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

Abstract

23Human Group IIA secreted phospholipase A 2 (hGIIA) is an acute phase protein with 24 bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic 25 mice have highlighted the importance of hGIIA as an innate defense mechanism against the 26 human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). 27Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To 28 identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon 29 library to recombinant human hGIIA and compared relative mutant abundance with library 30 input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the 31 output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA 32 susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included 33 two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene 34 cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented 35 strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in 36 the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is 37 conserved across different GAS serotypes. Increased resistance is associated with delayed 38 penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B 39Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. 40This suggests that the streptococcal Lancefield antigens, which are critical determinants for 41 streptococcal physiology and virulence, are required for the human bactericidal enzyme 42 hGIIA to exert its bactericidal function.

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides

“…We previously identified gacH in a GAS transposon library screen against the bactericidal activity of hGIIA 27 . Complementary to this susceptibility screen, we also exposed the Krmit GAS transposon library 14 to a lethal concentration of recombinant hGIIA to identify resistant mutants.…”

“…1b). M5005_Spy_1390 was also identified in the initial susceptibility screen as an artifact due to biased transposon insertions 27 and therefore not followed up further. To validate our finding for gacH, we generated a gacH deletion mutant in a GAS clinical isolate of the globally-disseminated serotype M1T1 clone 5448, creating 5448ΔgacH, and complemented the mutant with gacH on an expression plasmid, creating 5448ΔgacH:pgacH.…”

“…hGIIA susceptibility experiments were performed as described previously 27 . In short, mid-log suspensions (OD 600 = 0.4), of GAS and S. mutans were diluted 1,000 times in HEPES solution (20 mM HEPES, 2 mM Ca 2+ , 1% BSA [pH 7.4]) and 10 µL was added to sterile round-bottom 96…”

“…Similarly, the GacH/SccH-dependent GroP modifications on SRPs are required for hGIIA to exert its bactericidal effect against GAS and S. mutans, respectively. We have previously demonstrated that loss of the GlcNAc GAC side-chain strongly hampers trafficking of hGIIA through the GAS cell wall, with a minor contribution of reduced hGIIA binding to the cell surface 27 . Since GroPmodifications were also lost in the GlcNAc side-chain deficient mutant, 5448ΔgacI, described in this study, we now assume that the mechanisms of the hGIIA-dependent phenotype are similar in the gacI and gacH mutants.…”

“…Recently, we employed the Krmit GAS transposon mutant library 16 and identified gacI and gacH as factors that confer sensitivity of GAS to human secreted group IIA phospholipase A2 (hGIIA) (https://www.biorxiv.org/content/early/2018/02/23/269779) 30 , an important bactericidal protein of the innate immune defense against Gram-positive pathogens 31 .…”