Regulation of Autolysis-Dependent Extracellular DNA Release by Enterococcus faecalis Extracellular Proteases Influences Biofilm Development

Division of Biology, Kansas State University, 116 Ackert Hall, Manhattan, KS 66506, USA.
Journal of bacteriology (Impact Factor: 2.81). 07/2008; 190(16):5690-8. DOI: 10.1128/JB.00314-08
Source: PubMed


Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE(-) mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE(-) mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases--GelE and SprE--is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of E. faecalis biofilms.

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Available from: Vinai Chittezham Thomas, Jul 17, 2014
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    • "The significant contribution of extracellular DNA (eDNA) to biofilm structure and function, including structural integrity, recombination, and antibiotic resistance, has been also recently become apparent [6], [7]. Autolysis can underlie either biofilm formation or dispersal, and can release eDNA [6], [8], [9]. "
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    ABSTRACT: Campylobacter jejuni is a leading cause of foodbourne gastroenteritis, despite fragile behaviour under standard laboratory conditions. In the environment, C. jejuni may survive within biofilms, which can impart resident bacteria with enhanced stress tolerance compared to their planktonic counterparts. While C. jejuni forms biofilms in vitro and in the wild, it had not been confirmed that this lifestyle confers stress tolerance. Moreover, little is understood about molecular mechanisms of biofilm formation in this pathogen. We previously found that a ΔcprS mutant, which carries a deletion in the sensor kinase of the CprRS two-component system, forms enhanced biofilms. Biofilms were also enhanced by the bile salt deoxycholate and contained extracellular DNA. Through more in-depth analysis of ΔcprS and WT under conditions that promote or inhibit biofilms, we sought to further define this lifestyle for C. jejuni. Epistasis experiments with ΔcprS and flagellar mutations (ΔflhA, ΔpflA) suggested that initiation is mediated by flagellum-mediated adherence, a process which was kinetically enhanced by motility. Lysis was also observed, especially under biofilm-enhancing conditions. Microscopy suggested adherence was followed by release of eDNA, which was required for biofilm maturation. Importantly, inhibiting biofilm formation by removal of eDNA with DNase decreased stress tolerance. This work suggests the biofilm lifestyle provides C. jejuni with resilience that has not been apparent from observation of planktonic bacteria during routine laboratory culture, and provides a framework for subsequent molecular studies of C. jejuni biofilms.
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    • "The mechanisms by which the DNA is released in biofilms are poorly understood but autolysis of cells has been hypothesized to mediate DNA release (Bayles, 2007; Ma et al., 2009). In Enterococcus faecalis, the release of extracellular DNA by autolysis is regulated by the action of the two proteases GelE and SprE (Thomas et al., 2008) whereas in Staphylococcus aureus, a finely tuned holin/antiholin system is thought to mediate cell lysis and programmed cell death (Bayles, 2007; Rice and Bayles, 2008). A previous study has revealed no extracellular DNA in biofilms of both Bordetella bronchiseptica strain 276 and Escherichia coli strain ECL 17602 using confocal laser scanning microscopy (Wu et al., 2013a), indicating that extracellular DNA is not a component of all biofilms. "
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    • "Furthermore, no difference of the intracellular nucleoside triphosphate ATP was measured in the supernatant of the wild type vs. the SpxB mutant, further confirming that no general lysis is associated with eDNA release in both oral streptococci [10]. We concluded that both streptococcal species used in our studies did not lyse substantially under conditions known to cause lysis of other firmicutes, for example E. faecalis and S. aureus [10], [16], [40]. "
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