Antibacterial treatment of <em>Staphylococcus aureus

Abstract

Staphylococcus aureus, both a commensal organism and an important human pathogen, has been the objective of basic and clinical research for decades. S. aureus is the leading cause for a broad range of diseases, such as pneumonia, endocarditis or toxic shock syndrome. Especially nosocomial and community-acquired infections by methicillin-resistant S. aureus (MRSA) have become a major health issue. Since there has been an increased emergence of microorganisms resistant to various antibiotics, the development of new treatment strategies has become a key issue of modern biological and medical science and triggered the need for a fundamental knowledge on how these bacteria gain resistance to antibiotics.<br /> In the first part of this work, the response and putative resistance strategies of S. aureus to the lantibiotic mersacidin were studied. Mersacidin is an antimicrobial peptide of 20 amino acids that is ribosomally produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin acts by complexing the sugar phosphate head group of the peptidoglycan precursor lipid II, thereby inhibiting the transglycosylation reaction of peptidoglycan biosynthesis. First, the growth of S. aureus in the presence of subinhibitory concentrations of mersacidin was analyzed. Transcriptional data revealed an extensive induction of the cell wall stress response which is partly controlled by the two-component regulatory system (TCRS) VraSR and which predominantly included the transcription of cell wall biosynthesis genes. In contrast to other cell wall-active antibiotics, such as the glycopeptide vancomycin, lower concentrations of mersacidin were sufficient for induction, probably, because the efficacy of mersacidin is not affected by an increased cell wall thickness. However, the cell wall stress response was equally induced in the more resistant S. aureus strains SA137/93A and SA137/93G as well as in the highly susceptible strain SG511-Berlin. Therefore, the cell wall stress response may not account for the different susceptibilities of these strains to mersacidin, but it appears to be a general accelerator system of cell wall biosynthesis, thereby contributing to a common resistance strategy of S. aureus to cell wall-active antibiotics. Since the transcription of the VraDE ABC transporter genes was induced up to 1700-fold in these experiments, the role of VraDE in the response to mersacidin was examined. However, a vraE knock-out phenotype did not exhibit an increased susceptibility to mersacidin compared to the wild type strain. <br /> In order to gain further insights into the mechanisms that S. aureus uses to counteract antimicrobials like mersacidin, the features of S. aureus SG511-Berlin were identified that contribute to its high susceptibility to antimicrobial peptides (AMPs) compared to other S. aureus strains. The fairly susceptible strain SG511-Berlin has been extensively used in the field of basic research on staphylococci and has represented a standard strain for antimicrobial susceptibility testing for many years. Comparative expression profiling of S. aureus SG511-Berlin versus the more resistant S. aureus strain SA137/93A revealed a divergent regulation of the dltB, mprF and vraFG genes, which are under the control of the TCRS GraRS. These transcripts showed significantly lower abundance in strain SG511-Berlin. Sequence analysis of graS in strain SG511-Berlin revealed a native nucleotide insertion that generates a stop codon at position 64 of the sensor histidine kinase GraS, thereby deleting the entire cytoplasmic part of the protein. Quantitative RT-PCR and determination of the whole cell surface charge of graS complemented S. aureus SG511-Berlin directly linked its decreased dltB transcript level and the resulting increased negative cell surface charge to the nucleotide insertion in graS. MIC determinations identified the GraRS TCRS as a resistance factor to the lantibiotics mersacidin, nisin and Pep5. In conclusion, mersacidin appears to be a strong inducer of the cell wall stress response of S. aureus at very low concentrations, which reflects its general mode of action as a cell wall-active peptide as well as its use of a unique target site on lipid II. Additionally, mersacidin appeared not to be a substrate for the ABC transporter VraDE and therefore may provide directions for the design of future antimicrobials that circumvent the action of resistance transporters. Furthermore, the GraRS system represents an important resistance factor of S. aureus to counteract AMPs and, due to these findings, the use of S. aureus SG511-Berlin for research purposes should be carefully considered, since this strain does not reflect the normal response of S. aureus against antibiotics.<br /> In the second part of this work, the lysis genes of the bacteriophages phi11 and phi12 of S. aureus NCTC8325 were characterized to evaluate the potential of endolysins as a novel treatment strategy for S. aureus biofilms. Knowledge about the lytic activities of both endolysins is limited. Their nucleotide sequences have been published and the phi11 endolysin has been shown to possess a D-alanyl-glycyl endopeptidase and an N-acetylmuramyl-L-alanine amidase activity on crude cell walls of S. aureus OS2. In this approach, the lytic activities of heterologously overexpressed enzymes and their single subdomains were tested on isolated cell walls, whole cells and biofilms of staphylococci. The recombinant phi11 endolysin hydrolyzed heat-killed staphylococci as well as staphylococcal biofilms. Cell wall targeting appeared to be a prerequisite for lysis of whole cells and the combined action of the endopeptidase and amidase domains was necessary for maximum activity. In contrast, the phi12 endolysin was inactive and caused aggregation of the cells. Thus, endolysins may provide directions for the development of new biofilm treatment strategies to combat S. aureus nosocomial infections.