Streptococcal Lancefield polysaccharides are critical cell wall determinants for human Group IIA secreted phospholipase A2 to exert its bactericidal effects.

Vincent P Van Hensbergen,Vincent De Maat,Britta Brügger,Nina M Van Sorge,Christian Lüchtenborg,Victor Nizet,Anna Henningham,Yoann Le Breton,Gérard Lambeau,Christine Payré,Kevin S Mciver,Fredric Carlsson,Jos A G Van Strijp,Elin Movert

doi:10.1371/journal.ppat.1007348

Abstract

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the bactericidal enzyme hGIIA to exert its bactericidal function.

Highlights

Many important human bacterial pathogens are common colonizers of mucosal barriers
HGIIA requires binding to negatively charged surface structures before it can penetrate through the thick peptidoglycan layer and reach the target phospholipid membrane
We demonstrate that the Lancefield cell wall polysaccharides that are expressed by these bacteria, the Group A Carbohydrate (GAC) for Group A Streptococcus (GAS) and the Group B Carbohydrate (GBC) for Group B Streptococcus (GBS), are required for optimal human Group IIA phospholipase A2 (hGIIA) bactericidal efficacy by facilitating penetration through the peptidoglycan layer

Summary

Introduction

Many important human bacterial pathogens are common colonizers of mucosal barriers Such pathogens penetrate these physical barriers to invade the underlying tissues and cause infections. One of the most potent bactericidal molecules against Gram-positive bacteria is the secreted enzyme human Group IIA phospholipase A2 (hGIIA) [1,2]. Sterile inflammation or infection increases hGIIA expression with concentrations reaching up to 1 μg/ml in serum [7], which is sufficient to kill most Gram-positive pathogens in vitro. A unique feature of hGIIA compared to other secreted phospholipase A2 family members is its high cationic charge, which is required for binding to negatively-charged surface structures and for penetration of the thick peptidoglycan layer surrounding Gram-positive bacteria [2,8,9]. The potent bactericidal activity of hGIIA against Gram-positive bacteria has been demonstrated in vitro, using recombinant hGIIA, and is suggested by infection experiments that show increased protection from infection using hGIIA transgenic mice [10,11,12,13,14,15,16]

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