Other major factors
Several DNA binding response regulators, phosphotransferase system transporter proteins, Autolysin, D-alanylation of lipoteichoic acid (dltABCD), etc. were also found to be more than two-fold upregulated in biofilm stages. Of these, autolysin is well known to be associated with the eDNA release in the matrix and structural integrity of the biofilm. Previous report showed that autolysin deficient mutants had defects in primary attachment and eDNA release which are mainly associated with the accumulative phase for maturation and structural stability of biofilm in E. faecalis [5]. These mutants lack DNase1-sensitive fibrous network which is found to have a role in biofilm stability. D-alanylation of lipoteichoic acid (dltC and dltD) was also found to be abundant in biofilm mode (2.36 to 4.17 fold), possibly incorporating lipoteichoic acid into the EPS matrix. Lack of d-alanine esters results in a stronger negative net charge on the bacterial cell surface and dlt mutants showed a reduction in biofilm formation on polystyrene surfaces. Furthermore, dltABCD operon is involved in the pathogenesis of E. faecalis leading to enhanced biofilm formation, host tissue attachment and increased resistance to antimicrobial peptides [6].
STRING analysis revealed that upregulated genes including stress response factors, major glycolytic enzymes, arginine metabolism and rhamnose biosynthesis were either directly or indirectly poses a close molecular interaction (p-value < 1.0e-16), thereby regulating each other in stressful environment prevailing during biofilm development. Among the upregulated cellular processes, rfbB (Rhamnose biosynthesis), arcA (Arginine deiminase), luxS (Quorum sensing), cAD1 (Pheromone cAD1 lipoprotein) and fbn (Fibrinogen/Fibronectin binding protein associated with adhesion) were selected for gene expression analysis and were found to be enhanced by 2.29, 4.03, 2.35, 1.5 and 13.87 fold respectively in biofilm stages. Of these, luxS mediated quorum sensing system is attributed to play a major role in E. faecalis SK460 biofilm which is devoid of fsr two-component signal transduction system. Accordance of the proteome data with RT-PCR results confirms the reliability of the analysis, serving as a validation for the identified determinants of Enterococcal biofilm development.