Focal targeting by human β-defensin 2 disrupts localized virulence factor assembly sites in Enterococcus faecalis

Abstract

Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human β-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.

Keywords: MprF; focal localization; immunofluorescence; microscopy.

Fig. 1.

SecA and SrtA localize at discrete foci near the equator of E. faecalis. E. faecalis bacteria were grown to midlog phase, fixed, subjected to lysozyme degradation of the cell wall with (A–C) or without (D–F) subsequent triton permeabilization of the cytoplasmic membrane. Wild-type OG1RF was immunolabeled with α-SecA (A–C), and OG1RF ΔsrtA pAK1::srtA-HA was immunolabeled with α-HA for the localization of SrtA (D–F). (Scale bar, 0.5 µm.) Representative images of at least three independent experiments are shown. Cartoons depict a representative localization pattern observed over many cells.

Fig. 2.

hBD2 interacts focally with E. faecalis. (A and B) Representative images (Top, Cy3 fluorescence; Middle, phase contrast; Bottom, merge of fluorescence and phase contrast) of live E. faecalis OG1RF (A) and 0852 (B) cells incubated with hBD2-Cy3 and imaged without fixation or processing. (Scale bar in A, 0.5 µm.) (C and D) Immunoelectron microscopy of E. faecalis OG1X incubated with a subinhibitory concentration of hBD2 (C) or untreated (D), followed by fixation and immune-labeling with α-hBD2 and a gold-labeled secondary antibody. (Scale bars in C and D, 200 nm.)

Fig. 3.

Antimicrobial peptides disrupt SrtA and SecA focal localization. E. faecalis OG1RF ΔsrtA pAK1::srtA-HA cells were incubated with buffer (A–D) or subinhibitory concentrations of hBD2-Cy3 (E and F) before fixation and processing for fluorescent microscopy. (A and E) SrtA-HA was visualized via antibodies against the HA tag. (C and G) SecA was visualized via α-SecA antibodies. Representative images are shown. (Scale bar, 0. 5 µM.) The mean fluorescent intensity around the cell perimeter from at least 200 early-division cells from two independent experiments was determined by PSICIC software. Cells in which no fluorescence was detected were excluded from the analyses. Units are displayed in arbitrary units (AU). Cartoons (B and D) depict the predominant site of SrtA-HA and SecA localization in hBD2 untreated cells; the coordinates of the perimeter that correspond with the x-axes (B, D, F, and H) are shown also. In B, D, F, and H, the bold line indicates the mean and error bars indicate the SEM fluorescence at each point on the perimeter. All images were deconvolved using ZEN (blue edition) by Carl Zeiss Microimaging GmbH.

Fig. 4.

Antimicrobial peptide foci are more intense in mprF mutants. (A) Live E. faecalis OG1X were incubated with NAO, which specifically interacts with anionic lipids. (B) Relative survival after exposure to increasing concentrations of hBD2 was measured for OG1RF wild-type, ΔmprF2, and ΔmprF2/mprF2 cells. (C) E. faecalis OG1RF wild-type, ΔmprF1, or ΔmprF2 cells incubated with 0.2 µM Cy3-hBD2 throughout the cell cycle. (D) Mean fluorescent intensity around the cell perimeter of at least 340 early-division cells per strain, from two independent experiments. (E) Mean fluorescent intensity around the cell perimeter of at least 75 early-division cells per strain, grown in the presence of kanamycin to ensure plasmid maintenance. (D and E) Intensity analysis performed in PSICIC (MATLAB); error bars reflect the SEM.

Fig. 5.

Model for localized virulence factor assembly and CAMP targeting in E. faecalis. (A) Wild-type cells expressing MprF2 and aminoacylated phosphatidylglycerol in anionic lipid microdomains have limited focal interaction between CAMPs (pink stars) and the lipid domains. MprF2 aminoacylation facilitates the neutralization of anionic lipids in the outer leaflet of the membrane (positive and negative charges). (B) In cells lacking MprF2, anionic lipid domains are available for enhanced focal targeting by CAMPs. (A and B) The general secretion machinery, consisting of the integral membrane translocon SecYEG and SecA translocase, colocalizes with sortase enzymes at the septum of the cell. SrtC mediates the assembly of pilus subunits, including the major pilus subunit, endocarditis and biofilm-associated pilus subunit C (EbpC) (blue ovals), before attachment to the cell wall (red hatches). Sortase localization is mediated by the highly positively charged cytoplasmic tail of the enzyme that also may involve electrostatic interactions with focal anionic lipid domains (negative charge, inner leaflet). Cytoplasmic tail residues of SrtA, SrtC, and EbpC are indicated.