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

Abstract

AbstractHuman 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 AStreptococcus(GAS) and Group BStreptococcus(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 human 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, includingdltAandlytR,conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes,gacHandgacIthat are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenicgacImutant and gacI-complemented strains, we demonstrate that loss of the GACN-acetylglucosamine (GlcNAc) side chain in theΔgacImutant 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 human bactericidal enzyme hGIIA to exert its bactericidal function.Author summaryThe human immune system is capable of killing invading bacteria by secreting antimicrobial proteins. Cationic human Group IIA secreted phospholipase A2(hGIIA) is especially effective against Gram-positive bacteria by degrading the bacterial membrane. HGIIA requires binding to negatively charged surface structures before it can penetrate through the thick peptidoglycan layer and reach the target phospholipid membrane. HGIIA is constitutively expressed at high concentrations at sites of possible bacterial entry, e.g. in tears, skin and small intestine. In serum, normal concentrations are low but can increase up to 1,000-fold upon inflammation or infection.In vitro,ex vivoandin vivoexperiments suggest an important role for hGIIA in defense against two human pathogens, Group A and Group BStreptococcus(GAS, GBS). We demonstrate that the Lancefield cell wall polysaccharides that are expressed by these bacteria, the Group A Carbohydrate (GAC) for GAS and the Group B Carbohydrate (GBC) for GBS, are required for optimal hGIIA bactericidal efficacy by facilitating penetration through the peptidoglycan layer. Given the increased hGIIA resistance of antigen-modified or antigen-deficient streptococci, it will be of interest to determine potential regulatory mechanisms regarding expression of streptococcal Lancefield polysaccharides.