Dataset Information

Comparative transcriptome analysis of two S. aureus strains with different susceptibilities to mersacidin.

ABSTRACT: The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids that is produced by Bacillus sp. strain HIL Y-85,54728. Lantibiotics are antibiotics containing nonproteinogenic amino acids like lanthionine and/or 3-methyllanthionine. Mersacidin interferes with cell wall biosynthesis by targeting the peptidoglycan precursor lipid II and inhibits the growth of MRSA and other gram-positive bacteria. Therefore, mersacidin could be a lead substance for the development of new antibacterial agents. The clinical VISA isolates S. aureus 137/93A and its spontaneous mutant S. aureus 137/93G show reduced sensitivity towards mersacidin. This phenotype could not be traced to factors being responsible for vancomycinresistance (i.e. the thickened cell wall). Here, we focussed on the comparative transcriptome analysis of S. aureus 137/93A and S. aureus SG511 (sensitive strain) via full genome S. aureus microarrays to identify genes involved in the reduced sensitivity towards mersacidin. Overall design: In this experiment, the transcriptomes of S. aureus 137/93A and S. aureus SG511 (sensitive strain) should be compared via microarray analysis. To this end, S. aureus 137/93A and S. aureus SG511 were grown without mersacidin to determine the genes which are differently expressed in these strains. Each experiment was performed 2 times including a dye swap resulting in two chips per competitive comparison with the incorporated dye reversed. All hybridizations were done with equal amounts of fluorescence-labeled cDNA probes or picomols of dye.

INSTRUMENT(S): sciTracer Staphylococcus aureus N315 Scienion2005

SUBMITTER: Peter Sass

PROVIDER: GSE12016 | GEO | 2009-01-17

SECONDARY ACCESSION(S): PRJNA113289

REPOSITORIES: GEO

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Publications

Native graS mutation supports the susceptibility of Staphylococcus aureus strain SG511 to antimicrobial peptides.

Sass Peter P Bierbaum Gabriele G

International journal of medical microbiology : IJMM 20090109 5

Staphylococcus aureus strain SG511-Berlin has been used as a standard strain for antimicrobial susceptibility testing and basic research since the first half of the last century. This study aimed at the identification of features of S. aureus SG511-Berlin that contribute to its high susceptibility to antimicrobial peptides (AMPs) in order to give insights into the mechanisms that S. aureus uses to counteract antimicrobials and to evaluate the role of S. aureus SG511-Berlin as a standard test str ...[more]

PMID: 19138560

Similar Datasets

Project description:The lantibiotic 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. Here, we studied the transcriptomic response of S. aureus to subinhibitory concentrations of mersacidin using microarray and qRT-PCR techniques. The transcriptomics revealed an extensive induction of the cell wall stress response of S. aureus, which is partly controlled by the two-component regulatory system VraSR. In contrast to other cell wall-active antibiotics such as vancomycin, 0.2 x MIC of mersacidin was sufficient for induction. Interestingly, the cell wall stress response was equally induced in vancomycin intermediate resistant S. aureus (VISA) as well as in a highly susceptible strain. Furthermore, the efficiency of mersacidin was not affected by an increased cell wall thickness, which is part of the VISA-type resistance mechanism. 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 of S. aureus to mersacidin. Unexpectedly, the deletion of the vraE gene did not result in an increased susceptibility to mersacidin compared to the wild type strain. 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. Keywords: antibiotic induced response Overall design: In this experiment, the transcriptomic response of S. aureus was assayed via microarray analysis. To this end, S. aureus strains SA137/93A [mersacidin MIC 35 mg/L] and SA137/93G [mersacidin MIC 30 mg/L] were grown without or with mersacidin [16 mg/L] to determine the genes being induced or repressed by the lantibiotic. SA137/93G was additionally grown in the presence of 4 mg/L mersacidin. Furthermore, the highly susceptible S. aureus strain SG511 [mersacidin MIC 1 mg/L] was also grown without or with mersacidin [1 mg/L]. Each experiment was performed 4 times including a dye swap resulting in four chips per competitive comparison to increase reproducibility. Only strain SA137/93G incubated with 0.2 x MIC of mersacidin was reproduced in duplicate. All hybridizations were done with equal amounts of cDNA probes displaying similar picomoles of incorporated dye.

Project description:BACKGROUND: Lantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. 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) are organized in biosynthetic gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: Sequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides. CONCLUSIONS/SIGNIFICANCE: In conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains.

Project description:Staphylococcus aureus causes a wide variety of invasive human infections. However, delineation of the genes which are essential for the in vivo survival of this pathogen has not been accomplished to date. Using signature tag mutagenesis techniques and large mutant pool screens, previous investigators identified several major gene classes as candidate essential gene loci for in vivo survival; these include genes for amino acid transporters, oligopeptide transporters, and lantibiotic synthesis (W. R. Schwan, S. N. Coulter, E. Y. W. Ng, M. H. Langhorne, H. D. Ritchie, L. L. Brody, S. Westbrock-Wadman, A. S. Bayer, K. R. Folger, and C. K. Stover, Infect. Immun. 66:567-572, 1998). In this study, we directly compared the virulence of four such isogenic signature tag mutants with that of the parental strain (RN6390) by using a prototypical model of invasive S. aureus infection, experimental endocarditis (IE). The oligonucleotide signature tag (OST) mutant with insertional inactivation of the gene (putP) which encodes the high-affinity transporter for proline uptake exhibited significantly reduced virulence in the IE model across three challenge inocula (10(4) to 10(6) CFU) in terms of achievable intravegetation densities (P, <0.05). The negative impact of putP inactivation on in vivo survival in the IE model was confirmed by simultaneous challenge with the original putP mutant and the parental strain as well as by challenge with a putP mutant in which this genetic inactivation was transduced into a distinct parental strain (S6C). In contrast, inactivation of loci encoding an oligopeptide transporter, a purine repressor, and lantibiotic biosynthesis had no substantial impact on the capacity of OST mutants to survive within IE vegetations. Thus, genes encoding the uptake of essential amino acids may well represent novel targets for new drug development. These data also confirm the utility of the OST technique as an important screening methodology for identifying candidate genes as requisite loci for the in vivo survival of S. aureus.