YajC, a predicted membrane protein, promotes Enterococcus faecium biofilm formation in vitro and in a rat endocarditis model

Abstract

Biofilm formation is a critical step in the pathogenesis of difficult-to-treat Gram-positive bacterial infections. We identified that YajC, a conserved membrane protein in bacteria, plays a role in biofilm formation of the clinically relevant Enterococcus faecium strain E1162. Deletion of yajC conferred significantly impaired biofilm formation in vitro and was attenuated in a rat endocarditis model. Mass spectrometry analysis of supernatants of washed ΔyajC cells revealed increased amounts in cytoplasmic and cell-surface-located proteins, including biofilm-associated proteins, suggesting that proteins on the surface of the yajC mutant are only loosely attached. In Streptococcus mutans YajC has been identified in complex with proteins of two cotranslational membrane protein-insertion pathways; the signal recognition particle (SRP)-SecYEG-YajC-YidC1 and the SRP-YajC-YidC2 pathway, but its function is unknown. In S. mutans mutation of yidC1 and yidC2 resulted in impaired protein insertion in the cell membrane and secretion in the supernatant. The E. faecium genome contains all homologous genes encoding for the cotranslational membrane protein-insertion pathways. By combining the studies in S. mutans and E. faecium, we propose that YajC is involved in the stabilization of the SRP-SecYEG-YajC-YidC1 and SRP-YajC-Yid2 pathway or plays a role in retaining proteins for proper docking to the YidC insertases for translocation in and over the membrane.

Keywords: Biofilm; Enterococcus faecium; Streptococcus mutans; YajC; cotranslational membrane protein insertion pathway; rat endocarditis model.

Figure 1.

The effect of targeted yajC mutation on semistatic and flow cell biofilm model. (A) Confocal imaging of biofilm formation of wild-type, ∆yajC and ∆yajC+yajC in a semistatic biofilm model. Cells were grown for 24 h on poly-l-lysine-coated glass slides, in TSBg, at 120 rpm, at 37ºC, chemically fixed in 8% glutaraldehyde and stained with PI (red) (scale bars in A, 10 µm). The biomass and average thickness of biofilms was measured at five random positions of three biological replicates and analyzed with Comstat/Matlab software. Pictures were taken at 63x magnification with 2.5 optical zoom. Asterisks represent significant differences (***P < .001) with the wild-type strain as determined by an unpaired two-tailed Student’s t-test. (B) Biofilms of wild-type, ∆yajC and ∆yajC+yajC in a flow cell biofilm model. Biofilms were grown for 17 h in a Stovall flow cell system, in 1:10 diluted TSB with 1% glucose (0.13 ml/min), at 37ºC. Pictures were taken at 40x magnification with 2.5 optical zoom. Cells were stained with syto 9 and PI after 17 h of growth (scale bars in bright filter picture and confocal images, 30 µm and 10 µm, respectively).

Figure 2.

The effect of targeted yajC mutation on initial adhesion. E1162 wild-type, ∆yajC and ∆yajC+yajC were cultured overnight, washed two times with PBS, or not, 100 µl of bacterial suspension was added in triplicate to a polystyrene plate and incubated for 2 h at 37ºC without shaking. Cell attachment was measured by absorbance of crystal violet at 595 nm and repeated three times. Asterisks represent significant difference (***P < .001) with the wild-type strain as determined by an unpaired two-tailed Student’s t-test.

Figure 3.

The effect of targeted yajC mutation on protein attachment. (A) Proteins present in the supernatant of wild-type, ∆yajC and ∆yajC+yajC after wash with PBS pH 7 were separated through a 12.5% SDS page gel with Coomassie blue stain. (B) Presence of GAPDH, ATP synthase, and Tu in the supernatant of washed E. faecium E1162 wild-type, ∆yajC and ∆yajC+yajC was analyzed by western blot using α-GAPDH, α-ATP synthase, or α-Tu antibodies. (C) Cell surface exposure of GAPDH, ATP synthase and Tu on washed E. faecium wild-type, ∆yajC and ∆yajC+yajC was analyzed by confocal microscopy. Washed cells were incubated with α-GAPDH, α-ATP synthase, or α-Tu antibodies and goat α-rabbit or goat α-mouse Alexa 488 (green). Bacterial membranes were stained with FM 95–5 (red) (scale bars in A, 10 µm). (D) Ratio between green (protein of interested) and red (bacteria) was calculated in ImageJ software. Asterisks represent significant difference (***P < .001) with the wild-type strain as determined by an unpaired two-tailed Student’s t-test.

Figure 4.

The effect of yajC mutation in the rat endocarditis model. Endocarditis of E. faecium wild-type and ∆yajC was measured by determining CFU/ml heart valve vegetations (A) and by determining the mass of vegetations in milligrams (B) in a rat endocarditis model. Vegetations on the heart valve formed by wild-type (C) and ∆yajC (D) were visualized by Phenom World tabletop SEM with 10 000x magnification (scale bars in C and D, 10 µm). Arrows indicate E. faecium in a biofilm structure. For the mouse colonization model (E and F), mice were orally inoculated with wild-type E. faecium or ∆yajC. During 10 days, colonization of E. faecium was determined in stool pallets by CFU enumeration of the mice at different time points (E). After 10 days of CFU counts of E1162 and ∆yajC were also determined in the ileum, cecum, and colon (F). Data are expressed as CFU per gram of stool/fecal contents and means are shown for eight mice per group. Asterisk represents significant differences (*P < .05) as determined by an unpaired two-tailed Student’s t-test between the indicated samples.