Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Nov 26;200(24):e00361-18.
doi: 10.1128/JB.00361-18. Print 2018 Dec 15.

Planktonic Interference and Biofilm Alliance Between Aggregation Substance and Endocarditis- And Biofilm-Associated Pili in Enterococcus Faecalis

Affiliations
Free PMC article

Planktonic Interference and Biofilm Alliance Between Aggregation Substance and Endocarditis- And Biofilm-Associated Pili in Enterococcus Faecalis

Irina Afonina et al. J Bacteriol. .
Free PMC article

Abstract

Like many bacteria, Enterococcus faecalis encodes a number of adhesins involved in colonization or infection of different niches. Two well-studied E. faecalis adhesins, aggregation substance (AS) and endocarditis- and biofilm-associated pili (Ebp), both contribute to biofilm formation on abiotic surfaces and in endocarditis, suggesting that they may be expressed at the same time. Because different regulatory pathways have been reported for AS and Ebp, here, we examined if they are coexpressed on the same cells and what is the functional impact of coexpression on individual cells and within a population. We found that while Ebp are only expressed on a subset of cells, when Ebp and AS are expressed on the same cells, pili interfere with AS-mediated clumping and impede AS-mediated conjugative plasmid transfer during planktonic growth. However, when the population density increases, horizontal gene transfer rates normalize and are no longer affected by pilus expression. Instead, at higher cell densities during biofilm formation, Ebp and AS differentially contribute to biofilm development and structure, synergizing to promote maximal biofilm formation.IMPORTANCE Most bacteria express multiple adhesins that contribute to surface attachment and colonization. However, the network and relationships between the various adhesins of a single bacterial species are less well understood. Here, we examined two well-characterized adhesins in Enterococcus faecalis, aggregation substance and endocarditis- and biofilm-associated pili, and found that they exhibit distinct functional contributions depending on the growth stage of the bacterial community. Pili interfere with aggregation substance-mediated clumping and plasmid transfer under planktonic conditions, whereas the two adhesins structurally complement one another during biofilm development. This study advances our understanding of how E. faecalis, a ubiquitous member of the human gut microbiome and an opportunistic pathogen, uses multiple surface structures to evolve and thrive.

Keywords: Enterococcus faecalis; aggregation substance; biofilm; endocarditis- and biofilm-associated pili; horizontal gene transfer.

Figures

FIG 1
FIG 1
AS and Ebp are coexpressed on the same cells after pheromone induction. (A) IFM was performed with AS antiserum, and the percentage of AS+ cells within the population was quantified. (B) IFM with EbpC antiserum was performed on cCF10 pheromone-uninduced (black bars) and -induced (gray bars) OG1RF/pCF10 at the indicated time points, and the percentage of EbpC+ cells within the population was quantified. (C) IFM on pheromone-induced cells with Ebp antiserum (red), AS (green), and DNA (blue). Scale bars = 1 μm. For panels A and B, the mean values are shown from 3 independent experiments in which at least 300 cells were counted. Asterisks above the 30-min time point indicate a significant difference for each population compared to all other time points. Error bars represent the standard deviation. Statistical analysis was performed by the unpaired t test using GraphPad. *, P < 0.05; ****, P < 0.0001, n.s., P > 0.05.
FIG 2
FIG 2
Ebp interfere with AS-mediated clumping. (A) Representative image of OG1RF/pCF10 uninduced (left) and OG1RF/pCF10 induced (right) with pheromone cCF10 (0.12 ng/ml), 2 h after induction. (B) IFM was performed 2 h post-cCF10 induction with EbpC antiserum on the total population, suspension fraction (top 2 ml), and pellet fraction (clumped cells), and the percentage of EbpC+ cells was quantified. (C) RT-qPCR on RNA isolated from pheromone-induced cells 2 h post-cCF10 induction. Fold change indicates the change in ebpC and ebpR transcription in the pellet cells compared to the control (total). Mean values are shown from 3 independent experiments. Error bars represent the standard deviation. Each experiment was performed in triplicate using the ΔΔCT method to assess expression changes, with gyrA as the reference housekeeping gene. Statistical analysis was performed by unpaired t test using GraphPad.*, P < 0.05; **, P < 0.01, ***, P < 0.001; ****, P < 0.0001, n.s., P > 0.05.
FIG 3
FIG 3
Ebp impede AS-mediated HGT. (A) HGT rates from suspension and pellet cells of donor OG1SS/pCF10 (Str and Tet resistant, Rif sensitive) 2 h post-pheromone induction to plasmid-free Ebp-null recipient OG1RF ΔebpABC srtC (Rif resistant, Str and Tet sensitive). (B) HGT rates from OG1RF/pCF10 and ΔebpABC srtC/pCF10 mutant (both Rif and Tet resistant, Str sensitive) donor cells to plasmid-free recipient OG1X ΔsrtC (Str resistant, Rif and Tet sensitive). HGT rate is expressed as the number of transconjugants (Tc) per donor cell 30 and 120 min postmating. Error bars represent the standard deviation of the results from 3 independent experiments. Statistical analysis was performed by the unpaired t test using GraphPad. *, P < 0.05; **, P < 0.01, n.s., P > 0.05.
FIG 4
FIG 4
AS contributes to initial attachment and biofilm formation independent of Ebp. (A) IFM with Ebp antiserum was performed on cells attached to the biofilm chamber 2 h after induction. Error bars represent the standard deviation from 3 independent experiments. Statistical analysis was performed by unpaired t test using GraphPad. *, P < 0.05; **, P < 0.01, n.s., P > 0.05. (B) CV staining of biofilms formed by OG1RF, OG1RF/pCF10, OG1RF ΔebpABC srtC, and OG1RF ΔebpABC srtC/pCF10 in TSBG medium supplemented with 0.12 ng/ml cCF10 on plastic after 24 h. Error bars represent the standard deviation of the results from 3 independent experiments. Statistical analysis was performed by unpaired t test using GraphPad.
FIG 5
FIG 5
Ebp and AS differentially contribute to biofilm structure and development. Confocal laser scanning microscopy images of 24 h biofilms grown in plastic chambers in 40% TSBG with cCF10 (1.2 μg/ml). Cells were fixed and stained with Hoechst dye. (A) z-stack images represent biofilm depths of 0, 10, 20, and 30 μm, followed by z-view for the same samples. Scale bars are 10 μm. (B) Imaris software modeling of the confocal laser scanning microscopy images from panel A, where every cell is represented as a sphere based on Hoechst staining, and the z-depth is color-coded (0 to 25 μm), where purple is 0 μm and red is 25 μm. Both perspective (top) and side (bottom) views are shown. Scale bars are 20 μm.

Similar articles

See all similar articles

References

    1. Schlievert PM, Gahr PJ, Assimacopoulos AP, Dinges MM, Stoehr JA, Harmala JW, Hirt H, Dunny GM. 1998. Aggregation and binding substances enhance pathogenicity in rabbit models of Enterococcus faecalis endocarditis. Infect Immun 66:218–223. - PMC - PubMed
    1. Nallapareddy SR, Singh KV, Sillanpaa J, Garsin DA, Hook M, Erlandsen SL, Murray BE. 2006. Endocarditis and biofilm-associated pili of Enterococcus faecalis. J Clin Invest 116:2799–2807. doi:10.1172/JCI29021. - DOI - PMC - PubMed
    1. Flores-Mireles AL, Pinkner JS, Caparon MG, Hultgren SJ. 2014. EbpA vaccine antibodies block binding of Enterococcus faecalis to fibrinogen to prevent catheter-associated bladder infection in mice. Sci Transl Med 6:254ra127. doi:10.1126/scitranslmed.3009384. - DOI - PMC - PubMed
    1. Navarre WW, Schneewind O. 1994. Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in Gram-positive bacteria. Mol Microbiol 14:115–121. doi:10.1111/j.1365-2958.1994.tb01271.x. - DOI - PubMed
    1. Sillanpää J, Xu Y, Nallapareddy SR, Murray BE, Hook M. 2004. A family of putative MSCRAMMs from Enterococcus faecalis. Microbiology 150:2069–2078. doi:10.1099/mic.0.27074-0. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback