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Bioluminescence based biosensors for quantitative detection of enterococcal peptide-pheromone activity reveal inter-strain telesensing in vivo during polymicrobial systemic infection.

La Rosa SL, Solheim M, Diep DB, Nes IF, Brede DA - Sci Rep (2015)

Bottom Line: Enterococcus faecalis is a significant threat in the nosocomial setting due to the emergence of isolates that are multi-antibiotic resistant, refractory to the available therapies and equipped with a variety of pathogenicity determinants.The two E. faecalis reporters conditionally expressed bioluminescence in the presence of GBAP and CylL(S) both in the supernatants of liquid cultures and in an agar-overlay assay in as little as three hours, with a high level of sensitivity.Furthermore, we identified a clinical E. faecalis isolate that acted as a biological cheater, producing cytolysin only upon sensing CylL(S)-producers in its environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Aas, Norway.

ABSTRACT
Enterococcus faecalis is a significant threat in the nosocomial setting due to the emergence of isolates that are multi-antibiotic resistant, refractory to the available therapies and equipped with a variety of pathogenicity determinants. This bacterium uses quorum-sensing systems to regulate its physiological processes, including the expression of virulence traits, to adapt and proliferate within a host. Here, we describe the construction and application of two bioluminescence-based reporter systems for the direct detection of the quorum-sensing regulated expression of (i) the gelatinase biosynthesis-activating pheromone (GBAP) and (ii) the cytolysin small subunit (CylL(S)) in natural samples. The two E. faecalis reporters conditionally expressed bioluminescence in the presence of GBAP and CylL(S) both in the supernatants of liquid cultures and in an agar-overlay assay in as little as three hours, with a high level of sensitivity. Biosensors employed to investigate the interaction between the fsr and cyl systems revealed that fsr impeded CylL(S) activity by 75%. Furthermore, we identified a clinical E. faecalis isolate that acted as a biological cheater, producing cytolysin only upon sensing CylL(S)-producers in its environment. This isolate enhanced its virulence during polymicrobial systemic infection of Galleria mellonella.

No MeSH data available.


Related in: MedlinePlus

Bioluminescence-based detection of CylLS (A) and GBAP (B) producers.From the right to the left, uppermost row: V583, DS5, and E1Sol; central row: V583fsrB*, CH188, and ×98; bottom row: MHH594, T2, and V583ΔgelE. The E. faecalis isolates were cultured on GM17 agar plates, and the biosensor was overlaid after an overnight incubation. Plates were kept at 37°C for 3 hours and imaged with a Xenogen IVIS Lumina II Imaging System (Calipers Corp., CA). A 10-fold higher light emission was induced by CylLS than by GBAP.
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f2: Bioluminescence-based detection of CylLS (A) and GBAP (B) producers.From the right to the left, uppermost row: V583, DS5, and E1Sol; central row: V583fsrB*, CH188, and ×98; bottom row: MHH594, T2, and V583ΔgelE. The E. faecalis isolates were cultured on GM17 agar plates, and the biosensor was overlaid after an overnight incubation. Plates were kept at 37°C for 3 hours and imaged with a Xenogen IVIS Lumina II Imaging System (Calipers Corp., CA). A 10-fold higher light emission was induced by CylLS than by GBAP.

Mentions: To test the ability of the JH2-2 CBS and V583fsrB* GBS to sense the presence of CylLS and GBAP producers in the environment, we employed nine genome-sequenced E. faecalis strains of clinical and commensal origin and developed a screening method that utilised GM17-agar plates. The strains had been previously tested for their cytolytic phenotype and GBAP-production ability (S. Leanti La Rosa, L. G. Snipen, B. E. Murray, R. Willems, M. S. Gilmore, D. B. Diep, I. F. Nes, and D. A. Brede, submitted). The panel included the cytolysin-positive strains DS5 and ×98, the GBAP-positive isolates E1Sol, V583 and V583ΔgelE, the GBAP- and cytolysin-producer MMH594, the GBAP- and cytolysin-negative strains T2 and CH188, and V583fsrB*, which harbours a mutation in the fsrB gene and is thus unable to synthesise the GBAP pheromone. Cells of the above-mentioned strains were cultured on two GM17-agar plates that were individually overlaid with the biosensors. Induction of visible light emission by the CylLS- and GBAP-producers but not by the non-producers occurred by 3 hours following the application of the appropriate biosensor (Figure 2). These bioluminescently tagged E. faecalis strains may therefore offer a simple, cost-effective and rapid method for determining the presence of cytolysin or GBAP producers in food, water, faecal and clinical samples. Furthermore, the systems were highly specific in sensing CylLS and GBAP and therefore would effectively prevent the false positive assumption of virulence traits based on only the detection of genes that might not necessarily lead to the corresponding phenotype.


Bioluminescence based biosensors for quantitative detection of enterococcal peptide-pheromone activity reveal inter-strain telesensing in vivo during polymicrobial systemic infection.

La Rosa SL, Solheim M, Diep DB, Nes IF, Brede DA - Sci Rep (2015)

Bioluminescence-based detection of CylLS (A) and GBAP (B) producers.From the right to the left, uppermost row: V583, DS5, and E1Sol; central row: V583fsrB*, CH188, and ×98; bottom row: MHH594, T2, and V583ΔgelE. The E. faecalis isolates were cultured on GM17 agar plates, and the biosensor was overlaid after an overnight incubation. Plates were kept at 37°C for 3 hours and imaged with a Xenogen IVIS Lumina II Imaging System (Calipers Corp., CA). A 10-fold higher light emission was induced by CylLS than by GBAP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321189&req=5

f2: Bioluminescence-based detection of CylLS (A) and GBAP (B) producers.From the right to the left, uppermost row: V583, DS5, and E1Sol; central row: V583fsrB*, CH188, and ×98; bottom row: MHH594, T2, and V583ΔgelE. The E. faecalis isolates were cultured on GM17 agar plates, and the biosensor was overlaid after an overnight incubation. Plates were kept at 37°C for 3 hours and imaged with a Xenogen IVIS Lumina II Imaging System (Calipers Corp., CA). A 10-fold higher light emission was induced by CylLS than by GBAP.
Mentions: To test the ability of the JH2-2 CBS and V583fsrB* GBS to sense the presence of CylLS and GBAP producers in the environment, we employed nine genome-sequenced E. faecalis strains of clinical and commensal origin and developed a screening method that utilised GM17-agar plates. The strains had been previously tested for their cytolytic phenotype and GBAP-production ability (S. Leanti La Rosa, L. G. Snipen, B. E. Murray, R. Willems, M. S. Gilmore, D. B. Diep, I. F. Nes, and D. A. Brede, submitted). The panel included the cytolysin-positive strains DS5 and ×98, the GBAP-positive isolates E1Sol, V583 and V583ΔgelE, the GBAP- and cytolysin-producer MMH594, the GBAP- and cytolysin-negative strains T2 and CH188, and V583fsrB*, which harbours a mutation in the fsrB gene and is thus unable to synthesise the GBAP pheromone. Cells of the above-mentioned strains were cultured on two GM17-agar plates that were individually overlaid with the biosensors. Induction of visible light emission by the CylLS- and GBAP-producers but not by the non-producers occurred by 3 hours following the application of the appropriate biosensor (Figure 2). These bioluminescently tagged E. faecalis strains may therefore offer a simple, cost-effective and rapid method for determining the presence of cytolysin or GBAP producers in food, water, faecal and clinical samples. Furthermore, the systems were highly specific in sensing CylLS and GBAP and therefore would effectively prevent the false positive assumption of virulence traits based on only the detection of genes that might not necessarily lead to the corresponding phenotype.

Bottom Line: Enterococcus faecalis is a significant threat in the nosocomial setting due to the emergence of isolates that are multi-antibiotic resistant, refractory to the available therapies and equipped with a variety of pathogenicity determinants.The two E. faecalis reporters conditionally expressed bioluminescence in the presence of GBAP and CylL(S) both in the supernatants of liquid cultures and in an agar-overlay assay in as little as three hours, with a high level of sensitivity.Furthermore, we identified a clinical E. faecalis isolate that acted as a biological cheater, producing cytolysin only upon sensing CylL(S)-producers in its environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Aas, Norway.

ABSTRACT
Enterococcus faecalis is a significant threat in the nosocomial setting due to the emergence of isolates that are multi-antibiotic resistant, refractory to the available therapies and equipped with a variety of pathogenicity determinants. This bacterium uses quorum-sensing systems to regulate its physiological processes, including the expression of virulence traits, to adapt and proliferate within a host. Here, we describe the construction and application of two bioluminescence-based reporter systems for the direct detection of the quorum-sensing regulated expression of (i) the gelatinase biosynthesis-activating pheromone (GBAP) and (ii) the cytolysin small subunit (CylL(S)) in natural samples. The two E. faecalis reporters conditionally expressed bioluminescence in the presence of GBAP and CylL(S) both in the supernatants of liquid cultures and in an agar-overlay assay in as little as three hours, with a high level of sensitivity. Biosensors employed to investigate the interaction between the fsr and cyl systems revealed that fsr impeded CylL(S) activity by 75%. Furthermore, we identified a clinical E. faecalis isolate that acted as a biological cheater, producing cytolysin only upon sensing CylL(S)-producers in its environment. This isolate enhanced its virulence during polymicrobial systemic infection of Galleria mellonella.

No MeSH data available.


Related in: MedlinePlus