Project description:Understanding how pathogens respond to antimicrobial peptides, and how this compares to currently available antibiotics, is crucial to optimizing antibiotic therapy. Staphylococcus aureus has several known resistance mechanisms against human cationic antimicrobial peptides (CAMPs). We aim to determine how S. aureus responds to sheep and frog CAMPs, and whether this response is associated with resistance. Gene expression changes in Staphylococcus aureus Newman cells exposed to linear CAMPs were analyzed by DNA microarray. Three antimicrobial peptides were used in the analysis, two of them are derived from frog, temporin L and dermaseptin K4-S4(1-16), one is from sheep, ovispirin-1. The peptides induced the VraSR cell-wall regulon and several other genes which are also upregulated in cells treated with vancomycin and other cell wall-active antibiotics. In addition to this similarity, three genes/operons were particularly strongly induced by the peptides: vraDE, SA0205 and SAS016, encoding an ABC transporter, a putative membrane-bound lysostaphin-like peptidase and a small functionally unknown protein, respectively. Ovispirin-1 and dermaseptin K4-S4(1-16), which disrupt lipid bilayers by the carpet mechanism, were strong inducers of the vraDE operon. We show that high level induction by ovispirin-1 was dependent on the amide modification of the peptide C-terminus. This suggests that the amide group has a crucial role in the activation of the Aps sensory system, the regulator of vraDE. In contrast, temporin L, which disrupts lipid bilayers by forming pores, was clearly a weaker inducer of vraDE despite the C-terminal amide modification. Sensitivity testing with CAMPs and other antimicrobials suggested that VraDE is a transporter dedicated to resist bacitracin. We also showed that SA0205 belongs to the VraSR regulon. Furthermore, VraSR was shown to be important for resistance against a wide range of cell wall-active antibiotics and other antimicrobial agents including the amide-modified ovispirin-1, bacitracin, teicoplanin, cefotaxime and 10 other β-lactam antibiotics, chlorpromazine, thioridazine and EGTA. The effects of the three different antimicrobial peptides on gene expression of S. aureus Newman were studied by using whole genome oligo-DNA microarrays. Bacteria were grown in BHI medium to the early exponential phase (OD600=0.6) and antimicrobial peptides were added at sublethal concentrations. Samples were taken for RNA isolations after treating the cultures with the peptides for 10 minutes. Control cultures without peptide additions were treated similarly and in parallel.

Project description:Understanding how pathogens respond to antimicrobial peptides, and how this compares to currently available antibiotics, is crucial to optimizing antibiotic therapy. Staphylococcus aureus has several known resistance mechanisms against human cationic antimicrobial peptides (CAMPs). We aim to determine how S. aureus responds to sheep and frog CAMPs, and whether this response is associated with resistance. Gene expression changes in Staphylococcus aureus Newman cells exposed to linear CAMPs were analyzed by DNA microarray. Three antimicrobial peptides were used in the analysis, two of them are derived from frog, temporin L and dermaseptin K4-S4(1-16), one is from sheep, ovispirin-1. The peptides induced the VraSR cell-wall regulon and several other genes which are also upregulated in cells treated with vancomycin and other cell wall-active antibiotics. In addition to this similarity, three genes/operons were particularly strongly induced by the peptides: vraDE, SA0205 and SAS016, encoding an ABC transporter, a putative membrane-bound lysostaphin-like peptidase and a small functionally unknown protein, respectively. Ovispirin-1 and dermaseptin K4-S4(1-16), which disrupt lipid bilayers by the carpet mechanism, were strong inducers of the vraDE operon. We show that high level induction by ovispirin-1 was dependent on the amide modification of the peptide C-terminus. This suggests that the amide group has a crucial role in the activation of the Aps sensory system, the regulator of vraDE. In contrast, temporin L, which disrupts lipid bilayers by forming pores, was clearly a weaker inducer of vraDE despite the C-terminal amide modification. Sensitivity testing with CAMPs and other antimicrobials suggested that VraDE is a transporter dedicated to resist bacitracin. We also showed that SA0205 belongs to the VraSR regulon. Furthermore, VraSR was shown to be important for resistance against a wide range of cell wall-active antibiotics and other antimicrobial agents including the amide-modified ovispirin-1, bacitracin, teicoplanin, cefotaxime and 10 other β-lactam antibiotics, chlorpromazine, thioridazine and EGTA.

Project description:A promising alternative to antibiotics for treatment of Staphylococcus aureus infections is photodynamic inactivation (PDI), which employs a photosensitizer (PS) that produces cytotoxic reactive oxygen species (ROS) when exposed to molecular oxygen and antimicrobial blue light in the spectrum of 400-470 nm. Although the precise mechanistic basis of PDI has not been defined, the formation of ROS and free radicals that oxidize a number of cellular targets, including membrane lipids, damage to proteins and nucleic acids result in inactivation of essential cellular functions and subsequent cell death. Because PDI is non-selective and affects multiple cellular targets, development of resistance or tolerance to PDI has been considered to be unlikely and attempts to induce S. aureus resistance or tolerance upon repeated sub-lethal doses of PDI have not succeeded. However, multiple aspects of PDI suggest that development of tolerance is highly probable, in particular when PDI is used for treatment of infections where the environment at the infection site prevents penetration of PDI at a level sufficient to cause death of all bacteria and with tolerant phenotypes emerging from the surviving bacteria. In this study, we sought to identify the mechanisms that contribute to PDI tolerance in S. aureus. S. aureus HG003 and the isogenic HG003DmutSL strain with defects in DNA mismatch repair were used to evaluate the response of S. aureus and the roles of DNA mismatch repair and gene regulatory networks to repeated sublethal doses of PDI. Global transcriptome and genome analyses were used in an agnostic approach to identify the underlying transcriptional responses and genetic adaptations that occur as a result of repeated PDI and contribute to PDI tolerance. Our results reveal multiple metabolic, transport and cell wall biogenesis pathways that contribute to PDI tolerance, and a S. aureus regulatory gene likely responsible for the adaptive transcriptional response to PDI.

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GNPS - Staphylococcus aureus response to antimicrobial peptides

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