Pilin and sortase residues critical for endocarditis- and biofilm-associated pilus biogenesis in Enterococcus faecalis - PubMed (original) (raw)

Pilin and sortase residues critical for endocarditis- and biofilm-associated pilus biogenesis in Enterococcus faecalis

Hailyn V Nielsen et al. J Bacteriol. 2013 Oct.

Abstract

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.

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Figures

Fig 1

Fig 1

Pili expressed by the SrtA− strain incorporate all structural subunits and remain associated with protoplasts after depletion of cell wall material by treatment with cell wall hydrolases. (A) Culture supernatants of OG1RF and the SrtA− strain were assessed by Western blotting with the indicated antipilin immune sera. (B) OG1RF, SrtA−, or SrtC− SrtA− cells were treated with lysozyme and mutanolysin for the indicated time. Protoplasts were isolated by centrifugation, and protoplast-associated pili or EbpC monomers were detected on Western blots probed with anti-EbpC immune sera. HMW, high-molecular-weight ladders.

Fig 2

Fig 2

Pili accumulate in the cell membranes of strains lacking SrtA or strains with the predicted catalytic Cys 200 residue mutated to Ala. Western blots were performed with the noted antiserum on (A) membrane fractions and (B) cell lysates of OG1RF, the SrtA− strain, and the SrtA− strain carrying either p::AL1 (vector control), p::AL1-SrtA, or p::AL1-SrtAC200A. The arrow and caret denote SrtA and a nonspecific band, respectively.

Fig 3

Fig 3

EbpB monomer doublets migrate at a higher molecular mass in the absence of SrtA. Western blots on SDS-PAGE of (A) cell lysates or (B) supernatants of the SrtC− and SrtC− SrtA− strains and the SrtC− SrtA− strain carrying either pAL1 (vector control) or pAL1::SrtA were probed with the indicated anti-pilin sera. The symbol # shows the EbpB monomer released mainly to culture supernatants of the SrtC− strain. The asterisk shows the larger EbpB monomer primarily associated with cells in strains lacking SrtA.

Fig 4

Fig 4

EbpC's pilin motif Lys 186 and EbpB's pilin motif Lys 197 are necessary for EbpC polymerization and EbpB incorporation into pilus fibers, respectively. (A) Negative-stain immunogold EM studies were carried out with anti-EbpC sera to visualize pilus fibers expressed by the EbpABC− strain carrying either pGCP123 (vector control), p-ebpABC, p-ebpAB_K179A_C, or p-_ebpABC_K186A. Scale bars are 500 nm. (B to D) Western blots of the EbpABC− strain carrying either pGCP123 (vector control), p-ebpABC, p-ebpAB_K179A_C, or p-_ebpABC_K186A were performed after SDS-PAGE of the indicated cell fractions with (B) anti-EbpC, (C) anti-EbpB, and (D) anti-EbpA immune sera. The top blots show pilus HMWs (brackets), while the bottom blots show pilin monomers (#, EbpCK186A; arrow, EbpBK179A; asterisk, EbpA degradation product).

Fig 5

Fig 5

EbpB's sortase recognition motif is dispensable for EbpB incorporation into pilus fibers. (A) Negative-stain immungold EM using anti-EbpC sera and (B to D) Western blot analyses of cell lysates were performed on the EbpABC− SrtC− strain carrying either pGCP123 (vector control), p-ebpABCsrtC, or p-ebpAB_NTPLK_CsrtC. (A) Scale bars are 500 nm. Blots were probed with (B) anti-EbpC, (C) anti-EbpB, or (D) anti-EbpA immune sera. Pilus HMWs are indicated with brackets.

Fig 6

Fig 6

EbpA's sortase recognition motif is necessary for EbpA incorporation into pilus fibers and for EbpA's localization at the fiber. (A) Negative-stain immunogold EM studies were carried out with anti-EbpC sera to visualize pilus fibers expressed by the EbpABC− strain carrying either pGCP123 (vector control), p-ebpABC, or p-ebpA_GTPLE_BC. Scale bars are 500 nm. Western blots (B to D) of the EbpABC− strain carrying either pGCP123 (vector control), p-ebpABC, or p-ebpA_GTPLE_BC were performed after SDS-PAGE of the indicated cell fractions with (B) anti-EbpC, (C) anti-EbpB, and (D) anti-EbpA immune sera. The top blots show pilus HMWs (brackets), while the bottom blots show pilin monomers (arrowheads, EbpAGTPLE). (E) Deep-etch immunogold EM was performed with anti-EbpA sera to determinate the localization of EbpA in the pilus fiber. Solid arrowheads indicate gold beads. Scale bars are 100 nm.

Fig 7

Fig 7

EbpA is not necessary for elaboration of EbpB−- and EbpC−-containing pili with normal morphology. (A) Negative-stain immunogold EM studies were carried out with anti-EbpC sera to visualize pilus fibers expressed by the EbpABC− SrtC− strain carrying either p-ebpABCsrtC or p-ebpBCsrtC. Scale bars are 500 nm. Western blots (B and C) of the EbpABC− SrtC− strain carrying either p-ebpABCsrtC or p-ebpBCsrtC were performed after SDS-PAGE of the indicated cell fractions with (B) anti-EbpC or (C) anti-EbpB immune sera. Pilus HMWs are indicated with brackets.

Fig 8

Fig 8

The ebp operon in E. faecalis OG1RF encodes the Ebp pilus structural subunits EbpA (blue), EbpB (purple), and EbpC (orange) and the pilus-associated sortase SrtC (green). The housekeeping sortase SrtA (brown) is encoded elsewhere in the genome (A). Sortase recognition motif amino acid sequences (LPETG for EbpA, LPKTN for EbpB, and LPSTG for EbpC), pilin motif Lys residues (K), and the sortase catalytic Cys residues (C) are shown by the one-letter code on their respective subunits. Predicted Srt subunit thioacyl intermediates are faded (A to E). EbpA incorporation relies on cleavage of its sortase recognition motif by SrtC and resolution of the resultant intermediate by the EbpC pilin motif Lys residue (B). EbpC polymerization requires SrtC transpeptidation between the sortase recognition motif of the most recently incorporated EbpC subunit of a growing fiber and the pilin motif Lys residue of an incoming EbpC subunit (C). EbpB is incorporated into a growing fiber via its pilin motif Lys residue (D). SrtA participates in anchoring fully polymerized pilus fibers to the cell wall, likely via transpeptidation of the EbpB sortase recognition motif with a cell wall precursor molecule.

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