PubMed · 15134077
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2004. Structurally speaking.. https://doi.org/10.1038/nrmicro891
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Gram-positive bacteria display virulence-associated pili that facilitate adhesion and biofilm formation. These pili are covalently polymerized by class C sortase enzymes, which selectively recognize their cognate pilin substrates amid numerous cell wall sorting signal (CWSS)-bearing proteins. The molecular basis for this stringent substrate specificity has remained unclear. Here, we develop a rapid, quantitative fluorescence-activated cell sorting assay to monitor pilus assembly in Corynebacterium diphtheriae, enabling high-throughput analysis of SpaA pilin and SrtA sortase variants. Using this platform, together with molecular modeling and dynamics simulations, we show that SrtA engages nearly the entire SpaA CWSS to form a membrane-embedded complex that incorporates not only the LPXTG motif but also its connector and transmembrane helix elements. Formation of this interface displaces an inhibitory active-site lid and activates the enzyme to load the pilin substrate. Systematic CWSS swapping experiments and deep mutational scanning further support this model, demonstrating that noncognate pilins are excluded because they fail to form the required interface. Conversely, SrtA variants with an artificially unlatched lid bypass the need for this interface, indicating that membrane-driven complex formation is important for substrate licensing. Together, these findings define a "two-factor authentication" mechanism for pilus assembly in gram-positive bacteria: class C sortases first verify pilin identity by forming a membrane-embedded interface that activates the enzyme, then they recognize the LPXTG motif to initiate loading and crosslinking. This work provides a unified molecular framework for selective pilin incorporation in gram-positive bacteria and identifies potential vulnerabilities in the licensing machinery that may be exploited therapeutically.
Clostridium perfringens is a gram-positive, anaerobic, spore-forming bacterial pathogen of humans and animals. C. perfringens also produces type IV pili (T4P) and has two complete sets of T4P-associated genes, one of which has been shown to produce surface pili needed for cell adherence. One hypothesis about the second set of T4P genes is that they comprise a type II secretion system (TTSS) like those found in gram-negative bacteria, but for gram-positive bacteria, the TTSS would aid transit across the thick peptidoglycan (PG) layer. The secretome of mutants lacking type IV pilins was examined, and a single protein, BsaC (CPE0517), was identified as being dependent on pilin PilA3 for secretion. The bsaC gene is in an operon with genes encoding a SipW signal peptidase and two putative biofilm matrix proteins, BsaA and BsaB, both of which have remote homology to Bacillus subtilis biofilm protein TasA. Since BsaA forms long oligomers that are secreted, we analyzed BsaA monomer interactions with de novo modeling. These models projected that the monomers formed isopeptide bonds as part of a donor strand exchange process. Mutations in residues predicted to form the isopeptide bonds led to the loss of oligomerization, supporting an exchange and lock mechanism, and isopeptide bonds were detected by mass spectrometry methods. Phylogenetic analysis showed the BsaA family of proteins is widespread among bacteria and archaea, but only a subset is predicted to form isopeptide bonds.
Dynamic properties of type IV pili are essential for their function in bacterial infection, twitching motility and gene transfer. Laser tweezers are versatile tools to study the molecular mechanism underlying pilus dynamics at the single molecule level. Recently, these optical tweezers have been used to monitor pilus elongation and retraction in vivo at a resolution of several nanometers. The force generated by type IV pili exceeds 100 pN making pili the strongest linear motors characterized to date. The study of pilus dynamics at the single molecule level sheds light on kinetics, force generation, switching and mechanics of the Neisseria gonorrhoeae pilus motor.