supplementary Table 5) and the following results correspond to the experiments obtained with heat-inactivated serum. This condition was chosen because it may be a more reproducible in vitro environment, particularly since the interaction between the complement system and Gram-positive bacteria remains to be fully elucidated (41, 42).
We identified 37 genes that significantly contributed to growth of E745 in human serum (Fig. 2 and Supplementary table 6): twenty-nine genes were located on the chromosome and eight genes were present on plasmids (six genes on pE745-5, two genes on plasmid pE745-6). The relatively large number of genes identified indicates that growth of E. faecium in human serum is a multi-factorial process. The genes that conferred the most pronounced effect on growth of E. faecium in serum included genes that are part of a phosphotransferase systems (PTS) involved in carbohydrate uptake (manZ_3, manY_2, ptsL), a putative transcriptional regulator (algB) and genes involved in the biosynthesis of purine and pyrimidine nucleotides (guaB, purA, pyrF, pyrK_2, purD, purH, purL, purQ, purC) (Fig. 2). Notably, the purD, purH, and purL genes were found to exhibit higher expression upon growth in human serum in the RNA-seq analysis (Fig. 1). Nine genes were identified as negatively contributing to growth in serum, i.e. the transposon mutants in these genes were significantly enriched upon growth in serum. The effects of these mutations were relatively limited, compared to the major effects observed in the transposon mutants discussed above (Supplementary Table 6), but it is notable that five (clsA_1, ddcP, ldtfm, mgs, and lytA_2) of these genes have roles in cell wall and cytoplasmic membrane biosynthesis.
E. faecium genes involved in nucleotide biosynthesis or carbohydrate metabolism contribute to growth in human serum
We developed a PCR-based method (Supplementary Fig. 3) to selectively isolate five transposon mutants (in the purine metabolism genes purD and purH, the pyrimidine metabolism genes pyrF and pyrK_2 and the PTS gene manY_2) from the transposon library. Growth in rich medium of these transposon insertion mutants was equal to the parental strain. However, all mutants were significantly impaired in their growth in human serum (Fig. 3A), confirming the results of the Tn-seq experiments.
E. faecium pyrK_2 and manY_2 contribute to intravenous infection of zebrafish
Next, we investigated whether the transposon insertion mutants in the manY_2 and pyrK_2 genes were attenuated in vivo (Fig. 3B). The mutants in these genes were selected because they represent the mutants in nucleotide and carbohydrate metabolism genes that were previously shown to contribute to the growth of E. faecium in human serum. As a model for intravenous infection, we used a recently described model in which E. faecium was injected into the circulation of zebrafish embryos to mimic systemic infections (43). We showed that both the manY_2 and the pyrK_2 mutant were significantly less virulent than the parental strain. The overall survival at 92 hours post infection (hpi) of zebrafish embryos infected with WT strain was 53%, compared to 88% and 83% respectively, for zebrafish embryos that were infected with the transposon insertion mutants in manY_2 and pyrK_2.
Discussion
E. faecium can contaminate the skin and from there colonize indwelling devices such as intravenous catheters, or it can translocate from the gastrointestinal tract in immunosuppressed patients, leading to the development of bacteremia and endocarditis. E.faecium infections are often difficult to treat, due to the multi-drug resistant character of the strains causing nosocomial infections (3, 4). However, the bloodstream poses challenges for the proliferation and survival of E. faecium, including a scarcity of nutrients.
In the present study we sequenced the complete genome of a vancomycin-resistant E. faecium strain, and identified E. faecium genes that were essential for growth in human serum. A total of 37 genes were found to be required for fitness of E. faecium E745 in serum, among which genes with roles in carbohydrate uptake and nucleotide biosynthesis. Previously, fitness determinants for growth in human serum have been identified through large-scale screening of mutant libraries in both a Gram-negative (Escherichia coli) and a Gram-positive (Streptococcus pyogenes) pathogen (44, 45). Notably, these studies have also identified the ability for de novo synthesis of purines and pyrimidines as a crucial factor for growth in serum. In addition, in diverse bacteria (including Burkholderia cepacia, Pasteurella multocida, Acinetobacter baumannii, Salmonella enterica serovar Typhimurium, Bacillus anthracis, and Streptococcus pneumoniae), the ability to synthesize nucleotides contributes importantly to virulence (46–51). The data presented here indicate that de novo biosynthesis of nucleotides is also required for E. faecium growth in serum and virulence. The nucleotide biosynthesis pathway of E. faecium may be a promising target for the development of novel antimicrobials for the treatment of E. faecium bloodstream infections. Indeed, compounds that target guanine riboswitches, thereby inhibiting nucleotide biosynthesis, have already shown their efficacy in a Staphylococcus aureus infection model (52).
Three genes (ptsL, manY_2 and manZ_3) encoding subunits of PTSs were found to contribute to growth in serum in our Tn-seq experiments. Previously, PTSs have been associated with gut colonization (5) and endocarditis (7) in another clinical E. faecium strain. The PTSs identified in this study are different from these previously characterized systems, suggesting that the remarkably large number of genes encoding for PTSs in a typical E. faecium genome (4) provide metabolic flexibility for growth in a wide variety of environments.
It is notable that among the nine genes that exhibited increased fitness upon inactivation by transposon insertion, five genes are predicted to have a role in cell wall or cytoplasmic membrane biosynthesis. The protein encoded by ddcP was previously characterized as a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity (19), while ldtfm acts as a peptidoglycan L, D transpeptidase (53). The predicted α-monoglucosyldiacylglycerol synthase gene mgs is orthologous (73% amino acid identity) to bgsB in E. faecalis, which is required for the biosynthesis of membrane glycolipids (54). The clsA_1 gene is predicted to be responsible for the synthesis of cardiolipin (bisphosphatidylglycerol) and its inactivation may modulate the physical properties of the cytoplasmic membrane (55). Finally, lytA_2 is predicted to encode an autolysin, which may be involved in the turnover of peptidoglycan in the cell wall (56). The transposon mutants in these genes were not further characterized in this study, but our findings suggest that genes involved in cell wall or cytoplasmic membrane remodeling may confer subtle fitness defects to E. faecium when growing in serum.
Our RNA-seq-based transcriptional profiling of E. faecium E745 during mid-exponential growth in serum showed pervasive changes in gene expression compared to exponential growth in rich medium. The purine metabolism genes purL, purH, purD, which were found to be required for growth in serum in our Tn-seq experiments, were among those that were significantly upregulated during growth in serum compared to growth in rich medium. Notably, a single prophage was expressed at higher levels during growth in serum than in rich medium. The abundance of prophage elements in the genome of E. faecium has been noted before (4, 57, 58). Interestingly, in the related bacterium Enterococcus faecalis prophages encode platelet-binding proteins (59) and may have a role in intestinal colonization (60). The contribution of E. faecium prophages to traits that are important for colonization and infection may provide important insights into the success of E. faecium as a nosocomial pathogen.
Collectively our data indicates that nucleotide biosynthesis and carbohydrate metabolism are critical metabolic pathways for the proliferation and survival of E. faecium in the bloodstream. The proteins encoded by the genes required for growth in human serum that were identified in this study, could serve as candidates for the development of novel anti-infectives for the treatment of bloodstream-infections by multi-drug resistant E. faecium.
Author contributions
X. Z, R. J. L. W. and W.v. S. designed the study. X. Z., A. M. G. P., T. K. P., M. B., J. R. B., M. R. and S. M. performed experiments. All authors contributed to data interpretation. The manuscript was written by Z. X., A. M. G. P., M. J. M. B., R. J. L. W. and W.v. S.
Acknowledgements
This work was supported by the European Union Seventh Framework Programme (FP7-HEALTH-2011-single-stage) “Evolution and Transfer of Antibiotic Resistance” (EvoTAR) under grant agreement number 282004 and by an NWO-VENI grant (916.86.044) and NWO-VIDI grant (917.13.357) to W.v. S.