ExPortal and the LiaFSR Regulatory System Coordinate the Response to Cell Membrane Stress in Streptococcus pyogenes

Abstract

LiaFSR is a gene regulatory system important for response to cell membrane stress in Gram-positive bacteria but is minimally studied in the important human pathogen group A Streptococcus (GAS). Using immunofluorescence and immunogold electron microscopy, we discovered that LiaF (a membrane-bound repressor protein) and LiaS (a sensor kinase) reside within the GAS membrane microdomain (ExPortal). Cell envelope stress induced by antimicrobials resulted in ExPortal disruption and activation of the LiaFSR system. The only human antimicrobial peptide whose presence resulted in ExPortal disruption and LiaFSR activation was the alpha-defensin human neutrophil peptide 1 (hNP-1). Elimination of membrane cardiolipin through targeted gene deletion resulted in loss of LiaS colocalization with the GAS ExPortal and activation of LiaFSR, whereas LiaF membrane localization was unaffected. Isogenic mutants lacking either LiaF or LiaS revealed a critical role of LiaF in ExPortal integrity. Thus, LiaF and LiaS colocalize with the GAS ExPortal by distinct mechanisms, further supporting codependence. These are the first data identifying a multicomponent signal system within the ExPortal, thereby providing new insight into bacterial intramembrane signaling in GAS that may serve as a paradigm for Gram-positive bacteria. IMPORTANCE Bacterial two-component systems sense and induce transcriptional changes in response to environmental stressors, including antimicrobials and human antimicrobial peptides. Since the stresses imposed by the host’s defensive responses may act as markers of specific temporal stages of disease progression or host compartments, pathogens often coordinately regulate stress response programs with virulence factor expression. The mechanism by which bacteria recognize these stresses and subsequently induce transcriptional responses remains not well understood. In this study, we showed that LiaFSR senses cell envelope stress through colocalization of LiaF and LiaS with the group A Streptococcus (GAS) ExPortal and is activated in direct response to ExPortal disruption by antimicrobials or human antimicrobial peptides. Our studies shed new light on the sensing of cell envelope stress in Gram-positive bacteria and may contribute to the development of therapies targeting these processes.

LiaR-dependent gene expression contributes to antimicrobial responses in group AStreptococcus

“…Disruption of the LiaR operator of spxA2 reduced GAS survival to challenge by hNP-1, the major AMP produced by human neutrophils. Combined with the specific response of LiaFSR to α-defensins such as hNP-1 (Lin et al 2020; M. A. Sanson et al 2021), our data support a critical role of LiaR-mediated SpxA2 activity in GAS pathogenesis.…”

“…Inasmuch as GAS is a human-specific pathogen that responds to human molecules, we next performed the same CFUv determinations following exposure to hNP-1 – the host AMP known to activate the LiaFSR system (Lin et al 2020). Strains in which LiaR was absent or phosphorylation was reduced showed a significant decrease in CFUv following exposure to hNP-1 ( P <0.05, Figure 3C ).…”

“…The primers used in this study are listed in Supplemental Table S4. Isogenic mutants lacking LiaF and LiaR proteins, as well as the LiaS-R135G substitution mutant lacking in LiaR phosphorylation were generated in previous studies (Lin et al 2020; Flores et al 2015). Isogenic mutants in which the pattern of LiaR phosphorylation was altered were generated in the emm3 strain background (MGAS10870) by the in-frame replacement of nucleotides in the liaS ( SpyM3_1367 ) coding sequence resulting in the following residue substitutions: glutamine to alanine at position 146 (Q146A), aspartate to alanine at position 142 (D142A).…”

“…The concentration of bacitracin chosen for isogenic mutant comparisons was determined from previous experimentation indicating consistently reproducible activation of the LiaFSR system upon GAS exposure to 1µg/ml bacitracin (M. A. Sanson et al 2021; Lin et al 2020). Each strain was tested four times (biological replicates) on separate days by starting with overnight cultures inoculated from different TSA-II/SB plates.…”

“…Three biological replicates of strains MGAS10870 (low LiaR~P), LiaS-Q146A (high LiaR~P), and ∆liaR (control) were chromatin immunoprecipitated using a previously generated anti-LiaR antibody (Lin et al 2020). Briefly, strains were grown to mid-logarithmic phase (OD600 ~0.4) in 40 mL THY, proteins crosslinked to DNA with 1% formaldehyde, cells harvested by centrifugation, flash-frozen, and stored at -80 o C until processed.…”