The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis - PubMed (original) (raw)

The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis

Diana Panesso et al. BMC Microbiol. 2011.

Abstract

Background: Plasmids containing hylEfm (pHylEfm) were previously shown to increase gastrointestinal colonization and lethality of Enterococcus faecium in experimental peritonitis. The hylEfm gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated E. faecium, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the hylEfm-region and we evaluated their effect on virulence using a murine peritonitis model.

Results: Five mutants of the hylEfm-region of pHylEfmTX16 from the sequenced endocarditis strain (TX16 [DO]) were obtained using an adaptation of the PheS* system and were evaluated in a commensal strain TX1330RF to which pHylEfmTX16 was transferred by mating; these include i) deletion of hylEfm only; ii) deletion of the gene downstream of hylEfm (down) of unknown function; iii) deletion of hylEfm plus down; iv) deletion of hylEfm-down and two adjacent genes; and v) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by cat. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHylEfmTX16Δ7,534 was transferred to the TX1330RF background, the transconjugant was affected in in vitro growth versus TX1330RF(pHylEfmTX16) and was attenuated in virulence; however, neither hylEfm nor hylEfm-down restored wild type function. We did not observe any in vivo effect on virulence of the other deletions of the hylEfm-region

Conclusions: The four genes of the hylEfm region (including hylEfm) do not mediate the increased virulence conferred by pHylEfmTX16 in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of E. faecium in the future.

PubMed Disclaimer

Figures

Figure 1

Physical map of the hyl Efm -region in pHyl EfmTX16. The annotated predicted function of the corresponding genes is shown above the genes. The genes were divided into three groups (metabolism, transport [in gray] and regulation based on putative functions). Strain nomenclature follows that specified in Table 1. Black arrows above the genes indicate the position of the primers used to obtain DNA fragments for mutagenesis and follow the nomenclature of Table 2. The crosses depict the genes that were deleted. The asterisks indicate only partial deletion of the gene was obtained. _a_The number refers to the glycosyl hydrolase family with hyl Efm depicted in bold; _b_allelic replacement with the chloramphenicol acetyl transferase gene (cat) was performed. NA, not applicable.

Figure 2

Physical map of the plasmids pHOU1 and pHOU2 for targeted mutagenesis of E. faecium. A, plasmid used for construction of TX1330RF (pHylEfmTX16Δ4genes), TX1330RF(pHylEfmTX16Δ_hyl_), TX1330RF(pHylEfmTX16Δ_hyl-down_) and TX1330RF (pHylEfmTX16Δ_down_) deletion mutants (Figure 1); B, plasmid used for construction of the TX1330RF(pHylEfmTX16Δ7,534) deletion mutant (Figure 1)

Figure 3

Transcriptional analysis of genes in the hyl Efm region using reverse transcriptase (RT)-PCR. A, physical map of the hyl Efm region and primers used for RT-PCR experiments. Black arrows above the genes indicate the position of the primers used to amplify DNA sequences from the cDNA obtained after reverse transcription. B, RT-PCR using primers A1-A2; C, RT-PCR using primes B1-B2; D, RT-PCR using primers C1-C2; E, RT-PCR using primers D1-D2; F, RT-PCR with ddl as the target gene using primers E1-E2 (Table 2) [32,33]. Lanes 1 and 2, TX1330RF (RT-PCR reaction and control without RT enzyme, respectively); lanes 3 and 4, TX1330RF(pHylEfm16) (RT-PCR reaction and control without RT enzyme, respectively); lanes 5 and 6 TX16(pHylEfm16) (RT-PCR reaction and control without RT enzyme respectively). The molecular weight of the bands is indicated to the right.

Figure 4

Growth and survival curves in the mouse peritonitis model of E. faecium TX0016(pHyl EfmTX16 ) and TX1330RF(pHyl EfmTX16 ), carrying an intact hyl Efm -region, and pHyl EfmTX16Δ7,534 (6 gene mutant of the hyl Efm -region). A, Survival curve of representative inoculum (5 inocula per experiment in two independent experiments) of TX0016(pHylEfmTX16) vs TX0016(pHylEfmTX16Δ7,534) in mouse peritonitis; B, growth curves of TX1330RF(pHylEfmTX16) vs TX1330RF(pHylEfmTX16Δ7,534) and a second transconjugant [TX1330RF(pHylEfmTX16Δ7,534)-TCII] obtained from the same mating experiment between TX16(pHylEfmTX16Δ7,534) and TX1330RF, expressed as optical density (_A_600) in brain heart infusion (BHI) broth (results of at least three experiments per strain). C and D, survival curves of TX1330RF(pHylEfmTX16) vs TX1330RF(pHylEfmTX16Δ7,534) obtained in the peritonitis model at different inocula in independent experiments performed at different days.

Figure 5

Survival curves in the mouse peritonitis model of E. faecium TX1330RF and derivatives. A and B show survival curves of the TX1330RF(pHylEfmTX16Δ7,534) (6 gene mutant in the hyl Efm region) complemented with pAT392-derivatives (which include pAT392::hyl Efm and pAT392::hyl Efm -down) obtained in the peritonitis model at different inocula in independent experiments performed at different days. The asterisk indicates that the lines are superimposed since values are identical.

Figure 6

Survival curves in the mouse peritonitis model of E. faecium TX1330RF(pHyl EfmTX16 ) and deletion mutants (Figure 1 and Table 1) showing representative inocula (5 inocula per each experiment). A, TX1330RF(pHylEfmTX16) vs TX1330RF(pHylEfmTX16Δ4genes); B, TX1330RF(pHylEfmTX16) vs TX1330RF (pHylEfmTX16Δhyl); C, TX1330RF(pHylEfmTX16) vs TX1330RF(pHylEfmTX16Δ_hyl-down_); D, TX1330RF(pHylEfmTX16) vs TX1330RF(pHylEfmTX16Δ_down_)

References

1. Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK. NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infect Control Hosp Epidemiol. 2008;29(11):996–1011. doi: 10.1086/591861. -DOI -PubMed
1. Willems RJ, van Schaik W. Transition of Enterococcus faecium from commensal organism to nosocomial pathogen. Future Microbiol. 2009;4:1125–1135. doi: 10.2217/fmb.09.82. -DOI -PubMed
1. van Schaik W, Top J, Riley DR, Boekhorst J, Vrijenhoek JE, Schapendonk CM, Hendrickx AP, Nijman IJ, Bonten MJ, Tettelin H. et al. Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island. BMC Genomics. 2010;11:239. doi: 10.1186/1471-2164-11-239. -DOI -PMC -PubMed
1. Willems RJ, Top J, van Santen M, Robinson DA, Coque TM, Baquero F, Grundmann H, Bonten MJ. Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex. Emerg Infect Dis. 2005;11(6):821–828. -PMC -PubMed
1. Heikens E, Bonten MJ, Willems RJ. Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162. J Bacteriol. 2007;189(22):8233–8240. doi: 10.1128/JB.01205-07. -DOI -PMC -PubMed

The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis - PubMed (original) (raw)