Structural Basis of Pilus Anchoring by the Ancillary Pilin RrgC of Streptococcus pneumoniae

Abstract

Background: RrgC is a key component of the pneumococcal pilus, a virulence factor that plays an important role in pathogenesis. Results: RrgC folds into three independent domains and requires the housekeeping sortase for surface association. Conclusion: The rod-like structure of RrgC suggests that it stably bridges peptidoglycan and pilus fiber. Significance: A complete model of the pneumococcal pilus reveals a multidomain, flexible assembly.

The Sortase-Dependent Fimbriome of the Genus Bifidobacterium: Extracellular Structures with Potential To Modulate Microbe-Host Dialogue

“…Notably, these FimB/Q paralogs also encompass a CNA domain, suggesting that these pili promote adhesion of bifidobacterial cells to host tissue, such as the gut mucosa, as well as adhesion to carbohydrates present in the gut, including those present in mucin or introduced with the diet (35,36). More specifically, in addition to a CNA domain homologous to the product of the rrgC gene of S. pneumoniae (37), the products of the fimB/Q homologs that make up COPGs 8, 9, 10, 11, and 15 also include domains predicted to be involved in sugar binding. These encompass homologs of domains 4 and 5 of the S-layer sugar-binding protein SbsC of Geobacillus stearothermophilus (38), as well as, except for COPG 9, homologs of the CBM 25 of Bacillus halodurans, responsible for starch binding (39).…”

Noncanonical sortase‐mediated assembly of pilus type 2b in group B Streptococcus

“…Bacteria were grown at 30°C for 12 h, and recombinant protein expression was induced by the addition of 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at 25°C for 24 h. The soluble proteins were extracted with CelLytic Express (Sigma-Aldrich) and purified by immobilized metal-affinity chromatography (IMAC) on His-Trap FF-Crude columns (GE Healthcare, Piscataway, NJ, USA). 15 N-labeled samples, were eluted with 300 mM imidazole, and the buffer was exchanged with TEV cleavage buffer [50 mM Tris-HCl (pH 8), 1 mM DTT, and 0.5 mM EDTA] (continued from previous page) immobilized metal-affinity chromatography; IPTG, isopropyl b-D-1-thiogalactopyranoside; KO, knockout; MOLREP, molecular replacement; PDB, Protein Data Bank; PEG, polyethylene glycol; PI, pilus island; PIPE, polymerase incomplete primer extension; rmsd, root-mean-square deviation; SEC, size-exclusion chromatography; SOE, splicing by overlap extension; Spa, sortase-mediated pilus assembly; Srt2-2b, sortase 2 + pilus type 2b; SrtA or C, class A or C sortase; SrtC1-2b, class C sortase 1 + pilus type 2b; THB, Todd Hewitt broth; TM, transmembrane; WT, wild-type using a PD-10 desalting column (GE Healthcare). The HIS-tag was cleaved by AcTEV protease (12 h at room temperature) and then removed by a subtractive IMAC purification step, followed by size-exclusion chromatography (SEC).…”

“…Srt enzymes, with their active cysteinyl group, cleave the peptide bond between the threonine (T) and the glycine (G) residues of the LPXTG motif, joining proteins to an amino group located on the next pilin subunit (6,7). SrtA is generally involved in anchoring of pilus to the cell wall in different species: C. diphtheriae (13), Bacillus cereus (14), Streptococcus pneumoniae (15), and S. agalactiae (16,17). The assembly and cell wall-anchoring mechanisms of GBS type 1 and 2a pili have been characterized (10,16,17), whereas those of pilus type 2b are not yet understood.…”

Pneumococcal Pili and Adhesins

“…Domain stabilization by autocatalytically formed, internal Lys-Asn isopeptide bonds, as shown for the first time in GAS major pilin Spy0128 by Kang and coworkers in 2007, was detected for RrgB D4, D3, and D2 domains [98À102]. In both variants, full-length, properly folded RrgA seems to be essential for RrgA-mediated pilus binding abilities [104]. Importantly, this indicates that certain structural features of pilus-building blocks most likely take place upon interaction of individual subunits during assembly of the polymeric pilus structure and are getting lost from studies using distant and individual recombinant domains.…”