A putative phosphatase regulates surface attachment and biofilm formation in Enterococcus faecalis

Project: Research


  • Julia Laura Elizabeth Willett (PI)


Enterococcus faecalis is a common nosocomial pathogen and is the third leading cause of hospital-acquired endocarditis. E. faecalis readily infects surgically implanted devices such as catheters and drive lines of left ventricular assist devices, providing direct access of this pathogen to the cardiac chamber. The ability of E. faecalis to thrive as a cardiovascular pathogen is due in part to its ability to form biofilms, or surface-associated clusters of cells enmeshed in a complex extracellular matrix. Biofilms render bacteria more resistant to antibiotics and are difficult to eliminate, leading to persistent and recurring infections. Because E. faecalis has developed resistance to nearly all clinically relevant antibiotics, biofilm-associated enterococcal endocarditis is a serious health concern. As part of an innovative comprehensive effort to characterize all unknown and hypothetical E. faecalis proteins, we identified a transposon mutant in the gene OG1RF_10435 with severely attenuated biofilm formation. Extensive preliminary work shows that this gene contributes to biofilm formation and initial surface attachment in vitro and attachment to ex vivo porcine cardiac valves. At least one protein appears to be differentially expressed in the absence of OG1RF_10435. Structural modeling suggests that OG1RF_10435 is a nucleotide phosphatase, and we hypothesize that this activity regulates biofilm formation and attachment. The specific aims of this proposal are to 1) determine the molecular mechanism of OG1RF_10435 phosphatase activity and 2) identify the cellular targets of OG1RF_10435. We will do this using an in vitro colorimetric assay to assess phosphatase activity of purified wild-type and mutant protein as well as mass spectrometry to identify differentially expressed proteins. Reporter constructs will be built using candidate genes identified from mass spec analysis in order to understand whether OG1RF_10435 influences gene expression or protein stability. Biofilm formation and attachment phenotypes will be assessed using in vitro substrates as well as ex vivo porcine heart valves. We aim to achieve an understanding of how OG1RF_10435 regulates biofilm formation and attachment and how this contributes to the development of enterococcal endocarditis. This will advance our objective of discovering the function of conserved hypothetical Enterococcus proteins and identifying new drug or vaccine targets for improved endocarditis therapies.
Award amount$106,532.00
Award date01/01/2019
Program typePostdoctoral Fellowship
Award ID19POST34450124
Effective start/end date01/01/201912/31/2020