Abstract
Analysis of the culture supernatant exoproteins produced by two PFGE clusters of high-level gentamicin and ciprofloxacin-resistant clinical isolates of Enterococcus faecalis from the UK and Ireland revealed two distinct protein profiles. This grouping distinguished OG1RF and GelE metalloprotease-expressing isolates from JH2-2 and other GelE-negative isolates. The integrity of the fsrABDC operon was found to determine the exoproteome composition, since an fsrB mutant of strain OG1RF appeared very similar to that of strain JH2-2, and complementation of the latter with the fsrABDC operon produced an OG1RF-like exoproteome. The proteins present in the supernatant fraction of OG1RF were separated using 2D gels and identified by mass spectrometry and comprised many mass and pI variants of the GelE and SprE proteases. In addition cell wall synthesis and cell division proteins were identified. An OG1RF fsrB mutant had a distinct exoprotein fraction with an absence of the Fsr-regulated proteases and was characterised by general stress and glycolytic proteins. The exoproteome of the OG1RF fsrB mutant resembles that of a divIVA mutant of E. faecalis, suggestive of a stress phenotype.
Highlights
Recent years have seen greater study of medically important opportunistic bacterial pathogens due to increased levels of nosocomial infection and antibiotic resistance [1,2]
Distinct exoprotein patterns in E. faecalis strains Stationary phase exoproteins were purified from culture supernatants of clinical isolates bearing high level gentamicin and ciprofloxacin resistance that were previously divided into two separate PFGE clusters [24]
In contrast with OG1RF, strain JH2-2 is negative for gelatinase activity (Zn-metalloprotease) [26] and the clinical isolates were tested for proteolysis on casein agar
Summary
Recent years have seen greater study of medically important opportunistic bacterial pathogens due to increased levels of nosocomial infection and antibiotic resistance [1,2] Of these pathogens, Enterococcus faecalis is prominent due to the frequency of disease and its implication in antibiotic resistance transfer to Staphylococcus aureus and Listeria species [3,4,5,6]. Transcription of the gelE-sprE operon is temporally regulated via FsrA, the response regulator of the density-dependent two-component system. Insertional inactivation of this locus ablates expression of these proteases and the Fsr system is the only known regulatory locus for the genes [10]. Density-dependent activation of the Fsr system occurs via extracellular accumulation of gelatinase biosynthesis activating pheromone (GBAP, the product of fsrD) [14]
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