Use of recombinase-based in vivo expression technology to characterize Enterococcus faecalis gene expression during infection identifies in vivo-expressed antisense RNAs and implicates the protease Eep in pathogenesis

Kristi L Frank; Aaron M T Barnes; Suzanne M Grindle; Dawn A Manias; Patrick M Schlievert; Gary M Dunny

doi:10.1128/iai.05964-11

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Use of recombinase-based in vivo expression technology to characterize Enterococcus faecalis gene expression during infection identifies in vivo-expressed antisense RNAs and implicates the protease Eep in pathogenesis

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Use of recombinase-based in vivo expression technology to characterize Enterococcus faecalis gene expression during infection identifies in vivo-expressed antisense RNAs and implicates the protease Eep in pathogenesis

Kristi L Frank, Aaron M T Barnes, Suzanne M Grindle, Dawn A Manias, Patrick M Schlievert and Gary M Dunny

Infection and immunity, Vol.80(2), pp.539-549

02/2012

DOI: 10.1128/iai.05964-11

PMCID: PMC3264308

PMID: 22144481

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Abstract

Enterococcus faecalis is a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades. E. faecalis must be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-based in vivo expression technology (RIVET) to identify promoters on the E. faecalis OG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putative in vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected in in vitro- and in vivo-grown cells, providing the first evidence of in vivo-expressed antisense RNAs in E. faecalis. Deletions in the in vivo-activated genes that encode glutamate 5-kinase (proB [EF0038]), the transcriptional regulator EbrA (ebrA [EF1809]), and the membrane metalloprotease Eep (eep [EF2380]) did not hinder biofilm formation in in vitro assays. In a rabbit model of endocarditis, the ΔebrA strain was fully virulent, the ΔproB strain was slightly attenuated, and the Δeep strain was severely attenuated. The Δeep virulence defect could be complemented by the expression of the wild-type gene in trans. Microscopic analysis of early Δeep biofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

Abscess - microbiology

Rabbits

Enterococcus faecalis - pathogenicity

Recombinases - metabolism

Membrane Proteins - genetics

Enterococcus faecalis - genetics

Bacterial Proteins - genetics

Genetic Techniques

Gene Expression Regulation, Bacterial - physiology

Animals

Biofilms

Enterococcus faecalis - metabolism

RNA, Antisense - metabolism

Gram-Positive Bacterial Infections - microbiology

Endocarditis, Bacterial - microbiology

Bacterial Proteins - metabolism

Membrane Proteins - metabolism

RNA, Bacterial - metabolism

RNA, Antisense - genetics

Details

Title: Subtitle: Use of recombinase-based in vivo expression technology to characterize Enterococcus faecalis gene expression during infection identifies in vivo-expressed antisense RNAs and implicates the protease Eep in pathogenesis
Creators: Kristi L Frank - Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA. fran0616@umn.edu
Aaron M T Barnes
Suzanne M Grindle
Dawn A Manias
Patrick M Schlievert
Gary M Dunny
Resource Type: Journal article
Publication Details: Infection and immunity, Vol.80(2), pp.539-549
Publisher: United States
DOI: 10.1128/iai.05964-11
PMID: 22144481
PMCID: PMC3264308
ISSN: 1098-5522
eISSN: 1098-5522
Grant note: R01AI58134 / NIAID NIH HHS F32AI082881 / NIAID NIH HHS R01 AI058134 / NIAID NIH HHS T32GM008244 / NIGMS NIH HHS T32 DE007288 / NIDCR NIH HHS HHSN266200400042C / PHS HHS F32 AI082881 / NIAID NIH HHS T32DE007288 / NIDCR NIH HHS T32 GM008244 / NIGMS NIH HHS
Language: English
Date published: 02/2012
Academic Unit: Microbiology and Immunology; Internal Medicine
Record Identifier: 9984001136502771

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