Regulation of innate immunity by the pathogen Staphylococcus aureus

About This Grant

Project Summary The goal of this proposal is to identify how the Gram-positive pathogen Staphylococcus aureus manipulates inflammasome activities that are important for host defense. With no vaccine in place and antibiotic resistance on the rise, understanding immunological aspects of S. aureus infections is of paramount importance. S. aureus strains of diverse genetic and epigenetic phenotypes cause infections, which can be difficult to treat and are subject to relapse. Acute infections are associated with so-called toxigenic strains of bacteria, which produce pore-forming toxins that stimulate the inflammasome regulatory protein NLRP3. Non- toxigenic bacterial strains are increasingly recognized for their association with difficult-to-clear infections including persistent bacteremia, endocarditis, and cystic fibrosis-associated pneumonia. Published work from us and others have demonstrated that non-toxigenic S. aureus retain the ability to stimulate inflammasomes, yet the mechanisms to explain these activities are unclear. This proposal is based on our recent discovery that Pattern Recognition Receptor AIM2 (but not NLRP3) is the primary mediator of inflammasome activities induced by diverse species and strains of Staphylococcus. Commonly used laboratory strains and those derived from S. aureus USA300, which accounts for >97% of skin infections in the United States, all induced AIM2-dependent inflammasome activities in macrophages. A genome-wide genetic screen in S. aureus identified the enzyme TarM as a mediator of AIM2 activation. TarM is an enzyme that glycosylates wall teichoic acid (WTA), a component of peptidoglycan (PGN). Interestingly, the WTA attachment site on PGN can be acetylated by a negative regulator of inflammasome activities, the S. aureus enzyme O- acetyltransferase A (OatA). TarM and OatA therefore display competing activities towards PGN modifications and towards AIM2 inflammasome activities. No current model of AIM2 functions can explain how TarM promotes or how OatA inhibits AIM2 during S. aureus infections. Based on our discoveries, we hypothesize that glycosylated WTA is a direct or indirect mediator of AIM2 activation during S. aureus infection, and that OatA prevents AIM2 activation by reducing the presence of glycosylated WTA on the bacterial cell wall. The relationship between these activities and how bacterial or host DNA activates AIM2 will be explored. We plan to define how TarM mediates AIM2 activation during infection in cultured cells, and determine the impact of these activities on acute inflammation and long term adaptive immunity in mice (specific aim 1). We further plan to determine the molecular mechanism of AIM2 activation in macrophages and how these events are coordinated with phagosome activities that may mediate DNA release into the cytosol (specific aim 2). Central to our mechanistic work is stoichiometric tandem-mass tagging (TMT) mass spectrometry, which will define kinetic interaction modules that regulate AIM2 activation events during S. aureus infection. Such analyses will be coupled with direct visualization of AIM2-inflammasomes within living cells, which will provide a comprehensive analysis of inflammasome activities during a bacterial infection.