Abstract
Group A Streptococcus (GAS) responds to subinhibitory concentrations of LL-37 by up-regulation of virulence factors through the CsrRS (CovRS) two-component system. The signaling mechanism, however, is unclear. To determine whether LL-37 signaling reflects specific binding to CsrS or rather a nonspecific response to LL-37-mediated membrane damage, we tested LL-37 fragments for CsrRS signaling and for GAS antimicrobial activity. We identified a 10-residue fragment (RI-10) of LL-37 as the minimal peptide that retains the ability to signal increased expression of GAS virulence factors, yet it has no detectable antimicrobial activity against GAS. Substitution of individual key amino acids in RI-10 reduced or abrogated signaling. These data do not support the hypothesis that CsrS detects LL-37-induced damage to the bacterial cell membrane but rather suggest that LL-37 signaling is mediated by a direct interaction with CsrS. To test whether LL-37 binds to CsrS, we used the purified CsrS extracellular domain to pull down LL-37 in vitro, a result that provides further evidence that LL-37 binds to CsrS. The dissociation of CsrS-mediated signaling from membrane damage by LL-37 fragments together with in vitro evidence for a direct LL-37-CsrS binding interaction constitute compelling evidence that signal transduction by LL-37 through CsrS reflects a direct ligand/receptor interaction.
Highlights
Group A Streptococcus responds to the human antimicrobial peptide LL-37 by up-regulating virulence factors controlled by the CsrRS system
Antimicrobial activity for Group A Streptococcus (GAS) was assayed by determination of the minimum concentration of peptide that prevented growth during overnight incubation in 0.5ϫ Todd-Hewitt broth (MIC) and the minimum concentration of peptide that resulted in 99.9% killing after 1 h of incubation in 0.5ϫ phosphate-buffered saline (MBC)
Using qRT-PCR, we found that exposure of GAS 854 to 300 nM mCRAMP resulted in increased expression of the CsrRS-regulated genes hasB and sda1 (Fig. 1)
Summary
Group A Streptococcus responds to the human antimicrobial peptide LL-37 by up-regulating virulence factors controlled by the CsrRS system. To determine whether LL-37 signaling reflects specific binding to CsrS or rather a nonspecific response to LL-37-mediated membrane damage, we tested LL-37 fragments for CsrRS signaling and for GAS antimicrobial activity. Gryllos et al [24] first described the signaling relationship between LL-37 and the CsrS sensor They found that exposure of GAS to subinhibitory concentrations of LL-37 resulted in increased expression of multiple virulence factors including the has operon, responsible for capsular polysaccharide biosynthesis, and that CsrS was essential for this effect. We identified a 10-residue fragment of LL-37 (RI-10) that has similar activity to full-length LL-37 with respect to its ability to signal through CsrS This short peptide lacks all antimicrobial activity against GAS and exhibits specific amino acid and chirality requirements for its signaling effect. These data provide strong evidence that LL-37 signaling through CsrS reflects a specific binding interaction between LL-37 and the extracellular domain of CsrS
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