SgrA, a nidogen-binding LPXTG surface adhesin implicated in biofilm formation, and EcbA, a collagen binding MSCRAMM, are two novel adhesins of hospital-acquired Enterococcus faecium - PubMed (original) (raw)
SgrA, a nidogen-binding LPXTG surface adhesin implicated in biofilm formation, and EcbA, a collagen binding MSCRAMM, are two novel adhesins of hospital-acquired Enterococcus faecium
Antoni P A Hendrickx et al. Infect Immun. 2009 Nov.
Abstract
Hospital-acquired Enterococcus faecium isolates responsible for nosocomial outbreaks and invasive infections are enriched in the orf2351 and orf2430 genes, encoding the SgrA and EcbA LPXTG-like cell wall-anchored proteins, respectively. These two surface proteins were characterized to gain insight into their function, since they may have favored the rapid emergence of this nosocomial pathogen. We are the first to identify a surface adhesin among bacteria (SgrA) that binds to the extracellular matrix molecules nidogen 1 and nidogen 2, which are constituents of the basal lamina. EcbA is a novel E. faecium MSCRAMM (microbial surface component recognizing adhesive matrix molecules) that binds to collagen type V. In addition, both SgrA and EcbA bound to fibrinogen; however, SgrA targeted the alpha and beta chains, whereas EcbA bound to the gamma chain of fibrinogen. An E. faecium sgrA insertion mutant displayed reduced binding to both nidogens and fibrinogen. SgrA did not mediate binding of E. faecium cells to biotic materials, such as human intestinal epithelial cells, human bladder cells, and kidney cells, while this LPXTG surface adhesin is implicated in E. faecium biofilm formation. The acm and scm genes, encoding two other E. faecium MSCRAMMs, were expressed at the mRNA level together with sgrA during all phases of growth, whereas ecbA was expressed only in exponential and late exponential phase, suggesting orchestrated expression of these adhesins. Expression of these surface proteins, which bind to extracellular matrix proteins and are involved in biofilm formation (SgrA), may contribute to the pathogenesis of hospital-acquired E. faecium infections.
Figures
FIG. 1.
Schematic representation of the SgrA LPXTG surface protein and the EcbA MSCRAMM. Organization of the SgrA and EcbA proteins identified from the E. faecium TX0016 genome sequence as published at DDBJ/EMBL/GenBank is shown. Signal sequences are depicted by an S, a putative ligand binding domain is indicated by an A, and the Ser-Ser-Glu-Ser-Ser-Thr repeats are numbered. The cell wall sorting signal is depicted as a striped box, starting at the LPETG or FPQTG sortase substrate motif. The putative latching regions are depicted in gray, the Cna B domains are indicated by white boxes, the phenylalanine box is indicated in black, and the proline rich region is indicated by “Pro.”
FIG. 2.
mRNA expression of sgrA, ecbA, acm, and scm in different stages of growth. Panels A to D show mRNA expression of sgrA, ecbA, acm, and scm in E1162 (left part) or E135 (right part) cells isolated in early exponential (EE), exponential (E), late-exponential (LE), or stationary (S) phase of growth in BHI broth at 37°C. E135 lacks both the sgrA and ecbA genes. The acm and scm genes are expressed in all stages of growth in E1162 (panels C and D), while acm is not expressed in the E135 strain. Control ddl PCRs (E) with total mRNA preparations in which the RT reaction was omitted were all negative, demonstrating an absence of DNA contamination. Control ddl RT-PCRs (internal housekeeping control) (F) with _E. faecium_-specific ddl primers were all positive. The results are presented as amplified PCR products electrophoresed on an ethidium bromide-stained 1.5% agarose gel.
FIG. 3.
Binding of rSgrA and rEcbA to ligands of the ECM as assessed by ELISA. Panel A shows concentration-dependent binding of rSgrA to immobilized fibrinogen (black circles) and not to fibronectin (black ×), vitronectin (open circles), or BSA (black squares). The OD450s were corrected for the response of six-His IgG-HRP antibodies with the respective ECM proteins. Data points represent the means of OD450s ± standard deviations for three independent experiments with two different purified rSgrA protein batches. Panel B demonstrates concentration-dependent binding of biotinylated nidogen 1 (black squares) and nidogen 2 (black circles) to immobilized rSgrA in a saturable manner and not to BSA (black inverse triangles). Bound proteins were detected using streptavidin peroxidase conjugate. The data points are representative values of three independent experiments with two different purified rSgrA protein batches. Panel C indicates concentration-dependent binding of rEcbA to immobilized collagen type V (black triangles) and fibrinogen (black squares) and not to collagen types I to IV, vitronectin, laminin, or BSA. The data points are representative values of four independent experiments with three different purified rEcbA protein batches.
FIG. 4.
Ligand affinity blotting demonstrated binding of rSgrA to nidogen and fibrinogen. (A) Human fibrinogen and fibronectin were separated through SDS-PAGE, while nidogen 1 and nidogen 2 were separated through native PAGE followed by Coomassie blue staining (indicated by “C”). The ligand affinity blots were probed with rSgrA (fibrinogen; left part) or biotinylated rSgrA (nidogen 1 and nidogen 2) and are indicated by an “L.” (B) Reciprocal ligand affinity binding assays. rSgrA was separated through SDS-PAGE and stained with Coomassie (lane C). Anti-His monoclonal antibodies (L1) or biotinylated ligands including fibrinogen (L2), nidogen 1 (L3), nidogen 2 (L4), and a negative control (biotinylated fibronectin; L5) were allowed to bind to rSgrA and were detected using a streptavidin conjugate. (C) Ligand affinity blotting under reducing conditions demonstrated binding of rEcbA to collagen type V and fibrinogen and not to fibronectin.
FIG. 5.
Adherence of E1162 and the isogenic mutant E1162Δ_sgrA_ to ECM molecules by whole-cell ELISA. (A) Fibrinogen, nidogen 1, nidogen 2, and laminin (negative control) were immobilized on a microtiter plate, and E1162 and E1162Δ_sgrA E. faecium_ cells were added to the wells and allowed to bind to these components. Adherent bacteria were detected using an antienterococcus serum followed by goat anti-rabbit IgG-HRP antibodies. Black bars represent wild-type E. faecium E1162, and white bars indicate the E1162Δ_sgrA_ mutant. *, P < 0.005; **, P < 0.05. (B) Cell surface hydrophobicities of wild-type E1162 and an sgrA isogenic mutant (E1162Δ_sgrA_). The experiments were performed three times with similar results, and values represent means ± standard deviations of triplicate measurements. *, P = 0.0169.
FIG. 6.
Biofilm formation on polystyrene. The ability of an sgrA_-negative strain, E135 (gray bar), wild-type E1162 (black bar), or an sgrA isogenic mutant, E1162Δ_sgrA (white bar) to form a 24-h biofilm on a polystyrene surface is shown. The experiments were performed twice with similar results, and values represent means ± standard deviations of 10 measurements. *, P < 0.002; **, P = 0.03.
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