Mechanism for Sortase Localization and the Role of Sortase Localization in Efficient Pilus Assembly in Enterococcus faecalis

Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8230, Saint Louis, MO 63110-1093, USA.
Journal of bacteriology (Impact Factor: 2.81). 04/2009; 191(10):3237-47. DOI: 10.1128/JB.01837-08
Source: PubMed


Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and
secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently
attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion
and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The
SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of
secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in
single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC
abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively
charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

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Available from: Jerome Pinkner, Oct 08, 2015
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    • "Secretion of Ebp pilus subunits through the Sec system and their processing by either Bps or SrtA were recently shown to be spatially coupled to specific loci on the bacterial cell surface [48], similar to the ExPortal model proposed for secretion of streptococcal proteins [49]. We can extend this to a model in which membrane-bound EbpA first forms an acyl-enzyme intermediate with Bps. "
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    ABSTRACT: The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.
    PLoS ONE 07/2013; 8(7):e68813. DOI:10.1371/journal.pone.0068813 · 3.23 Impact Factor
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    • "The model presented here for the anchoring of surface proteins to the cell wall of S. pyogenes differs substantially from the model proposed for Streptococcus mutans (Hu et al., 2008) and E. faecalis (Kline et al., 2009). These studies showed that sortase A colocalizes with SecA in a single membranal microdomain termed ExPortal. "
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    ABSTRACT: Wall-anchored surface proteins are critical for the in vivo survival of Streptococcus pyogenes. Cues in the signal sequence direct the membrane translocation of surface proteins: M protein to the septum, and SfbI to the poles. Both proteins are subsequently anchored to the wall by the membrane bound enzyme sortase A. However, the cellular features of these pathways are not fully understood. Here we show that M protein and SfbI are anchored simultaneously throughout the cell cycle. M protein is rapidly anchored at the septum, and in part of the cell cycle, is anchored simultaneously at the mother and daughter septa. Conversely, SfbI accumulates gradually on peripheral peptidoglycan, resulting in a polar distribution. Sortase is not required for translocation of M protein or SfbI at their respective locations. Methicillin-induced unbalanced peptidoglycan synthesis diminishes surface M protein but not SfbI. Furthermore, overexpression of the division regulator DivIVA also diminishes surface M protein but increases SfbI. These results demonstrate a close connection between the regulation of cell division and protein anchoring. Better understanding of the spatial regulation of surface anchoring may lead to the identification of novel targets for the development of anti-infective agents, given the importance of surface molecules for pathogenesis.
    Molecular Microbiology 04/2012; 84(4):631-47. DOI:10.1111/j.1365-2958.2012.08047.x · 4.42 Impact Factor
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    • "This accumulation decays once cell division is complete. Surprisingly, by applying electron microscopy and immunogold labelling techniques, sortase A could be observed at a single site within the plasma membrane of E. faecalis [58]. As this location coincided with the location of pilin-specific sortase, sortase substrates and SecA, sortase A has been proposed to associate with the aforementioned ExPortal structure [58]. "
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    ABSTRACT: The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions.
    Philosophical Transactions of The Royal Society B Biological Sciences 04/2012; 367(1592):1123-39. DOI:10.1098/rstb.2011.0210 · 7.06 Impact Factor
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