Structural Basis of Host Cell Recognition by the Pilus Adhesin from Streptococcus pneumoniae

Abstract

Pili are fibrous virulence factors associated directly to the bacterial surface that play critical roles in adhesion and recognition of host cell receptors. The human pathogen Streptococcus pneumoniae carries a single pilus-related adhesin (RrgA) that is key for infection establishment and provides protection from bacterial challenge in animal infection models, but details of these roles remain unclear. Here we report the high-resolution crystal structure of RrgA, a 893-residue elongated macromolecule whose fold contains four domains presenting both eukaryotic and prokaryotic origins. RrgA harbors an integrin I collagen-recognition domain decorated with two inserted "arms" that fold into a positively charged cradle, as well as three "stalk-forming" domains. We show by site-specific mutagenesis, mass spectrometry, and thermal shift assays that intradomain isopeptide bonds play key roles in stabilizing RrgA's stalk. The high sequence similarity between RrgA and its homologs in other Gram-positive microorganisms suggests common strategies for ECM recognition and immune evasion.

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

“…It is of note that these bonds had been previously shown to confer thermal stability and resistance to proteolysis to RrgC (22). This arrangement is distinct from that seen for RrgA, where isopeptide bonds stabilize the different halves of a ␤-sandwich (26). As in the case for other pilus-forming proteins, however, the Lys-Asn isopeptide bonds present in RrgC are located within hydrophobic cores and include Leu-163, Val-231, and Val-250 for D2, and Tyr-329, Leu-332, Val-352 for D3.…”

“…Cell fractionation, transmission electron microscopy, in vitro polymerization tests, and cell wall sorting signal domain swapping experiments have confirmed that covalently linked repeating units of RrgB form the S. pneumoniae pilus backbone whereas RrgA is present at the tip of the pilus (18, 20 -25). Notably, RrgA was shown to be able to recognize extracellular matrix elements in vitro, a finding confirmed by its three-dimensional structure, which revealed the presence of an integrin collagen-recognition domain (9,25,26). These elements thus collectively suggest that RrgA plays the role of pilus adhesin.…”

“…Notably, RrgA and RrgC were suggested as being located at the extremities of the fiber, with RrgC at the base and RrgA at the free end (25). The structure of RrgA revealed a four-domain flexible molecule carrying a von Willebrand factor (VWF) domain capable of recognizing extracellular matrix components (26), whereas RrgB displayed a threedomain fold (24,59). Until recently, however, the structure of the third structural component of the pneumococcal pilus, RrgC, was still lacking, impeding a more complete understanding of the architecture of the fiber from S. pneumoniae.…”

“…Isopeptide bonds have now been identified in a number of pilin subunits from Gram-positive bacteria, including RrgA and RrgB, which display such bonds in almost all domains (24,26,59). In RrgC, the isopeptide bonds of domains D2 and D3 had been identified through sequence comparisons and mutagenesis studies; the latter indicated that both bonds play a key role in protein stability (22).…”

“…RrgA, the four-domain adhesin that binds elements of the eukaryotic extracellular matrix, is covalently associated with the backbone fiber through its YPRTG motif, exposing its von Willebrand factor region as far as possible from the bacterial surface to promote adhesion (26) (Fig. 6, cyan).…”