High Throughput Screen of Group A Streptococcus Clinical Isolates to Identify Conserved Strain-Specific Polymorphisms in Two-Component Systems Associated with Susceptibility to Membrane-Targeting Antimicrobials
Authors: Bui Dalton , Vega Luis , Guy Madeline , Morrison Gretchen , Mukherjee Piyali , Singh Bharat , Flores Anthony 
Published: November 2023

Abstract

Background
Group A Streptococcus (GAS) is a common human pathogen that causes various diseases including pharyngitis, impetigo, and rheumatic fever. Although penicillin remains an effective first-line treatment for GAS infections, GAS strains are becoming increasingly resistant to second-line treatments used when patients have beta-lactam allergies. Two-component systems (TCS) in bacteria sense and respond to environmental cues like cell envelope stresses, which include oxidative and antibiotic stress. The TCS known as LiaFSR is specific to Gram-positive bacteria and associated with daptomycin resistance in Enterococcus spp. A major target of LiaFSR regulation is SpxA2, a known regulator of GAS virulence associated with tolerance to oxidative stress. LiaFSR single nucleotide polymorphisms (SNPs) that potentially influence GAS cell envelope stress response are present in a strain-specific pattern. We sought to define SNPs in LiaFSR that affect the ability of GAS to resist and respond to cell envelope stress.

Hypothesis
We hypothesized that conserved strain-specific polymorphisms in the LiaFSR two-component system influence GAS response to cell envelope stress.

Methods
We identified conserved strain-specific polymorphisms in LiaFSR in a collection of pediatric GAS isolates from Texas Children’s Hospital and Children’s Memorial Hermann Hospital in Houston, TX. 55 clinical isolates were chosen from eight emm (M protein gene) types for testing. Isolates were screened for susceptibility to oxidative stress on plates containing diamide, a thiol oxidizing agent. Survival of serially diluted isolate cultures was assessed relative to an emm3 wild-type strain. Representative isolates from emm types showing differences in diamide tolerance were subjected to varying concentrations of bacitracin, polymyxin B, LL-37, nisin, or daptomycin and compared to the emm3 wild-type strain. Survival in the presence of antimicrobials was measured on a 96-well plate reader and compared to calibration growth curves to obtain virtual colony counts (CFUV). Differences in isolate CFUV following antimicrobial exposure relative to the emm3 wild-type strain were validated by colony counts (CFU) on blood agar plates of selected isolate cultures following exposure to inhibitory concentrations of antimicrobials.

Results
A P170S mutation in LiaF (emm25 and emm81) appeared to increase susceptibility to diamide-induced oxidative stress independent of genomic background. In the emm75 background, an H210N mutation in LiaS showed decreased diamide susceptibility relative to wild-type LiaS emm75 isolates. The emm75 isolate analyzed showed reduced susceptibility to nisin relative to an emm3 wild-type strain.

Conclusion
Strain-specific polymorphisms in LiaF and LiaS are worth pursuing to further our understanding of the influence LiaFSR has on cell envelope stress phenotypes. The findings of this screen provide a starting point for future research that will enable the production of targeted therapies against LiaFSR to optimize clinical outcomes and reduce the burden of multidrug resistance in GAS.