The Interaction of Antimicrobial Peptides with the Membrane and Intracellular Targets of Staphylococcus aureus Investigated by ATP Leakage, DNA-Binding Analysis, and the Expression of a LexA-Controlled Gene, recA.

The analysis of how antimicrobial peptides (AMPs) interact with bacterial membranes and intracellular targets is important for our understanding of how these molecules affect bacteria. Increased knowledge may aid the design of AMPs that work on their target bacterium without inducing bacterial resistance. Here, we describe different methods to investigate the mode of action of peptides against the Gram-positive bacterium Staphylococcus aureus. ATP leakage analysis can be used to evaluate the ability of AMPs to perturb bacteria. DNA-binding and SOS response induction can be analyzed to investigate intracellular targets.

The lysine-peptoid hybrid LP5 maintain activity under physiological conditions and affects virulence gene expression in Staphylococcus aureus.

The antimicrobial peptide, LP5, is a lysine-peptoid hybrid, with antimicrobial activity against clinically relevant bacteria. Here, we investigated how various environmental conditions affect the antimicrobial activity of LP5 against Staphylococcus aureus (S. aureus). We found that LP5 maintained activity under host physiological conditions of NaCl, MgCl2 and pH. However, when exposed to serum, LP5 lost activity. Furthermore, when increasing NaCl concentration and lowering pH, the peptide showed reduces activity. When investigating the tolerance mechanisms of S. aureus toward antimicrobial peptides, we found that LP5 was protease resistant. However, the dltA and vraF genes, involved in reducing the net anionic charge of the bacterial cell envelope and sensing of antimicrobial peptides, respectively, played a role in the tolerance of S. aureus against LP5. In addition, the exposure of S. aureus to sub-inhibitory concentrations of LP5 affected the expression of the major virulence factors of S. aureus, revealing a potential as anti-virulence compound. Thus, these results show how environmental factors affect the peptide efficiency and further add to the knowledge on how the peptide affects S. aureus, which is crucial information for designing new peptides for optimizing antimicrobial therapy.

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus.

The rapid rise in antibiotic-resistant pathogens is causing increased health concerns, and consequently there is an urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs), which have been isolated from a wide range of organisms, represent a very promising class of novel antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl(2) concentrations and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus-encoded natural tolerance mechanisms included peptide cleavage and the addition of positive charge to the cell surface, both of which minimized the antimicrobial activity of FL9. Our results add new information about FL9 and its effect on S. aureus, which may aid in the future development of analogues with improved therapeutic potential.

The antimicrobial lysine-peptoid hybrid LP5 inhibits DNA replication and induces the SOS response in Staphylococcus aureus.

BACKGROUND: The increase in antibiotic resistant bacteria has led to renewed interest in development of alternative antimicrobial compounds such as antimicrobial peptides (AMPs), either naturally-occurring or synthetically-derived. Knowledge of the mode of action (MOA) of synthetic compounds mimicking the function of AMPs is highly valuable both when developing new types of antimicrobials and when predicting resistance development. Despite many functional studies of AMPs, only a few of the synthetic peptides have been studied in detail. RESULTS: We investigated the MOA of the lysine-peptoid hybrid, LP5, which previously has been shown to display antimicrobial activity against Staphylococcus aureus. At concentrations of LP5 above the minimal inhibitory concentration (MIC), the peptoid caused ATP leakage from bacterial cells. However, at concentrations close to the MIC, LP5 inhibited the growth of S. aureus without ATP leakage. Instead, LP5 bound DNA and inhibited macromolecular synthesis. The binding to DNA also led to inhibition of DNA gyrase and topoisomerase IV and caused induction of the SOS response. CONCLUSIONS: Our data demonstrate that LP5 may have a dual mode of action against S. aureus. At MIC concentrations, LP5 binds DNA and inhibits macromolecular synthesis and growth, whereas at concentrations above the MIC, LP5 targets the bacterial membrane leading to disruption of the membrane. These results add new information about the MOA of a new synthetic AMP and aid in the future design of synthetic peptides with increased therapeutic potential.

Searching for small σB-regulated genes in Staphylococcus aureus.

In recent years, small RNAs (sRNAs) have been identified as important regulators of gene expression in bacteria. Most sRNAs are encoded from intergenic regions and are only expressed under highly specific growth conditions. In Staphylococcus aureus, the alternative sigma factor, σ(B), is known to contribute to the overall stress response, antibiotic resistance, and virulence. The σ(B) regulon in S. aureus is well described and comprises approximately 200 annotated genes, including several genes encoding virulence factors. In the present study, we have identified three novel σ(B)-dependent transcripts encoded from genomic regions previously annotated as intergenic. All three transcripts, named SbrA, SbrB, and SbrC, are highly conserved in S. aureus, and we confirmed their presence in four different isolates (SH1000, Newman, COL, and UAMS-1). Curiously, two of these genes (sbrA and sbrB) were found to contain open reading frames encoding small, highly basic peptides that are conserved among Staphylococci. The third transcript (SbrC) did not contain any likely open reading frame and thus constitute a genuine non-coding sRNA. The functions of these genes are currently unknown but are likely to be important for the σ(B)-mediated response of S. aureus to adverse conditions.

The heme sensing response regulator HssR in Staphylococcus aureus but not the homologous RR23 in Listeria monocytogenes modulates susceptibility to the antimicrobial peptide plectasin.

BACKGROUND: Host defence peptides (HDPs), also known as antimicrobial peptides (AMPs), have emerged as potential new therapeutics and their antimicrobial spectrum covers a wide range of target organisms. However, the mode of action and the genetics behind the bacterial response to HDPs is incompletely understood and such knowledge is required to evaluate their potential as antimicrobial therapeutics. Plectasin is a recently discovered HDP active against Gram-positive bacteria with the human pathogen, Staphylococcus aureus (S. aureus) being highly susceptible and the food borne pathogen, Listeria monocytogenes (L. monocytogenes) being less sensitive. In the present study we aimed to use transposon mutagenesis to determine the genetic basis for S. aureus and L. monocytogenes susceptibility to plectasin. RESULTS: In order to identify genes that provide susceptibility to plectasin we constructed bacterial transposon mutant libraries of S. aureus NCTC8325-4 and L. monocytogenes 4446 and screened for increased resistance to the peptide. No resistant mutants arose when L. monocytogenes was screened on plates containing 5 and 10 fold Minimal Inhibitory Concentration (MIC) of plectasin. However, in S. aureus, four mutants with insertion in the heme response regulator (hssR) were 2-4 fold more resistant to plectasin as compared to the wild type. The hssR mutation also enhanced resistance to the plectasin-like defensin eurocin, but not to other classes of HDPs or to other stressors tested. Addition of plectasin did not influence the expression of hssR or hrtA, a gene regulated by HssR. The genome of L. monocytogenes LO28 encodes a putative HssR homologue, RR23 (in L. monocytogenes EGD-e lmo2583) with 48% identity to the S. aureus HssR, but a mutation in the rr23 gene did not change the susceptibility of L. monocytogenes to plectasin. CONCLUSIONS: S. aureus HssR, but not the homologue RR23 from L. monocytogenes, provides susceptibility to the defensins plectasin and eurocin. Our data suggest that a functional difference between response regulators HssR and RR23 is responsible for the difference in plectasin susceptibility observed between S. aureus and L. monocytogenes.

The two-component system CesRK controls the transcriptional induction of cell envelope-related genes in Listeria monocytogenes in response to cell wall-acting antibiotics.

The two-component system CesRK of Listeria monocytogenes responds to cell wall-acting antibiotics. We show here that CesRK controls the transcription of several cell envelope-related genes. The CesRK-dependent induction of these genes may be viewed as an attempt by L. monocytogenes to protect itself against the damaging effects of cell wall-acting antibiotics.