A Highly Unusual Thioester Bond in a Pilus Adhesin Is Required for Efficient Host Cell Interaction*

Jonathan A Pointon,Wendy D Smith,Gerhard Saalbach,Allister Crow,Michael A Kehoe,Mark J Banfield

doi:10.1074/jbc.m110.149385

Jonathan A Pointon, Wendy D Smith + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.149385

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Abstract

Many bacterial pathogens present adhesins at the tips of long macromolecular filaments known as pili that are often important virulence determinants. Very little is known about how pili presented by Gram-positive pathogens mediate host cell binding. The crystal structure of a pilus adhesin from the important human pathogen Streptococcus pyogenes reveals an internal thioester bond formed between the side chains of a cysteine and a glutamine residue. The presence of the thioester was verified using UV-visible spectroscopy and mass spectrometry. This unusual bond has only previously been observed in thioester domains of complement and complement-like proteins where it is used to form covalent attachment to target molecules. The structure also reveals two intramolecular isopeptide bonds, one of these formed through a Lys/Asp residue pair, which are strategically positioned to confer protein stability. Removal of the internal thioester by allele-replacement mutagenesis in S. pyogenes severely compromises bacterial adhesion to model host cells. Although current paradigms of bacterial/host cell interaction envisage strong non-covalent interactions, the present study suggests cell adhesion could also involve covalent bonds.

Highlights

Quently referred to as the “shaft” or “major” pilus subunit); this structure is attached to the bacterial surface, frequently through a second minor pilus subunit that acts as a cell wall linker
Pili presented on the surface of the serotype M1 Group A Streptococcus strain SF370 (GAS)3 are required for efficient adhesion to model host cells and clinically relevant tissues (18 –20, 51)
Deletion of residues 53- 285 in GAS does not affect the ability of these bacteria to bind cultured host cells [51], and in some GAS serotypes this region has been lost from the genome, revealing it is dispensable for pilus assembly and Spy0125 function

Summary

EXPERIMENTAL PROCEDURES

Protein Production—The cloning of Spy0125 residues Asn286 —Thr-723 (Spy0125-CTR) has been described elsewhere [51]. Data used to solve the Spy0125-CTR structures were collected on BM14 at the European Synchrotron Radiation Facility, Grenoble, France from crystals grown using selenomethionine-labeled protein. The B-form crystal structure from the European Synchrotron Radiation Facility data comprises residues Thr-290 —Pro-719 in chain A (with the exception of residues Asn-318 —Ser-321 and Ile-373—Lys-376, which are disordered) and Pro-288 —Val720 in chain B (residues Asp-317—Asn-320 and Ile-373—Lys376 are disordered in this chain). Excitation and emission spectra for both wild-type and C426A variant Spy0125-CTR proteins were found to be essentially identical (supplemental Fig. 5A). For production of recombinant C426A Spy0125-CTR protein, the appropriate coding sequence was amplified from mutagenized GAS genomic DNA by PCR and cloned into the pOPIN-F expression vector [39]. Statistical significance was determined using Student’s t tests (two-tailed, unequal variance between samples within each experiment), with p values of 0.013, 1.46 ϫ 10Ϫ6, and 3.35 ϫ 10Ϫ5 for each experiment respectively (comparing wildtype to the C426A variant)

RESULTS

Expected mass

DISCUSSION