Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular
infections represent a significant clinically challenging subset of invasive, life-threatening S. aureus
infections. We have recently demonstrated that
purine biosynthesis plays an important role in such
persistent infections.
Cyclic di-AMP (
c-di-AMP) is an essential and ubiquitous second messenger that regulates many cellular pathways in bacteria. However, whether there is a regulatory connection between the
purine biosynthesis pathway and
c-di-AMP impacting persistent outcomes was not known. Here, we demonstrated that the
purine biosynthesis mutant MRSA strain, the ΔpurF strain (compared to its isogenic parental strain), exhibited the following significant differences in vitro: (i) lower
ADP,
ATP, and
c-di-AMP levels; (ii) less biofilm formation with decreased extracellular
DNA (eDNA) levels and Triton X-100-induced
autolysis paralleling enhanced expressions of the biofilm formation-related two-component regulatory system lytSR and its downstream gene lrgB; (iii) increased
vancomycin (VAN)-binding and VAN-induced lysis; and (iv) decreased wall teichoic
acid (WTA) levels and expression of the WTA biosynthesis-related gene, tarH. Substantiating these data, the dacA (encoding diadenylate cyclase
enzyme required for
c-di-AMP synthesis) mutant strain (dacAG206S strain versus its isogenic wild-type MRSA and dacA-complemented strains) showed significantly decreased
c-di-AMP levels, similar in vitro effects as seen above for the purF mutant and hypersusceptible to VAN treatment in an experimental biofilm-related MRSA endovascular
infection model. These results reveal an important intersection between
purine biosynthesis and
c-di-AMP that contributes to biofilm-associated persistence in MRSA endovascular
infections. This signaling pathway represents a logical therapeutic target against persistent MRSA
infections. IMPORTANCE Persistent endovascular
infections caused by MRSA, including vascular graft
infection syndromes and
infective endocarditis, are significant and growing public health threats. A particularly worrisome trend is that most MRSA isolates from these patients are "susceptible" in vitro to conventional anti-MRSA
antibiotics, such as VAN and
daptomycin (DAP), based on Clinical and Laboratory Standards Institute breakpoints. Yet, these
antibiotics frequently fail to eliminate these
infections in vivo. Therefore, the persistent outcomes in MRSA
infections represent a unique and important variant of classic "antibiotic resistance" that is only disclosed during in vivo
antibiotic treatment. Given the high morbidity and mortality associated with the
persistent infection, there is an urgent need to understand the specific mechanism(s) of this syndrome. In the current study, we demonstrate that a functional intersection between
purine biosynthesis and the second messenger
c-di-AMP plays an important role in VAN persistence in experimental MRSA
endocarditis. Targeting this pathway may represent a potentially novel and effective strategy for treating these life-threatening
infections.