Mechanisms of Resistance Against the Human Group IIA Secreted Phospholipase A2 in Group B Streptococcus

Antimicrobial peptides play a major role in humoral innate immunity against microorganisms. Multiple studies highlight the important role human group IIA secreted phospholipase A2 (hGIIA) plays against Gram-positive bacterial infection. hGIIA kills bacteria by catalyzing the hydrolysis of the membrane glycerophospholipids. The Gram-positive cell wall, consisting of multiple peptidoglycan layers decorated with a variety of glycopolymers, represents a substantial barrier to hGIIA and it is poorly understood how hGIIA gains access to the bacterial plasma membrane to produce lethal damage. In vitro and in vivo data support an important role for hGIIA in resistance to Group B Streptococcus (GBS), a leading cause of neonatal sepsis and meningitis. We have recently identified novel bacterial resistance mechanisms against hGIIA in Group A Streptococcus (GAS). Our work provided new insights into bacterial virulence, revised the current models of GAS cell wall architecture and highlighted the importance of these structural determinants for resistance to hGIIA. Interestingly, GBS is killed by hGIIA at concentrations that are approximately 500-fold lower, than closely-related GAS strains. The experiments described in this proposal are designed to understand the underlying mechanisms for hGIIA potency against GBS. To accomplish our goal, we will use two approaches. In the first approach we will conduct a Tn-seq screen of a recently constructed, highly saturated GBS transposon library using lethal concentrations of hGIIA. After construction of deletion mutants in the identified genes we will perform antimicrobial assays and mechanistic assays to confirm the hGIIA susceptibility phenotype of the mutants and understand the mechanisms of resistance/susceptibility. In the second approach, we will investigate role of GBS peptidoglycan-attached glycopolymers in hGIIA susceptibility by testing specific glycopolymer-deficient mutants for permeability and resistance to small bactericidal proteins and cationic antimicrobial peptides. Successful outcomes will guide future efforts for new drug development against GBS