Decoding the proteomic changes involved in the biofilm formation of Enterococcus faecalis SK460 to elucidate potential biofilm determinants

Abstract

Background: Enterococcus faecalis is a major clinically relevant nosocomial bacterial pathogen frequently isolated from polymicrobial infections. The biofilm forming ability of E. faecalis attributes a key role in its virulence and drug resistance. Biofilm cells are phenotypically and metabolically different from their planktonic counterparts and many aspects involved in E. faecalis biofilm formation are yet to be elucidated. The strain E. faecalis SK460 used in the present study is esp (Enterococcal surface protein) and fsr (two-component signal transduction system) negative non-gelatinase producing strong biofilm former isolated from a chronic diabetic foot ulcer patient. We executed a label-free quantitative proteomic approach to elucidate the differential protein expression pattern at planktonic and biofilm stages of SK460 to come up with potential determinants associated with Enterococcal biofilm formation.

Results: The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of proteomic data revealed that biofilm cells expressed higher levels of proteins which are associated with glycolysis, amino acid biosynthesis, biosynthesis of secondary metabolites, microbial metabolism in diverse environments and stress response factors. Besides these basic survival pathways, LuxS-mediated quorum sensing, arginine metabolism, rhamnose biosynthesis, pheromone and adhesion associated proteins were found to be upregulated during the biofilm transit from planktonic stages. The selected subsets were validated by quantitative real-time PCR. In silico functional interaction analysis revealed that the genes involved in upregulated pathways pose a close molecular interaction thereby coordinating the regulatory network to thrive as a biofilm community.

Conclusions: The present study describes the first report of the quantitative proteome analysis of an esp and fsr negative non gelatinase producing E. faecalis. Proteome analysis evidenced enhanced expression of glycolytic pathways, stress response factors, LuxS quorum signaling system, rhamnopolysaccharide synthesis and pheromone associated proteins in biofilm phenotype. We also pointed out the relevance of LuxS quorum sensing and pheromone associated proteins in the biofilm development of E. faecalis which lacks the Fsr quorum signaling system. These validated biofilm determinants can act as potential inhibiting targets in Enterococcal infections.

Keywords: Biofilm determinants; Enterococcus faecalis; Metabolic pathways; Quantitative proteomics; Stress response; luxS.

Fig. 1

Confocal laser scanning microscopy images of biofilm formation of E. faecalis SK460 at 12 h and 24 h. The images are processed using NIS-Element AR software, version 4.00.04

Fig. 2

KEGG pathways and molecular functions assigned to categorize the upregulated proteins obtained in biofilm stages of E. faecalis SK460

Fig. 3

STRING analysis (Version 10.5) showing the predicted molecular action among the upregulated proteins in biofilm stages. The gene nomenclatures are depicted in Table 1. Colored nodes: query proteins and first shell of interactors. Empty nodes: proteins of unknown 3D structure. Filled nodes: 3D structure is known/predicted. Edges represent protein-protein associations

Fig. 4

Real time qPCR validation of selected biofilm associated genes. Fold change expression of genes observed in biofilm stage with respect to its corresponding planktonic stage