Identifying LiaFSR Residues Contributing to ExPortal Integrity and Response to Antimicrobials in Group A Streptococcus
Authors: Guy Madeline , Vega Luis A , Flores Anthony R 
Published: November 2023

Abstract

Background
Group A Streptococcus (GAS), or Streptococcus pyogenes, is responsible for a myriad of diseases (pharyngitis, scarlet fever, impetigo, toxic shock syndrome) of which several are prevalent in the pediatric population. The LiaFSR two-component regulatory system (TCS) and the ExPortal functional membrane microdomain (FMM) of GAS have been shown to coordinate the response to cell membrane stress when the bacterium is exposed to antimicrobials or antimicrobial peptides (AMPs). The ExPortal coordinates translocation and processing of secreted GAS proteins and contributes to sensing membrane. In an unstressed state, LiaF acts as an inhibitor of LiaS activity, both residing within the ExPortal. In a stressed state, such as exposure to antimicrobial agents, LiaF and S dissociate, allowing LiaS to phosphorylate LiaR, resulting in subsequent activation and expression of downstream target genes. Genomic analysis indicates a strict conservation of specific protein domains and certain amino acid residues in LiaF, S, and R, which suggests their critical involvement in the LiaFSR mechanism of action. We hypothesize that conserved amino acid residues in LiaFSR, such as the LiaF C-terminal domain (CTD) and a 3xK residue motif, are essential to ExPortal formation and maintenance and may contribute to differences in antimicrobial susceptibility and virulence.

Methods
Two different mutant strains were analyzed and compared to (wild-type) WT: one lacking the LiaF CTD (LiaF_∆CTD) and one mutagenized in the 3xK motif (LiaF_K91-93A). Five different assays were used to examine phenotypes associated with LiaFSR activity: SpeB protease activity through bacterial growth on milk plates, ExPortal integrity via fluorescence microscopy, diamide-induced oxidative stress tolerance, antimicrobial/antimicrobial peptide (AM/AMP) susceptibility, and a direct bactericidal assay in human blood (Lancefield). For the AM/AMP susceptibility, we tested 6 different antimicrobial agents with varying mechanisms of action: bacitracin, polymyxin B, LL-37, nisin, daptomycin, and HNP-1.

Results
Milk plate analysis revealed a significant decrease in SpeB production in the LiaF-∆CTD vs. WT, but no difference in LiaF-K91-93A relative to WT. During fluorescence microscopy, no significant differences were detected between foci of fluorescence of either mutant strain relative to WT. When exposed to diamide-induced oxidative stress, both mutants exhibited increased tolerance relative to WT. AM/AMP susceptibility assays did not reveal any significant differences for any antimicrobial agent between either mutant strain vs. WT. Finally, the Lancefield assay revealed a significant decrease in survival in human blood between both mutant strains and WT. Mutant strains also had significantly decreased survival relative to a strain lacking LiaF (∆LiaF).

Conclusions
The results of the AM/AMP susceptibility assay suggest that, unlike the LiaFSR system in Enterococcus, LiaF function does not contribute to LiaFSR-dependent susceptibility. Significant differences in survival in blood indicate that the LiaF residues studied are physiologically relevant in GAS infection. However, their exact mechanism underlying this contribution is being investigated. Future research will examine downstream gene regulation to elucidate the LiaFSR-associated mechanism of decreased survival in human blood, with the aim of defining determinants of GAS bacteremia.