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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

Determination of CylLS-Inducing Units (CIU) in overnight (ON) cultures and during the growth (at increasing OD620 units) of cultures of E. faecalis ×98 pMG36c (red; control) and ×98 pCG (blue; GelE-overexpressing strain).The plot displays the averages of the results of triplicate independent experiments. * p-value < 0.05.
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f5: Determination of CylLS-Inducing Units (CIU) in overnight (ON) cultures and during the growth (at increasing OD620 units) of cultures of E. faecalis ×98 pMG36c (red; control) and ×98 pCG (blue; GelE-overexpressing strain).The plot displays the averages of the results of triplicate independent experiments. * p-value < 0.05.

Mentions: Interestingly, we observed a 2-fold difference in the levels of CylLS production in ×98 and MMH594 exponential-phase cultures, and a 4-fold higher level of CylLS in the supernatants of late exponential- and stationary-phase cultures of ×98 compared with those of MMH594 (P < 0.001, according to the results of the Mann-Whitney test) (Figure 4). In a recent study in which we investigated the pathogenicity of E. faecalis in the nematode Caenorhabditis elegans, statistical analyses showed that the concomitant expression of both gelatinase and cytolysin through the E. faecalis genome significantly increased its virulence (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). Interestingly, the effect of the co-presence of these two virulence traits was much less than the sum of their main individual effects, described as saturation or antagonistic effects. It was previously reported that GelE contribute to the virulence of E. faecalis by triggering the proteolytic degradation of a broad range of host substrates262728, and gelE expression was found to have a profound effect on the secretome of E. faecalis2930. GelE activity was found to be required for a variety of processes, such as the regulation of the display of the surface adhesin Ace31 and the activation of the primary autolysin AtlA and its contribution to biofilm formation32. Additionally, the production of gelatinase was reported to impair other cellular activities, such as conjugation due to the degradation of sex pheromone-related peptides33. Taken together with the results of utilising the CylLS-biosensor detection system, we considered the possibility that gelatinase might also have an impact on cytolysin. To test this hypothesis, we employed the JH2-2 CBS biosensor to detect CylLS accumulation in the supernatants of exponential- and stationary-phase cultures of the gelatinase-overproducing E. faecalis strain ×98::pCG (Figure 5). Under both of the tested conditions, our data showed that introducing gelatinase into the Cyl producer ×98 leads to a 4- to 8-fold reduction of CylLS activity (p < 0.05, according to the results of Mann-Whitney test).


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)

Determination of CylLS-Inducing Units (CIU) in overnight (ON) cultures and during the growth (at increasing OD620 units) of cultures of E. faecalis ×98 pMG36c (red; control) and ×98 pCG (blue; GelE-overexpressing strain).The plot displays the averages of the results of triplicate independent experiments. * p-value < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Determination of CylLS-Inducing Units (CIU) in overnight (ON) cultures and during the growth (at increasing OD620 units) of cultures of E. faecalis ×98 pMG36c (red; control) and ×98 pCG (blue; GelE-overexpressing strain).The plot displays the averages of the results of triplicate independent experiments. * p-value < 0.05.
Mentions: Interestingly, we observed a 2-fold difference in the levels of CylLS production in ×98 and MMH594 exponential-phase cultures, and a 4-fold higher level of CylLS in the supernatants of late exponential- and stationary-phase cultures of ×98 compared with those of MMH594 (P < 0.001, according to the results of the Mann-Whitney test) (Figure 4). In a recent study in which we investigated the pathogenicity of E. faecalis in the nematode Caenorhabditis elegans, statistical analyses showed that the concomitant expression of both gelatinase and cytolysin through the E. faecalis genome significantly increased its virulence (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). Interestingly, the effect of the co-presence of these two virulence traits was much less than the sum of their main individual effects, described as saturation or antagonistic effects. It was previously reported that GelE contribute to the virulence of E. faecalis by triggering the proteolytic degradation of a broad range of host substrates262728, and gelE expression was found to have a profound effect on the secretome of E. faecalis2930. GelE activity was found to be required for a variety of processes, such as the regulation of the display of the surface adhesin Ace31 and the activation of the primary autolysin AtlA and its contribution to biofilm formation32. Additionally, the production of gelatinase was reported to impair other cellular activities, such as conjugation due to the degradation of sex pheromone-related peptides33. Taken together with the results of utilising the CylLS-biosensor detection system, we considered the possibility that gelatinase might also have an impact on cytolysin. To test this hypothesis, we employed the JH2-2 CBS biosensor to detect CylLS accumulation in the supernatants of exponential- and stationary-phase cultures of the gelatinase-overproducing E. faecalis strain ×98::pCG (Figure 5). Under both of the tested conditions, our data showed that introducing gelatinase into the Cyl producer ×98 leads to a 4- to 8-fold reduction of CylLS activity (p < 0.05, according to the results of Mann-Whitney test).

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