Dataset Information

VirB8-like protein TraH is crucial for DNA transfer in Enterococcus faecalis.

ABSTRACT: Untreatable bacterial infections caused by a perpetual increase of antibiotic resistant strains represent a serious threat to human healthcare in the 21(st) century. Conjugative DNA transfer is the most important mechanism for antibiotic resistance and virulence gene dissemination among bacteria and is mediated by a protein complex, known as type IV secretion system (T4SS). The core of the T4SS is a multiprotein complex that spans the bacterial envelope as a channel for macromolecular secretion. We report the NMR structure and functional characterization of the transfer protein TraH encoded by the conjugative Gram-positive broad-host range plasmid pIP501. The structure exhibits a striking similarity to VirB8 proteins of Gram-negative secretion systems where they play an essential role in the scaffold of the secretion machinery. Considering TraM as the first VirB8-like protein discovered in pIP501, TraH represents the second protein affiliated with this family in the respective transfer operon. A markerless traH deletion in pIP501 resulted in a total loss of transfer in Enterococcus faecalis as compared with the pIP501 wild type (wt) plasmid, demonstrating that TraH is essential for pIP501 mediated conjugation. Moreover, oligomerization state and topology of TraH in the native membrane were determined providing insights in molecular organization of a Gram-positive T4SS.

SUBMITTER: Fercher C

PROVIDER: S-EPMC4840375 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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VirB8-like protein TraH is crucial for DNA transfer in Enterococcus faecalis.

Fercher Christian C Probst Ines I Kohler Verena V Goessweiner-Mohr Nikolaus N Arends Karsten K Grohmann Elisabeth E Zangger Klaus K Meyer N Helge NH Keller Walter W

Scientific reports 20160422

Untreatable bacterial infections caused by a perpetual increase of antibiotic resistant strains represent a serious threat to human healthcare in the 21(st) century. Conjugative DNA transfer is the most important mechanism for antibiotic resistance and virulence gene dissemination among bacteria and is mediated by a protein complex, known as type IV secretion system (T4SS). The core of the T4SS is a multiprotein complex that spans the bacterial envelope as a channel for macromolecular secretion. ...[more]

PMID: 27103580

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Project description:Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transferred. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens.

Project description:Tn5385 is a ca. 65-kb element integrated into the chromosomes of clinical Enterococcus faecalis strains CH19 and CH116. It confers resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, tetracycline-minocycline, and penicillin via beta-lactamase production. Tn5385 is a composite structure containing regions previously found in staphylococcal and enterococcal plasmids. Several transposons and transposon-like elements within Tn5385 have been identified, including conjugative transposon Tn5381, composite transposon Tn5384, and elements indistinguishable from staphylococcal transposons Tn4001 and Tn552. The divergent regions of Tn5385 are linked by a series of insertion sequence (IS) elements (IS256, IS257, and IS1216) of staphylococcal and enterococcal origin. The ends of Tn5385 consist of directly repeated copies of enterococcal IS1216. Within the chromosomes of strains CH19 and CH116, Tn5385 has interrupted an open reading frame with substantial homology to previously described alkyl hydrogen peroxide reductase genes. Segments of this open reading frame in both CH19 and CH116 have been deleted, but the amount of deleted DNA differs for the two insertions. Transfer of Tn5385 from both donors into E. faecalis recipients occurs at a low frequency. Two types of transconjugants have been identified. In one type, the target alkyl hydrogen peroxide reductase open reading frame has been deleted, and sequences flanking Tn5385 in the respective donors are carried over to the transconjugants. These data suggest that the mechanism of Tn5385 insertion into the recipient chromosome in these transconjugants was recombination across flanking regions in the donors and homologous sequences in the recipients. The second type of transconjugant appears to have resulted from excision of Tn5385 from the CH19 chromosome by recombination across the terminal IS1216 elements and insertion into the recipient chromosome by recombination across Tn5381 (within Tn5385) and a previously transferred Tn5381 copy in the recipient chromosome. These data confirm that Tn5385 is a composite structure with genetic material from diverse genera and suggest that it is a functional transposon. They also suggest that chromosomal recombination is a mechanism of genetic exchange in enterococci.

Project description:Antibiotic-resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Enterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in E. faecalis is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this "CRISPR tolerance." We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR-assisted genome-editing scheme. Our results suggest that E. faecalis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.IMPORTANCE CRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently nonlethal. We uncover novel phenotypes associated with this "CRISPR tolerance" and, after determining its genetic basis, develop a genome-editing platform in E. faecalis with negligible off-target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR-induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.

Dataset Information

VirB8-like protein TraH is crucial for DNA transfer in Enterococcus faecalis.

Publications

VirB8-like protein TraH is crucial for DNA transfer in Enterococcus faecalis.

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