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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

(A) Alignment of the cyl operons of E. faecalis MMH594 and T2. A BlastN comparison was performed using EasyFig version 2.1 software. The levels of similarity ranged from 100 to 87%, as shown in the grey gradient scale. The light blue arrows indicate the genes. (B) Comparison of CylA sequences of MMH594 and T2. The conserved residues are highlighted in blue. The alignment was conducted using the MAFFT alignment program.
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f6: (A) Alignment of the cyl operons of E. faecalis MMH594 and T2. A BlastN comparison was performed using EasyFig version 2.1 software. The levels of similarity ranged from 100 to 87%, as shown in the grey gradient scale. The light blue arrows indicate the genes. (B) Comparison of CylA sequences of MMH594 and T2. The conserved residues are highlighted in blue. The alignment was conducted using the MAFFT alignment program.

Mentions: Studies have shown that the cyl locus is subject to genetic instability both in vitro and in vivo3738. A number of investigators have reported phenotypic instabilities with no obvious explanations at the genetic level, indicating that silent or non-functional cyl genes occur in the genomes of enterococcal isolates of different origins394041. In a previous report, we identified an apparent incongruence between the cyl genotype and phenotype in the clinical isolate T242 (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). Here, we performed a detailed comparative sequence analysis of the cyl locus of a subset of genome-sequenced E. faecalis strains, including the strain MMH594, and we detected the presence of an IS6770 element that was integrated into the 3′-end of the cylA gene in T2 (Fig. 6A). The insertion causes a premature stop and the production of a truncated, and presumably non-functional, CylA (Fig. 6B).


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)

(A) Alignment of the cyl operons of E. faecalis MMH594 and T2. A BlastN comparison was performed using EasyFig version 2.1 software. The levels of similarity ranged from 100 to 87%, as shown in the grey gradient scale. The light blue arrows indicate the genes. (B) Comparison of CylA sequences of MMH594 and T2. The conserved residues are highlighted in blue. The alignment was conducted using the MAFFT alignment program.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: (A) Alignment of the cyl operons of E. faecalis MMH594 and T2. A BlastN comparison was performed using EasyFig version 2.1 software. The levels of similarity ranged from 100 to 87%, as shown in the grey gradient scale. The light blue arrows indicate the genes. (B) Comparison of CylA sequences of MMH594 and T2. The conserved residues are highlighted in blue. The alignment was conducted using the MAFFT alignment program.
Mentions: Studies have shown that the cyl locus is subject to genetic instability both in vitro and in vivo3738. A number of investigators have reported phenotypic instabilities with no obvious explanations at the genetic level, indicating that silent or non-functional cyl genes occur in the genomes of enterococcal isolates of different origins394041. In a previous report, we identified an apparent incongruence between the cyl genotype and phenotype in the clinical isolate T242 (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). Here, we performed a detailed comparative sequence analysis of the cyl locus of a subset of genome-sequenced E. faecalis strains, including the strain MMH594, and we detected the presence of an IS6770 element that was integrated into the 3′-end of the cylA gene in T2 (Fig. 6A). The insertion causes a premature stop and the production of a truncated, and presumably non-functional, CylA (Fig. 6B).

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