The lantibiotic mersacidin is a strong inducer of the cell wall stress response of Staphylococcus aureus

<p>Abstract</p> <p>Background</p> <p>The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids that is ribosomally produced by <it>Bacillus </it>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.</p> <p>Results</p> <p>Here, we studied the growth of <it>Staphylococcus aureus </it>in the presence of subinhibitory concentrations of mersacidin. Transcriptional data revealed an extensive induction of the cell wall stress response, which is partly controlled by the two-component regulatory system VraSR. In contrast to other cell wall-active antibiotics such as vancomycin, very low concentrations of mersacidin (0.15 × MIC) were sufficient for induction. Interestingly, the cell wall stress response was equally induced in vancomycin intermediately resistant <it>S. aureus </it>(VISA) and in a highly susceptible strain. Since the transcription of the VraDE ABC transporter genes was induced up to 1700-fold in our experiments, we analyzed the role of VraDE in the response to mersacidin. However, the deletion of the <it>vraE </it>gene did not result in an increased susceptibility to mersacidin compared to the wild type strain. Moreover, the efficacy of mersacidin was not affected by an increased cell wall thickness, which is part of the VISA-type resistance mechanism and functions by trapping the vancomycin molecules in the cell wall before they reach lipid II. Therefore, the relatively higher concentration of mersacidin at the membrane might explain why mersacidin is such a strong inducer of VraSR compared to vancomycin.</p> <p>Conclusion</p> <p>In conclusion, mersacidin appears to be a strong inducer of the cell wall stress response of <it>S. aureus </it>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 does not seem to be a substrate for the resistance transporter VraDE.</p

Generation of a vancomycin-intermediate Staphylococcus aureus (VISA) strain by two amino acid exchanges in VraS

Objectives Staphylococcus aureus is a notorious bacterial pathogen and antibiotic-resistant isolates complicate current treatment strategies. We characterized S. aureus VC40, a laboratory mutant that shows full resistance to glycopeptides (vancomycin and teicoplanin MICs ≥32 mg/L) and daptomycin (MIC = 4 mg/L), to gain deeper insights into the underlying resistance mechanisms. Methods Genomics and transcriptomics were performed to characterize changes that might contribute to development of resistance. The mutations in vraS were reconstituted into a closely related parental background. In addition, antimicrobial susceptibility testing, growth analyses, transmission electron microscopy, lysostaphin-induced lysis and autolysis assays were performed to characterize the phenotype of resistant strains. Results Genome sequencing of strain VC40 revealed 79 mutations in 75 gene loci including genes encoding the histidine kinases VraS and WalK that control cell envelope-related processes. Transcriptomics indicated the increased expression of their respective regulons. Although not reaching the measured MIC for VC40, reconstitution of the L114S and D242G exchanges in VraS(VC40) into the susceptible parental background (S. aureus NCTC 8325) resulted in increased resistance to glycopeptides and daptomycin. The expression of VraS(VC40) led to increased transcription of the cell wall stress stimulon, a thickened cell wall, a decreased growth rate, reduced autolytic activity and increased resistance to lysostaphin-induced lysis in the generated mutant. Conclusions We show that a double mutation of a single gene locus, namely vraS, is sufficient to convert the vancomycin-susceptible strain S. aureus NCTC 8325 into a vancomycin-intermediate S. aureu

Production of capsular polysaccharide does not influence Staphylococcus aureus vancomycin susceptibility

Background: Diverse mechanisms (increased cell wall thickness, low cross linking, decreased autolysis, etc.) have been reported for Staphylococcus aureus strains with intermediate vancomycin susceptibility (VISA). This study was conducted to identify common mechanisms responsible for decreased vancomycin susceptibility in a VISA strain pair. Results: Transcriptional profiling of the clinical heterogeneous VISA isolate SA137/93A and its spontaneous homogeneous mutant strain SA137/93G pointed to an increased capsule production in the strain pair compared to a susceptible control. Furthermore, transcript quantification of the gene cap5E, which is essential for capsule biosynthesis, revealed elevated levels in the VISA strains SA137/93A, SA137/93G and Mu50 in comparison with susceptible strains Reynolds, Newman and SA1450/94. The increased expression was observed in bacteria from exponential as well as stationary growth phase. However, suppression of type 5 capsule formation by expression of antisense RNA did not increase vancomycin susceptibility in the VISA strain SA137/93G. Likewise, construction of inducible mutants of S. aureus Newman or repair of capsule biosynthesis of S. aureus HG001 and S. aureus 1450/94 did not influence resistance to vancomycin. Furthermore, purified type 5 polysaccharide did not protect indicator strains from the action of vancomycin. Conclusions: The VISA strain tested in this study displayed an increased production of type 5 capsular polysaccharide. However, the production of capsule material did not protect strain SA137/93G and three vancomycin sensitive strains in the presence of vancomycin and thus is not part of the resistance mechanism; however it may represent a by-product of VISA life style that is often characterized by a high sigma factor B activity

Marine Myxobacteria as a Source of Antibiotics—Comparison of Physiology, Polyketide-Type Genes and Antibiotic Production of Three New Isolates of Enhygromyxa salina

Three myxobacterial strains, designated SWB004, SWB005 and SWB006, were obtained from beach sand samples from the Pacific Ocean and the North Sea. The strains were cultivated in salt water containing media and subjected to studies to determine their taxonomic status, the presence of genes for the biosynthesis of polyketides and antibiotic production. 16S rDNA sequence analysis revealed the type strain Enhygromyxa salina SHK-1T as their closest homolog, displaying between 98% (SWB005) and 99% (SWB004 and SWB006) sequence similarity. All isolates were rod-shaped cells showing gliding motility and fruiting body formation as is known for myxobacteria. They required NaCl for growth, with an optimum concentration of around 2% [w/v]. The G + C-content of genomic DNA ranged from 63.0 to 67.3 mol%. Further, the strains were analyzed for their potential to produce polyketide-type structures. PCR amplified ketosynthase-like gene fragments from all three isolates enhances the assumption that these bacteria produce polyketides. SWB005 was shown to produce metabolites with prominent antibacterial activity, including activity towards methicillin resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE)

Expression of the Lantibiotic Mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent

Purification and Activity Testing of the Full-Length YycFGHI Proteins of Staphylococcus aureus

Background: The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. Methodology/Principal Findings: Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposomemodel indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. Conclusions/Significance: The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies an

Gram-positive bacteria with orthologous <i>yyc</i> (<i>wal</i>/<i>vic</i>) genes.

<p>The upper part of the figure shows the structural organization of orthologous <i>yyc</i> operons in Gram-positive bacteria with low G+C-content adopting the classification into class I and II proposed by Szurmant et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030403#pone.0030403-Szurmant1" target="_blank">[5]</a>. To distinguish between genera that contain 5 or 6 cistrons, the class I operon was divided into two subclasses. For a clear overview, orthologous genes are named according to the corresponding <i>yyc</i> gene in <i>Bacillus subtilis</i> in all genera and synonymous terms commonly used in a genus are given in brackets. The lower part of the figure illustrates the organization of the <i>mtrAB</i>/<i>lpqB</i> operon in Gram-positive Actinobacteria with high G+C-content. The operons are drawn to scale from representative species: <i>Bacillus subtilis</i> 168 (NC_000962), <i>Staphylococcus aureus</i> N315 (NC_002745), <i>Streptococcus pneumoniae</i> R6 (NC_003098), <i>Lactococcus lactis</i> Il1403 (NC_002662), <i>Corynebacterium glutamicum</i> ATCC 13032 (NC_006958). Essential genes are highlighted. TM coding regions were determined utilizing the TMHMM 2.0 server 2.0 web interface (<a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM/</a>) and marked as black bars. Functions are indicated below the arrows: RR = response regulator, SK = sensor kinase, NR = negative regulator of kinase function, ‘β-lac’ = similarities to an enzyme super-family containing metallo-β-lactamases, protease = serine protease, LP = conserved lipoprotein. Within the class II of <i>yyc</i> operons, the YycG kinase of <i>Lactococcus lactis</i> is an exception of the rule, because it possesses two transmembrane domains (instead of one), which flank a very short extracytoplasmic loop comprising four amino acids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030403#pone.0030403-Szurmant3" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030403#pone.0030403-Winkler1" target="_blank">[8]</a>.</p