Association of RrgA and RrgC into the Streptococcus pneumoniae Pilus by Sortases C-2 and C-3

Abstract

Pili are surface-exposed virulence factors involved in the adhesion of bacteria to host cells. The human pathogen Streptococcus pneumoniae expresses a pilus composed of three structural proteins, RrgA, RrgB, and RrgC, and requires the action of three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3, to covalently associate the Rrg pilins. Using a recombinant protein expression platform, we have previously shown the requirement of SrtC-1 in RrgB fiber formation and the association of RrgB with RrgC. To gain insights into the substrate specificities of the two other sortases, which remain controversial, we have exploited the same robust strategy by testing various combinations of pilins and sortases coexpressed in Escherichia coli. We demonstrate that SrtC-2 catalyzes the formation of both RrgA-RrgB and RrgB-RrgC complexes. The deletion and swapping of the RrgA-YPRTG and RrgB-IPQTG sorting motifs indicate that SrtC-2 preferentially recognizes RrgA and attaches it to the pilin motif lysine 183 of RrgB. Finally, SrtC-2 is also able to catalyze the multimerization of RrgA through the C-terminal D4 domains. Similar experiments have been performed with SrtC-3, which catalyzes the formation of RrgB-RrgC and RrgB-RrgA complexes. Altogether, these results provide evidence of the molecular mechanisms of association of RrgA and RrgC with the RrgB fiber shaft by SrtC-2 and SrtC-3 and lead to a revised model of the pneumococcal pilus architecture accounting for the respective contribution of each sortase.

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

“…We then set out to investigate the transpeptidation roles of SrtC-1 and SrtC-3, which had been previously linked to RrgC functionality within the pilus (19,23,52). To do so, we expressed different combinations of pilus-related proteins in S. pneumoniae R6 and R6⌬srtA strains and tested for the presence of RrgC in total, cell wall, and membrane extracts (Fig.…”

“…23 was used for mutagenesis experiments. Mutants were designed based on a surface entropy reduction (SER) method through the employment of the SER-prediction (SERp) server which suggested the modification of Glu-179, Lys-180, and Glu-181 to Ala, and were generated by using the QuikChange mutagenesis kit (Stratagene).…”

Pneumococcal Pili and Adhesins

“…After cytoplasmic expression of the P-1 subunits and Sec-dependent secretion, pilins are anchored to the membrane by C-terminal hydrophobic stretches. Polymerization of the major subunit RrgB that contains an IPQTG sorting motif and the conserved DVVDAHVYPKN pilin motif (with the ε-amino group of Lys 183 ) was described as being catalyzed by SrtC-1 and SrtC-2, resulting in covalent intersubunit linkages of the pilus backbone structure [48,101,109,110,113,114] ( Figure 17.2D). Sortase cysteine-transpeptidase activity mediates cleavage between threonine and glycine and catalyzes the subsequent ligation of the new C-terminus to a lysine ε-amino group of the next subunit to be incorporated into the pilus.…”

“…Pilus assembly occurs in the extracellular space by sortases that recognize LPxTG cell-wall sorting motifs (or variants thereof) near the C-terminal of individual subunits. Incorporation of ancillary protein RrgA at the pilus tip (RrgA sorting motif: YPRTG2Lys 183 of subsequent RrgB backbone subunit) is suggested by redundant SrtC-1 and SrtC-2 activity [48,101,109]. PI-1, TIGR4-specific sortases SrtC-1, SrtC-2, and SrtC-3 diverge in sequence from the housekeeping sortase (SrtA) and exhibit functional redundancy concerning pilus assembly [48,109,110,113À115].…”

“…S. pneumoniae possesses an arsenal of sortase-anchored lytic enzymes, such as hyaluronidases (SpnHL), glycosyl hydrolyases (neuraminidase A, NanA, β-galactosidase, BgaA, Exo-β-N-acetylglucosaminidase, StrH, pullulanase, SpuA, endo-β-N-acetylglucosaminidase, EndoD, and Endo-α-N-acetylgalactosaminidase, Eng), and proteases (zinc metalloproteases, serine protease PrtA), with a broad substrate specificity enabling the degradation of human glycoproteins and glycosaminoglycans present in body fluids and cellular surfaces [31,40,41]. Importantly, class C sortases catalyze the assembly and anchorage of pili, which are large multi-subunit hair-like fibers on the surface of S. pneumoniae and other Gram-positive bacteria [38,39,48]. In addition, several LPxTG-anchored surface proteins have been implicated in pneumococcal adhesion and colonization, such as PavB, pneumococcal collagen-like protein A (PclA), plasmin-and fibronectin-binding protein A (PfbA), and BgaA, as well as biofilm formation, such as the pathogenicity island (PI)-encoded pneumococcal serine-rich repeat protein (PsrP) (Figure 17.4B) [28,44À47].…”