Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

Melanie A Higgins,Garrett E Whitworth,Nahida El Warry,Mialy Randriantsoa,Eric Samain,Robert D Burke,David J Vocadlo,Alisdair B Boraston

doi:10.1074/jbc.m109.024067

Abstract

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

Highlights

Large scale screening of pneumococcal virulence factors has revealed a large complement of genes devoted to complex carbohydrate metabolism that contribute to pneumococcal virulence [7,8,9]
We discuss these results in the context of the recent association of the pneumococcal fucose utilization operon with the virulence of S. pneumoniae [7, 12] and the possible strain-specific dependence of pneumococcal virulence on the carbohydrate antigens presented by different hosts
S. pneumoniae GH98 Enzymes—The GH98 enzymes from S. pneumoniae TIGR4 (Sp4GH98) and SP3-BS71 (Sp3GH98) are 1038- and 1005-amino acid, respectively, multimodular proteins with predicted classical N-terminal Gram-positive secretion signal sequences

Summary

Introduction

Substrate specificity assays of Sp3GH98 (18 mg/ml) and Sp4GH98 (34 mg/ml) were carried out using the LewisY tetrasaccharide and blood group A and B type 1, 2, and 4 pentasaccharides. GH98 Enzymes Are Active on Blood Group Antigens in Vitro and in Situ—Given the different modular structures of Sp3GH98 and Sp4GH98 and the presence of the genes encoding these divergent GH98 enzymes within fucose utilization operons having different gene contents and organizations [14], we suspected that these enzymes might have different, yet related, substrate specificities for human cell surface fucosecontaining glycans.

Results

Conclusion