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Role of Mobile DNA in the Evolution of Vancomycin-Resistant Enterococcus faecalis

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Science  28 Mar 2003:
Vol. 299, Issue 5615, pp. 2071-2074
DOI: 10.1126/science.1080613

Abstract

The complete genome sequence of Enterococcus faecalisV583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknownvanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.

The Gram-positive bacteriumEnterococcus faecalis is a natural inhabitant of the mammalian gastrointestinal tract and is commonly found in soil, sewage, water, and food, frequently through fecal contamination (1).E. faecalis can withstand oxidative stress, desiccation, and extremes of temperature and pH, and it has high endogenous resistance to salinity, bile acids, detergents, and antimicrobials (1).

E. faecalis is an opportunistic pathogen that is a major cause of urinary tract infections, bacteremia, and infective endocarditis (2). The intrinsic resistance of E. faecalis to many antibiotics and its acquisition of resistance to other antimicrobial agents, particularly vancomycin, which is used to treat serious infections by drug-resistant Gram- positive pathogens, has led to the emergence of E. faecalis as a nosocomial pathogen that is refractory to most therapeutic options (3). Recent reports of the long-predicted emergence of vancomycin-resistant Staphylococcus aureus clinical isolates from transfer of enterococcal genes is a serious health care concern (4). Here we report the complete genome sequence ofE. faecalis strain V583 (5), the first vancomycin-resistant clinical isolate reported in the United States (6). The genome sequence provides insight into the pathogenesis and biology of E. faecalis, the role of mobile elements in genome evolution, and the transfer of vancomycin resistance.

A total of 3337 predicted protein-encoding open reading frames (ORFs) were identified on the chromosome and three plasmids of E. faecalis V583 (Table 1; fig. S1) (7). Over a quarter of the E. faecalisV583 genome consists of mobile and/or exogenously acquired DNA, including seven probable integrated phage regions, 38 insertion elements (IS), multiple conjugative and composite transposons, a putative pathogenicity island, and integrated plasmid genes. To our knowledge, this represents one of the highest proportions of mobile elements observed in a bacterial genome. The plethora of mobile elements probably contributed to the accumulation of virulence and drug resistance factors by E. faecalis.

Table 1

General features of the E. faecalis genome. No., number; rRNA, ribosomal RNA; tRNA, transfer RNA.

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Vancomycin resistance in E. faecalis V583 appears to be encoded within a previously unknown mobile element (EF2282-EF2334) with some similarities to the probable E. faecalis vanBvancomycin-resistance conjugative transposon Tn1549(8). The vancomycin-resistance genes (EF1955-EF1963) encode vancomycin resistance via synthesis of modified peptidoglycan precursors terminating in d-lactate (9), and they are essentially identical to the vanB genes from Tn1549. The remainder of the element is very divergent from Tn1549 with multiple insertions, deletions, and rearrangements (Fig. 1); relatively low sequence similarity between conserved genes; and a different recombinational system (EF2283). Even though E. faecalisV583 is the earliest known vancomycin- resistant clinical isolate from the United States, the conjugative transposon-like features and atypical trinucleotide of this element indicate it was likely obtained as a cassette by lateral gene transfer. It is also flanked by Tn916-like genes (Fig. 1), which may have played a role in the acquisition of this element.

Figure 1

Linear representation of the E. faecalis V583 vanB vancomycin-resistance gene region and its relationship with the vancomycin-resistance transposon Tn1549 (8) and the E. faecalis V583vncRS/vex locus. Genes are shown as arrowheads (not to scale) colored by predicted function or transposable element: black, vanB vancomycin-resistance genes; magenta, Tn1549-like genes; orange, Tn916-like genes; green, transposases; red,vncRS/vex locus; brown, group II intron; light blue, transposon resolvase. Black lines connect best matches (BLAST P-value < 1 × 10−5), and red lines connect best matches with greater than 99% identity. Genes are labeled by name or by the appropriate ORF or locus numbers.

Highly similar Tn916-like genes are also found in association with a locus (EF1869-EF1863) encoding homologs of theStreptococcus pneumoniae VncRS two-component signal transduction system and Vex secretion proteins (Fig. 1). ThevncRS locus has been associated with vancomycin tolerance inS. pneumoniae via mutation of vncS(10), although recent evidence has cast doubt on this association (11). In E. faecalis V583, thevncS gene (EF1866) is disrupted by a nonfunctional ISL3 family insertion sequence. The possible role of this element in vancomycin tolerance in E. faecalis is unclear, but it is flanked by copies of IS256 and may also have been laterally acquired.

Thirty-eight IS elements were identified (table S1), with three types predominating: ISEf1, IS256, and IS1216. There are two clusters of IS elements on the chromosome (fig. S1). One is associated with a pathogenicity island and integrated plasmid genes. The second cluster includes several types of IS elements that flank a region of atypical trinucleotide composition (EF1860-EF1858) that may have been acquired by lateral gene transfer [Supporting Online Material (SOM) Text] and encodes three of the four steps of pantothenate biosynthesis.

A large pathogenicity island has previously been identified in E. faecalis V583 (12) (EF0479-EF0628), including genes for aggregation substance, cytolysin, and other possible virulence or adaptation genes. Trinucleotide composition analysis indicated that most of this island has highly atypical composition, except for a region containing integrated plasmid genes from a pTEF1-like plasmid (fig. S1). The presence of multiple IS elements and the integrated plasmid genes hints at a complex evolutionary history for this element. It is flanked at one end by an integrase gene, possibly responsible for integration of this element.

There are seven regions derived from probable integrated phage (fig. S1). These putative prophage are most closely related to phage from other low-GC Gram-positive bacteria. The integrated phage regions encode multiple homologs of Streptococcus mitis PblA and PblB, which have been implicated in binding human platelets, an interaction important in the pathogenesis of infective endocarditis (13). A ferrochetalase gene (EF1989) encoded within one of the phage regions may allow E. faecalis to utilize coproporphyrinogen III for heme synthesis (SOM Text).

A variety of diverse plasmids have been previously described inE. faecalis, particularly conjugative plasmids that encode a mating response to sex pheromone peptides secreted by plasmid-free recipient strains (14). Three plasmids are present in E. faecalis V583 (Table 1; fig. S2): pTEF1 and pTEF2 are structurally similar to the archetypal pheromone responsive plasmids pAD1 (15) and pCF10 (14), respectively, and pTEF3 belongs to the family of pAMβ1 broad host range plasmids.

The sex pheromone inhibitor (iAD1) and surface aggregation substance (Asa1) encoded by pTEF1 are identical to those of pAD1, and both plasmids share extensive regions of sequence similarity (fig. S2). There is a 31-kb inversion in pTEF1 relative to pAD1 that probably affects regulation of conjugation in E. faecalis V583 (SOM Text). The unique regions in pTEF1 include a Tn4001-like transposon encoding aminoglycoside resistance and another IS-flanked element carrying erythromycin resistance and multidrug resistance genes.

pTEF2 and the sex pheromone plasmid pCF10 share regions of similarity, including identical copies of the conjugation genesprgA-prgB-prgC, but pTEF2 lacks the pCF10 pheromone inhibitor prgQ gene, encoding a previously undescribed predicted pheromone inhibitor gene (EFB0005.1) in the equivalent position. pTEF3 is a nonconjugative plasmid but has acquired a pTEF2-like prgZ pheromone receptor, whose gene is adjacent to multiple IS elements. The occurrence of a novel pheromone inhibitor on pTEF2 suggests the possible occurrence of a broad diversity of different E. faecalis pheromones and pheromone inhibitors in nature. Five sex pheromones encoded within lipoprotein signal peptides have been identified in the genome of E. faecalis V583 (16). An additional 76 predicted lipoproteins were identified (table S2), some of which could represent previously unknown pheromone precursors.

The chromosome of E. faecalis contains at least three integrated plasmid remnants. Two of these regions encode homologs of aggregation substance (EF0485, EF0149) that may be important for virulence (fig. S3). Plasmid-encoded aggregation substance is a surface protein that enhances conjugative transfer and has been implicated in adhesion to colonic mucosal fibronectin and in translocation across intestinal epithelium (17). One of the integrated plasmid regions (EF0506-EF0485) is located within the probable pathogenicity island (12); the other two regions include genes of plasmid, phage, and conjugative transposon origin (fig. S3; SOM Text) and encode lipoproteins (EF0164 and EF2512) whose signal peptides resemble those of known pheromone precursors. The presence of multiple integrated plasmid remnants and three resident plasmids inE. faecalis V583 emphasizes the importance of plasmids in genome plasticity of the enterococci.

Comparison of the predicted protein set of E. faecalis with those of other sequenced genomes confirmed the relationship of E. faecalis within the low-GC Gram-positive bacteria. Over 85% ofE. faecalis ORFs with significant database matches had their best match to other sequenced low-GC Gram- positive organisms, and 519 E. faecalis ORFs were conserved in a set of 10 sequenced low-GC Gram-positive organisms (table S3) (5). The distribution of these conserved genes in the E. faecalisgenome revealed a number of regions with a low abundance of conserved genes (fig. S1), largely corresponding to the identified phage, integrated plasmid, pathogenicity island, and conjugative transposon regions. This set of conserved genes is involved in essential processes such as transcription, translation, and protein synthesis but also includes a considerable number of proteins from large paralogous families such as the PTS and ABC transporter families.

There is essentially no large-scale gene synteny between theE. faecalis genome and that of any sequenced low-GC Gram-positive bacterium. The multiplicity of mobile or foreign elements such as phage and IS elements suggests the E. faecalisgenome is highly malleable and may have undergone multiple rearrangement events, explaining the lack of gene synteny. Despite this lack of synteny, there is a strong transcriptional skew (fig. S1) as found in other sequenced low-GC Gram-positive bacteria. Genes encoded within the mobile elements also show a transcriptional skew; within the phage regions, for instance, 90% of the ORFs align with the direction of replication. Strong selective pressure for genes to be transcribed in the direction of replication appears to be a common feature of the low-GC Gram-positive bacteria.

Analysis of the transport and metabolic capabilities of E. faecalis V583 emphasizes the importance of fermentation of nonabsorbed sugars in the gastrointestinal tract in its life-style.E. faecalis has 35 probable PTS-type sugar transporters, comparable to Listeria species and considerably more than any other sequenced organism, as well as ABC-type and other sugar uptake systems (table S4). Consistent with these transport capabilities, E. faecalis encodes pathways for the utilization of more than 15 different sugars (table S4). Energy production from these substrates occurs via glycolysis or the pentose phosphate pathways, with the trichloroacetic acid (TCA) cycle absent.

E. faecalis is one of the few bacteria that are substantial producers of extracellular O2(18), and an array of oxidative stress resistance mechanisms are evident from its genome (table S5). E. faecalis is well endowed with cation homeostasis mechanisms (table S5), which probably contribute to its pH, salt, metal, and desiccation resistance, including 14 predicted metal ion P-type ATPases, more than any other currently sequenced bacterium. Despite its stress resistance capabilities, E. faecalis possesses a modest collection of regulatory genes (table S6), including only three alternate sigma factors.

E. faecalis is known to adhere to a variety of host cells or extracellular matrix components. Adhesins such as aggregation substance, MSCRAMM, hemagglutinin, and other virulence factors have been described for Enterococcus spp. (1). A comprehensive genome-wide exploration for signal sequences, lipoprotein motifs, and other potential host cell components or binding motifs yielded an additional 134 putative surface-exposed proteins that may be associated with early colonization stages or virulence (table S7).

Forty-seven candidates with potential choline- or integrin-binding motifs were identified that may play a role in adherence and internalization processes (table S7). A family of four probable adhesin lipoproteins was identified, including a characterized endocarditis-specific antigen (EF2076). A paralogous family of 21 LPxTG-motif cell wall surface anchor proteins is present, as are three corresponding sortases (EF3056, EF1094, and EF2524). Several of the genes for LPxTG proteins have an atypical nucleotide composition, suggesting a foreign origin. Translocation across the intestinal epithelium (19) may be facilitated by homologs of L. monocytogenes internalin A (EF2686), an S. aureusexfoliative toxin (EF0645), and probable secreted hyaluronidases (EF3023 and EF0818) and a protease (EF1818).

Antigenic or phase variation of surface structures is a common immune evasion tactic amongst pathogens and has been implicated in activating conjugation function in E. faecalis (20). Out of 134 putative surface- exposed proteins, 65 were found to contain stretches of homopolymeric sequence or iterative nucleotide motifs located within the predicted ORF or promoter region, which may enable phase variation via a slippage type mechanism (table S7).

An unprecedented amount of the E. faecalis V583 genome consists of intact or partial mobile elements. Many of these regions have complex mosaic structures comprised of different elements, suggesting they are “hotspots” or “graveyards” for mobile element insertion. This apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci and suggests that they act as a reservoir for the further dissemination of drug resistance traits such as vancomycin resistance via mobile elements and/or conjugative plasmids. The complete genome sequence ofE. faecalis V583 has enabled the identification of numerous predicted virulence factors and surface-exposed proteins that may facilitate the development of therapeutic approaches to combat this important nosocomial pathogen.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5615/2071/DC1

Materials and Methods

SOM Text

Figs. S1 to S3

Tables S1 to S7

  • * To whom correspondence should be addressed. E-mail: ipaulsen{at}tigr.org

  • Present address: Celera Genomics, 45 West Gude Drive, Rockville, MD 20850, USA.

  • Present address: Bristol-Myers Squibb PRI, 5 Research Parkway, Wallingford, CT 06492, USA.

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