Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Lamya El Mortaji,Remi Terrasse,Andrea Dessen,Thierry Vernet,Anne Marie Di Guilmi

doi:10.1074/jbc.m109.082776

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.

Highlights

ANR Grant 05-JC-JC-0049. □S The on-line version of this article contains supplemental Figs
The role played by the three different sortases in incorporation of the minor pilin subunits onto the fiber remains largely unresolved: both SrtC-1 and SrtC-2 have been suggested as playing a role in incorporation of RrgA, and both SrtC-1 and SrtC-3 have been proposed as participating in association of RrgC to the RrgB backbone [27, 29]
Co-expression of Pilin Subunits, Sortase, and Complex Purification—Bacterial cultures of Duet vectors transformed into E. coli STAR cells were performed, and protein expression was induced in 4 ml of Terrific Broth with 0.5 mM IPTG at 37 °C during 3 h

Summary

Pneumococcal Pilin Assembly

Investigation of the roles and relative contributions of the three sortases to the pilus formation process by genetic mutational approaches revealed that deletion of individual sortases does not compromise RrgB polymerization, indicating a redundancy in sortase functionality, deletion of all three sortases abolishes RrgB fiber formation [27, 29]. The role played by the three different sortases in incorporation of the minor pilin subunits onto the fiber remains largely unresolved: both SrtC-1 and SrtC-2 have been suggested as playing a role in incorporation of RrgA, and both SrtC-1 and SrtC-3 have been proposed as participating in association of RrgC to the RrgB backbone [27, 29]. We provide evidence that the intramolecular bonds stabilizing RrgB may play a role in its recognition by SrtC-1 and subsequent association to RrgC

EXPERIMENTAL PROCEDURES

NDa NDa GSSHHHH NDa NDa GSSHHHH NDa NDa

TrapTMHP columns to which only

Isopeptide bonds stabilize RrgB and

DISCUSSION