Isopeptide Bonds Block the Mechanical Extension of Pili in Pathogenic Streptococcus pyogenes

• Published in: The Journal of biological chemistry Vol. 285; no. 15; pp. 11235 - 11242
• Main Authors: Alegre-Cebollada, Jorge, Badilla, Carmen L., Fernández, Julio M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.04.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Atomic Force Microscopy; Bacteria; Bacterial Physiological Phenomena; Biophysics - methods; Crystallography, X-Ray - methods; Fimbriae, Bacterial - metabolism; Force Spectroscopy; Gram-positive; Hydrogen Bonding; Isopeptide; Mechanical Stability; Microbiology; Microscopy, Atomic Force - methods; Models, Biological; Mutation; Peptides - chemistry; Pilus; Protein Assembly; Protein Engineering - methods; Protein Folding; Protein Structure; Protein Structure and Folding; Protein Structure, Tertiary; Single Molecule Biophysics; Streptococcus pyogenes; Streptococcus pyogenes - metabolism; Streptococcus pyogenes - pathogenicity; Stress, Mechanical; Gram-positive; Single Molecule Biophysics; Bacteria; Force Spectroscopy; Isopeptide; Pilus; Atomic Force Microscopy; Protein Structure; Mechanical Stability; Protein Assembly
• DOI: 10.1074/jbc.M110.102962
• ISSN: 0021-9258; 1083-351X

In the early stages of an infection, pathogenic bacteria use long fibrous structures known as pili as adhesive anchors for attachment to the host cells. These structures also play key roles in colony and biofilm formation. In all those processes, pili must withstand large mechanical forces. The pili of the nasty Gram-positive human pathogen Streptococcus pyogenes are assembled as single, micrometer long tandem modular proteins of covalently linked repeats of pilin proteins. Here we use single molecule force spectroscopy techniques to study the mechanical properties of the major pilin Spy0128. In our studies, we engineer polyproteins containing repeats of Spy0128 flanked by the well characterized I27 protein which provides an unambiguous mechanical fingerprint. We find that Spy0128 is an inextensible protein, even when pulled at forces of up to 800 pN. We also found that this remarkable mechanical resilience, unique among the modular proteins studied to date, results from the strategically located intramolecular isopeptide bonds recently identified in the x-ray structure of Spy0128. Removal of the isopeptide bonds by mutagenesis readily allowed Spy0128 domains to unfold and extend, albeit at relatively high forces of 172 pN (N-terminal domain) or 250 pN (C-terminal domain). Our results show that in contrast to the elastic roles played by large tandem modular proteins such as titin and fibronectin, the giant pili of S. pyogenes evolved to abrogate mechanical extensibility, a property that may be crucial in the pathogenesis of this most virulent bacterium and, therefore, become the target of new therapeutic approaches against its infections.