The Enterococcus faecalis fsr two-component system controls biofilm development through production of gelatinase

Abstract

Bacterial growth as a biofilm on solid surfaces is strongly associated with the development of human infections. Biofilms on native heart valves (infective endocarditis) is a life-threatening disease as a consequence of bacterial resistance to antimicrobials in such a state. Enterococci have emerged as a cause of endocarditis and nosocomial infections despite being normal commensals of the gastrointestinal and female genital tracts. We examined the role of two-component signal transduction systems in biofilm formation by the Enterococcus faecalis V583 clinical isolate and identified the fsr regulatory locus as the sole two-component system affecting this unique mode of bacterial growth. Insertion mutations in the fsr operon affected biofilm formation on two distinct abiotic surfaces. Inactivation of the fsr-controlled gene gelE encoding the zinc-metalloprotease gelatinase was found to prevent biofilm formation, suggesting that this enzyme may present a unique target for therapeutic intervention in enterococcal endocarditis.

FIG. 1.

Genetic organization of the E. faecalis V583 fsr locus which includes fsrA (response regulator), fsrB (signaling peptide), fsrC (histidine kinase), gelE (gelatinase), and sprE (serine protease) (32). The open arrows specify the coding region of each gene. The gene designated orf1 encodes a putative N-acetylmuramidase; only the 3′ end of the gene is shown. The extents of the fragments cloned in the plasmids are indicated by the solid lines. For plasmids pML29 and pML30, the aphA-3 promoter was cloned upstream of the fragment shown, and it is depicted as an elevated arrow. The small solid arrows represent the position of the oligonucleotide primers used in the study. The positions of the promoters identified in this region are indicated by arrows and the symbols Pa, Pb, and Pe. Only restriction sites relevant to this study are shown. Restriction sites in parenthesis were introduced by the oligonucleotides used in PCR amplification reactions. Restriction enzyme symbols: EI, EcoRI; H, HincII; R, RsaI; Sa, Sau3A; Sp, SphI; Bs, BsmAI; N, NdeI; Ba, BamHI; Bg, BglII; X, XmnI; EV, EcoRV.

FIG. 2.

Biofilm formation by parental and mutant E. faecalis strains. The OD₅₅₀s of solubilized crystal violet from microtiter plate assays at 24 h (stippled) and 48 h (solid) are shown for the strains listed. (A) All strains are derivatives of the E. faecalis V583 clinical isolate. Indicated is the name of the strain, its relevant genotype, the name of the plasmid it carries, and the complementing gene present on the plasmid (in parentheses). Strains JML102 and JML103 were omitted, as the level of biofilm formation was comparable to the level observed with strain JML101. (B) All strains are derivatives of the E. faecalis FA2-2 laboratory strain and thus carry the fsrC mutation. Complementing plasmids are indicated as described above.

FIG. 3.

Confocal laser scanning microscopy of E. faecalis biofilms. The 24-h (A and B) and 48-h (C and D) biofilms were stained with acridine orange and visualized by confocal laser scanning microscopy as described in Materials and Methods. Standard projections of the biofilm through the x-y plane for strain V583 (A and C) and strain JML101 (fsrA) (B and D) are shown. Bars (A to D) indicate size in microns. Projections of the biofilms through the x-z plane at 48 h for strains V583 (E) and JML101 (fsrA) (F) are also shown. Bars (E and F), 10 μm.

FIG. 4.

Extracellular complementation of E. faecalis FA2-2 biofilm formation by purified gelatinase. OD₅₅₀s of solubilized crystal violet from the microtiter plate assay at 24 h are shown. Biofilm formation by E. faecalis strains V583 and FA2-2 at 24 h was measured as a control. Strain FA2-2 was complemented extracellularly with purified gelatinase (GelE) from E. faecalis culture supernatants or the catalytic site mutant E137A expressed in E. coli as a His tag fusion. The values indicated on the x axis correspond to the amount of purified protein added to 200 μl of culture. All assays were performed in triplicate and repeated at least twice. Representative data from two independent experiments are shown with mean values and standard deviations (error bars).