Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Abstract

Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process.

New Cell Surface Protein Involved in Biofilm Formation by Streptococcus parasanguinis

“…All the members of this family of putative pilin subunits have LPXTG-type cell wall sorting sequences that are presumably targeted by sortases (15). Sortase-mediated isopeptide bond cross-linking stabilizes and strengthens Gram-positive bacterial pili (18,20) and may mediate bacterial adhesion to host cells (1,8,34). In this regard, it is possible that the nine putative pilin isopeptide linkage domains of BapA1 may facilitate the assembly of BapA1 into a pilus-like structure on the bacterial surface.…”

NMR Spectroscopic and Theoretical Analysis of a Spontaneously Formed Lys–Asp Isopeptide Bond

“…[3] Subsequently isopeptides were identified in proteins known to form, or associated with, pili. [4][5][6][7][8] All bacterial isopeptides are found β-sheet domains resembling the CnaA or CnaB folds protein Cna from Staphylococcus aureus. [9,10] CnaA and CnaB domains are predicted to occur in thousands of bacterial surface proteins, and isopeptide bonds emerge as a very common posttranslational modification underpinning Gram-positive pilus formation and stability.…”

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

“…Notably, in both domains, the isopeptide bonds connect the first and last ␤-strand in the ␤-barrel (which are located side-by-side due to the unusual IgG fold described above), in analogous fashion to a belt buckle, indicating that in RrgC these bonds assist in the stability of the core ␤-barrel. 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).…”

“…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). Notably, in both RrgB and RrgC, the stabilization of the fold by intramolecular isopeptide bonds plays a significant role in the efficient recognition of the pilin subunits by their cognate sortases (22) underlining the key role played by these bonds for stability of the architecture of the entire pilus. Thus, the structuring of pilus-forming proteins as "beads on a string," with each domain stabilized by a covalent isopeptide bond, is a common signature in extracellular Gram-positive fibers.…”