Abstract
Due to the antibiotic resistance crisis, novel therapeutic strategies need to be developed against bacterial pathogens. Hydrophobic bacterial peptides (small proteins under 50 amino acids) have emerged as regulatory molecules that can interact with bacterial membrane proteins to modulate their activity and/or stability. Among them, the Salmonella MgtR peptide promotes the degradation of MgtC, a virulence factor involved in Salmonella intramacrophage replication, thus providing the basis for an antivirulence strategy. We demonstrate here that endogenous overproduction of MgtR reduced Salmonella replication inside macrophages and lowered MgtC protein level, whereas a peptide variant of MgtR (MgtR-S17I), which does not interact with MgtC, had no effect. We then used synthetic peptides to evaluate their action upon exogenous addition. Unexpectedly, upon addition of synthetic peptides, both MgtR and its variant MgtR-S17I reduced Salmonella intramacrophage replication and lowered MgtC and MgtB protein levels, suggesting a different mechanism of action of exogenously added peptides versus endogenously produced peptides. The synthetic peptides did not act by reducing bacterial viability. We next tested their effect on various recombinant proteins produced in Escherichia coli and showed that the level of several inner membrane proteins was strongly reduced upon addition of both peptides, whereas cytoplasmic or outer membrane proteins remained unaffected. Moreover, the α-helical structure of synthetic MgtR is important for its biological activity, whereas helix-helix interacting motif is dispensable. Cumulatively, these results provide perspectives for new antivirulence strategies with the use of peptides that act by reducing the level of inner membrane proteins, including virulence factors.
Highlights
Recent studies based on genomics and natural product discovery have led to a large increase in the number and diversity of small proteins identified to be produced by bacteria[1,2,3]
An MgtR variant carrying an S17I mutation, where a small residue of the Ala-coil motif is substituted by a large hydrophobic residue, was found to prevent the strong interaction detected between MgtC and MgtR using the bacterial adenylate cyclase two-hybrid (BACTH) system[6]
An increasing number of studies on membrane active peptides show that the peptides exert their biological activity by interacting with the cell membrane, either to disrupt it to lead to cell lysis or to translocate through it[44]
Summary
Recent studies based on genomics and natural product discovery have led to a large increase in the number and diversity of small proteins (size below 50 amino-acids, hereafter called peptides) identified to be produced by bacteria[1,2,3]. M. tuberculosis and P. aeruginosa lack mgtR gene in their genome, but ectopic expression of Salmonella mgtR in these bacteria mimicked the phenotypes reported for mgtC deletion mutants[24,29] These results showed an antivirulence action of MgtR upon endogenous production in various bacterial pathogens. The therapeutic interest in peptide modulators of protein-protein interactions in membrane has increased recently[30] With this context, the aim of the present study is to investigate the antivirulence effect and biological activity of a synthetic MgtR peptide added exogenously to Salmonella. Our results support an antivirulence effect of MgtR-derived synthetic peptides against Salmonella, through an action independent of the Ala-coil motif, which lowers levels of inner membrane proteins essential for virulence
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