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.
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
Project description:Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant S. aureus (MRSA) by Î²-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and Î²-lactams is not fully understood. In the present study we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs peptidoglycan biosynthesis at a stage that precedes transpeptidation. In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced Î²-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal peptidoglycan precursors with pentaglycine branches, the correct substrate of S. aureus penicillin-binding proteins. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards Î²-lactam antibiotics. Staphylococcus aureus USA300 was grown to early exponential phase and treated with TDZ (16 Âµg/ml) alone or in combination with DCX (0.125 Âµg/ml) for 30 min. Changes in global gene expression were analyzed using the untreated culture as control. Hybridizations were performed in triplicate using RNA isolated from independent cultures.
Project description:The phenotype “intermediate vancomycin resistance” in Staphylococcus aureus (CLSI: MIC = 4-8 mg/L) has been assigned to changes that lead to alterations in cell wall synthesis and morphology. Most vancomycin intermediately resistant S. aureus (VISA) strains are characterised by an increased cell wall thickness as a consequence of an activated cell wall biosynthesis and decreased autolysis. The purpose of this study was to analyse the genetic basis of the vancomycin resistance mechanism of the clinical VISA isolate SA137/93A and its spontaneous mutant strain SA137/93G. The methicillin-resistant S. aureus (MRSA) SA137/93A was isolated from a tracheal secretion and displays heterogeneous intermediate vancomycin resistance (hVISA strain, MIC: 8 mg/L in BHI). Subculturing in presence of 6 mg/L vancomycin generated a mutant with homogeneous intermediate vancomycin resistance, that showed an MIC value of 16 mg/L in BHI and was designated SA137/93G. PFGE profiles and phage typing of the strains showed that they were members of the Iberian clone (ST247), which was prevalent in Germany in the early nineties under the designation “Northern German epidemic strain”. Both strains possess a thickened cell wall. However, the vancomycin resistance of strain SA137/93A is most probably enhanced by an increased amount of free D-Ala-D-Ala termini in the cell wall, which is due to decreased crosslinking, whereas the mutant strain SA137/93G shows normal crosslinking. Moreover, strain SA137/93A displays an increased expression of the essential two-component system yycFGHI as a consequence of an IS256 insertion in the promoter region, while strain SA137/93G is characterised by an insertion of IS256 into the gene tcaA. Although both insertions were shown to correlate with a decrease in susceptibility to vancomycin, the difference in the vancomycin resistance level of the strain pair could be mainly attributed to the disruption of tcaA in the mutant.This study was conducted to identify resistance mechanisms that both strains might have in common. To this end we compared the transcriptomes of both strains with that of the closely related vancomycin susceptible MRSA/VSSA strain SA1450/94 (MIC: 2 mg/L). We found that the genes of the capsule biosynthesis were the only genes with higher expression in both VISA strains. Keywords: strain comparison
Project description:Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant S. aureus (MRSA) by β-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and β-lactams is not fully understood. In the present study we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs peptidoglycan biosynthesis at a stage that precedes transpeptidation. In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced β-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal peptidoglycan precursors with pentaglycine branches, the correct substrate of S. aureus penicillin-binding proteins. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards β-lactam antibiotics.
Project description:Ethanol is utilized widely in medicine for both its antiseptic and disinfectant capabilities. In an effort to understand the effects of ethanol on a very common human pathogen, we have determined the ethanol-stimulon of two well characterized unrelated clinical S. aureus methicillin resistant strains LP9 and MM66 with ethanol minimum inhibitory concentrations (MIC) of 10 and 7% respectively. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of LP9 and MM66 following 15 min exposure to 10% ethanol. Induction with 10 % ethanol led to the altered regulation of 797 genes in LP9 and 899 genes in MM66. There was an overlap of 600 commonly altered genes in both strains and of these, SAMMD identified 50 genes that are uniquely altered only in the ethanol stimulon compared to the other 93 transcriptomes used for comparison. SAMMD analysis further revealed that 259 of the 600 (43%) commonly Eth-altered genes, overlapped with the stringent response induced with the antibiotic mupirocin. Interestingly the gene SACOL2518 up-regulated 20.4 and 23 fold in LP9 and MM66 respectively has a RelA- and SpoT- like domain. We have also found the MICs and MBCs for both LP9 and MM66 to be affected by the addition of certain amino acids. Genes involved with the heat shock response were also among some of the highest similarly up-regulated genes. Overall, SAMMD found 30% (43 genes) in common between the heat shock and ethanol transcriptomes of LP9 and MM66. clpB encoding the clpB chaperone was the highest induced gene in both strains. groES, groEL, hrcA, grpE and dnaK were also some of the highest up-regulated genes in both strains.
Project description:Staphylococcus aureus is a worldwide pathogen that colonizes the human nasal cavity and is a major cause of respiratory and cutaneous infections. In the nasal cavity, S. aureus thrives with high concentrations of nitric oxide (NO) produced by the innate immune effectors and has available for growth slow metabolizing free hexoses, such as galactose. Here, we have used deep sequencing transcriptomic analysis (RNA-Seq) and 1H-NMR to uncover how S. aureus grown on galactose, a major carbon source present in the nasopharynx, survives the deleterious action of nitric oxide. We observed that, like on glucose, S. aureus withstands high concentrations of NO when using galactose. However, most likely this is achieved through a distinct metabolism that relies on the increased production of amino acids, such as glutamate, threonine and branched-chain amino acids. Moreover, we found that under these conditions the α-acetolactate synthase (ALS) enzyme, which converts pyruvate into α-acetolactate, plays a role in the resistance of S. aureus to NO. However, the role of ALS is not restricted to galactose but also extends to cells growing on glucose. The results suggest that ALS prevents intracellular acidification, promoting the production of branched-chain amino acids and activation of the TCA cycle. We show that ALS contributes to the successful infection of murine macrophages. Furthermore, ALS is also shown to contribute to the resistance of S. aureus to beta-lactam antibiotics such as methicillin and oxacillin.
Project description:Different transcriptome, metabolome, and phosphoproteome studies have already indicated a regulatory role of Ser/Thr phosphorylation in the central metabolism of S. aureus. However, it remains still unknown how this exactly tunes its metabolism. For that reason, Extreme Pathway Analysis was used to understand metabolic flux regulation by Ser/Thr phosphorylation. YANAsquare software and YANAvergence routine were chosen to calculate pathway activities of WT, ΔpknB, Δstp, and double mutant S. aureus NewmanHG strains. Gene expression data of S. aureus NewmanHG were collected taking the wild type and mutations of kinase (pknB), phosphatase (stp) or both (pknB/stp) phenotypes. The resulting flux changes were modelled with YANAvergence taking the different gene expression data and an established genome scale model of S. aureus metabolic modes as basis. The fluxes of the different strains were next calculated, taking the gene expression data into account. A concerted metabolic change of fluxes was observed. In particular the pathways for peptidoglycan, nucleotide, and aromatic amino acid synthesis as well as catabolism involving aspartate transaminase (GOT) changed significantly compared to the wild type. Single mutations of pknB and stp were compared to illustrate their different contribution to the phenotype, e.g. pyrimidine synthesis is dramatically impaired by pknB deletion but much less by loss of stp. In the double knock out strain, there is higher activity of peptidoglycan, purine, and aromatic amino acids synthesis from glucose, but lower activity of pyrimidine synthesis from glucose compared to WT. The results suggest a probable regulatory role of Ser/Thr phosphorylation in amino acid catabolism and the glycolysis/gluconeogenesis switch to provide the cell with sufficient cell wall components, nucleotides, and aromatic amino acids. Yvck is suggested to serve as regulatory protein linking Ser/Thr phosphorylation to the previous results of S. aureus metabolism. Moreover, Yvck seems in particular to be involved in the switch between glycolysis and gluconeogenesis. However, further studies based on experimental approaches are needed to demonstrate that Ser/Thr phosphorylation and Yvck are necessary for a correct cell growth under gluconeogenic conditions. The metabolic model allows not only a comprehensive representation of these changes for all involved pathways in the four strains, it further points out the function of the previously unannotated ycvk operon.. The relevance of these specific metabolic pathway adaptations regarding the pathogenicity of S.aureus and their contributions to the infection process will be analyzed and discussed.
Project description:Little is known about regulation of gene activity of the major pathogen Staphylococcus aureus during actual human infection. Here we characterize the transcriptome using deep RNA sequencing and the metabolome using NMR of S. aureus infected joint fluid derived from an acute human prosthetic joint infection, and compare them with the genome, transcriptome and metabolome of an isolate obtained from the sample grown in vitro (LB medium). The transcriptome indicated that the bacterial infection sustained on a versatile human-cell-based diet consisting of amino acids, glycans and nucleosides, since significant upregulation of genes involved in the catabolic degradation pathways of these compounds were observed in situ. This is consistent with metabolite analysis of the infected joint fluid and of S. aureus culture supernatants where the concentration of most amino acids and some amino sugars were found to be higher in the joint fluid, whereas the concentration of glucose was higher in culture supernatant. Furthermore, presumably because of oxygen limitations in the joint fluid, transcriptomic evidence for fermentation was observed, consistent with the presence of fermentation products (ethanol) in situ. Moreover, many, but not all, of the known virulence factor genes were upregulated in situ as well as the nine genes encoding the iron uptaking siderophore synthesis system.
Project description:The common research strain S. aureus NCTC 8325-4 was subjected to serial passage in increasing concentrations of ramoplanin. The resulting strain, RSPA16, had reduced susceptibility to not only ramoplanin but vancomycin and nisin as well. Electron microscopy revealed that the cell wall of RSPA16 was double the thickness of the susceptible progenitor strain, a phenotype commonly observed in vancomycin intermediate resistant S. aureus (VISA) strains. RSPA16 was also less susceptible to lysis induced by Triton X-100 than its progenitor strain. Transcriptional profiling experiments were performed with NCTC 8325-4 and RSPA16 without antibiotic exposure and exposed to ramoplanin. Increased expression of genes associated with cell wall stress was observed when either strain was treated with ramoplanin. We also observed that treatment with ramoplanin altered of the expression levels of numerous genes encoding proteins involved with amino acid biosynthesis, central metabolic pathways, nucleotide biosynthesis, iron acquisition, ABC transporters and regulation of transcription. Comparison of the transcriptional profiles of RSPA16 and NCTC 8325-4 not exposed to ramoplanin revealed alterations in the expression of levels of several genes involved with biosynthesis of teichoic acids, biosynthesis of peptidoglycan, central metabolism, DNA replication, nucleotide biosynthesis, iron acquisition, ABC transporters, and regulation of transcription. These transcriptional profiles provide insights into the possible sources of the reduced susceptibility of RSPA16 to peptide antibiotics. Mid log phase (OD620 = 0.4) batch TSB cultures of ramoplanin susceptible (NCTC 8325-4) and resistant (RSPA16) strains of S. aureus were exposed to ramoplanin for 30 minutes at 37 C. Untreated controls were performed for each strain as well. The transcriptional response was determined using Affymetrix Gene Chip Arrays. This study also examines the transcriptional alterations which confer reduced susceptibility to ramoplanin as the transcriptional profiles of NCTC 8325-4 and RSPA16 each untreated were compared.
Project description:Treatment of stationary growth phase Staphylococcus aureus SA113 with 100-fold of the MIC of the lipopeptide antibiotic daptomycin leaves alive a small fraction of drug tolerant albeit genetically susceptible bacteria. This study shows that cells of this subpopulation exhibit active metabolism even hours after the onset of the drug challenge. Isotopologue profiling using fully 13C-labeled glucose revealed de novo biosynthesis of the amino acids Ala, Asp, Glu, Ser, Gly and His. The isotopologue composition in Asp and Glu suggested an increased activity of the TCA cycle under daptomycin treatment compared to unaffected stationary growth phase cells. Microarray analysis showed differential expression of specific genes 10 minutes and 3 hours after addition of the drug. Besides factors involved in drug response, a number of metabolic genes appear to shape the signature of daptomycin-tolerant S. aureus cells. These observations will be useful towards the development of new strategies against persisters and related forms of bacterial cells with downshifted physiology.