Prediction driven functional annotation of hypothetical proteins in the major facilitator superfamily of S. aureus NCTC 8325.
ABSTRACT: Antibiotic resistance Staphylococcus aureus strains cause several life threatening infections. New drug treatment options are needed, but are slow to develop because 50% of the S. aureus genome is hypothetical. The goal of this is to aid in the annotation of the S. aureus NCTC 8325 genome by identifying hypothetical proteins related to the Major Facilitator Superfamily (MFS). The MFS is a broad protein group with members involved in drug efflux mechanisms causing resistance. To do this, sequences for three MFS proteins with x-ray crystal structures in E. coli were PSI-BLASTed against the S. aureus NCTC 8325 genome to identify homologs. Eleven identified hypothetical protein homologs underwent BLASTP against the non-redundant NCBI database to fit homologs specific to each hypothetical protein. ExPASy characterized the physiochemical features, CDD-BLAST and Pfam identified domains, and the SOSUI server defined transmembrane helices of each hypothetical protein. Based on size (300 - 700 amino acids), number of transmembrane helices (>7), CD06174 and MFS domains in CDD-BLAST and Pfam, respectively, and close relation to well-defined homologs, SAOUHSC_00058, SAOUHSC_00078, SAOUHSC_00952, SAOUHSC_02435, SAOUHSC_02752, and ABD31642.1 are members of the MFS. Further multiple-alignment and phylogeny analyses show SAOUHSC_00058 to be a quinolone resistance protein (NorB), SAOUHSC_00058 a siderophore biosynthesis protein (SbnD), SAOUHSC_00952 a glycolipid permease (LtaA), SAOUHSC_02435 a macrolide MFS transporter, SAOUHSC_02752 a chloramphenicol resistance (DHA1), and ABD31642.1 is a Bcr/CflA family drug resistance efflux transporter. These findings provide better annotation for the existing genome, and identify proteins related to antibiotic resistance in S. aureus NCTC 8325.
Project description:Staphylococcus aureus is one of the most common hospital acquired infections. It colonizes immunocompromised patients and with the number of antibiotic resistant strains increasing, medicine needs new treatment options. Understanding more about the proteins this organism uses would further this goal. Hypothetical proteins are sequences thought to encode a functional protein but for which little to no evidence of that function exists. About half of the genomic proteins in reference strain S. aureus NCTC 8325 are hypothetical. Since annotation of these proteins can lead to new therapeutic targets, a high demand to characterize hypothetical proteins is present. This work examines 35 hypothetical proteins from the chromosome of S. aureus NCTC 8325. Examination includes physiochemical characterization; sequence homology; structural homology; domain recognition; structure modeling; active site depiction; predicted protein-protein interactions; protein-chemical interactions; protein localization; protein stability; and protein solubility. The examination revealed some hypothetical proteins related to virulent domains and protein-protein interactions including superoxide dismutase, O-antigen, bacterial ferric iron reductase and siderophore synthesis. Yet other hypothetical proteins appear to be metabolic or transport proteins including ABC transporters, major facilitator superfamily, S-adenosylmethionine decarboxylase, and GTPases. Progress evaluating some hypothetical proteins, particularly the smaller ones, was incomplete due to limited homology and structural information in public repositories. These data characterizing hypothetical proteins will contribute to the scientific understanding of S. aureus by identifying potential drug targets and aiding in future drug discovery.
Project description:Antibiotic-resistant bacteria are a significant public health concern and motivate efforts to develop new classes of antibiotics. One such class of antibiotics is the arylomycins, which target type I signal peptidase (SPase), the enzyme responsible for the release of secreted proteins from their N-terminal leader sequences. Despite the essentiality, conservation, and relative accessibility of SPase, the activity of the arylomycins is limited against some bacteria, including the important human pathogen Staphylococcus aureus. To understand the origins of the limited activity against S. aureus, we characterized the susceptibility of a panel of strains to two arylomycin derivatives, arylomycin A-C16 and its more potent analog arylomycin M131. We observed a wide range of susceptibilities to the two arylomycins and found that resistant strains were sensitized by cotreatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. To further understand how S. aureus responds to the arylomycins, we profiled the transcriptional response of S. aureus NCTC 8325 to growth-inhibitory concentrations of arylomycin M131 and found that it upregulates the cell wall stress stimulon (CWSS) and an operon consisting of a putative transcriptional regulator and three hypothetical proteins. Interestingly, we found that mutations in the putative transcriptional regulator are correlated with resistance, and selection for resistance ex vivo demonstrated that mutations in this gene are sufficient for resistance. The results begin to elucidate how S. aureus copes with secretion stress and how it evolves resistance to the inhibition of SPase.
Project description:In most Staphylococcus aureus strains, inactivation of sarA increases hla transcription, indicating that sarA is a repressor. However, in S. aureus NCTC 8325 and its derivatives, used for most studies of hla regulation, inactivation of sarA resulted in decreased hla transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its rsbU mutation, which leads to sigma(B) deficiency. This has now been verified by the demonstration that sarA repressed hla transcription in an rsbU+ derivative of strain 8325-4 (SH1000). That sarA could act as a repressor of hla in an 8325-4 background was confirmed by the observation that inactivation of sarA in an agr sarS rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sarA as an activator of hla in 8325-4 was not a result of the rsbU mutation alone, as inactivation of sarA in another rsbU mutant, strain V8, led to increased hla transcription. Northern blot analysis revealed much higher levels of sarS mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the sarS activator, tcaR, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in sarS transcription upon the inactivation of sarA was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of sarA in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of sarA means a net decrease in repressor activity and, therefore, enhanced hla transcription.
Project description:Staphylococcus aureus is a notorious bacterial pathogen that causes a broad range of human diseases, and isolates that are resistant to several antibiotic classes including last resort antibiotics like vancomycin and daptomycin complicate the situation. We characterized S. aureus VC40, a strain that shows full resistance to vancomycin (MIC of 64 µg/ml) and daptomycin (MIC of 4 µg/ml) as well as a decreased susceptibility to further cell wall active agents. Genome sequencing revealed mutations in genes encoding the histidine kinases WalK and VraS that control cell envelope related processes and gene expression profiling indicated the induction of the respective regulons in strain VC40. Reconstitution of the mutations in walK or vraS into the susceptible S. aureus NCTC 8325 background resulted in a considerably increased resistance to vancomycin and daptomycin with MICs surpassing the clinical breakpoints for these antibiotics, thereby generating vancomycin-intermediate S. aureus (VISA) strains. As observed for S. aureus VC40, the walKwalk and vraS mutations also led to an increased expression of the respective regulons in the NCTC 8325 background. Phenotypic studies showed that S. aureus VC40 as well as the walKwalk and vraS mutants of strain NCTC 8325 were characterized by a significantly thickened cell wall, a decreased growth rate, a reduced autolytic activity and an increased resistance to lysostaphin-induced lysis. These results demonstrate that the WalK and VraS histidine kinases act as major switches which allow S. aureus to rapidly develop vancomycin resistance up to the VISA level via mutation of one single gene locus and concomitantly contribute to cross-resistance to other antibiotics including the last resort antibiotic daptomycin. Microarray was used to evaluate alteration in the transcriptome of mutS mutant and compared to the parental strain VC40
Project description:Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected ?11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (?63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin ?3 (PSM?3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.
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. Overall design: 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: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:Leishmaniasis is a neglected tropical disease caused by the pathogenic protozoan Leishmania donovani and it is transmitted by an infected sand fly. Approximately 0.4 million cases of Visceral Leishmaniasis are reported across the globe every year, of which 67% is from the Indian subcontinent. The currently available drugs have not been effective owing to their high toxicity levels, inadequate specificity, drug resistance, extended treatment periods and/or prohibitive prices. For this reason, hypothetical proteins in this pathogen, which constitute about 67% of its proteome, must be distinctly characterized and studied for their potential role as drug targets for Leishmaniasis. Domain information from PFAM and functional information from GO has been used to assign putative functions to 36 hypothetical membrane proteins in this protozoan. Furthermore, as a case study, we have performed a thorough sequence level characterization of a hypothetical protein E9BPD7 from the BT1 family of membrane proteins that transports folate/biopterin. Phylogenetic analyses of E9BPD7 have revealed interesting evolutionary correlations to BT1 family and MFS superfamily, which have significant roles in a number of diseases and drug resistance pathways.
Project description:An atypical two-cysteine peroxidase, SAOUHSC_01822, from the virulent Staphylococcus aureus strain NCTC 8325 plays a major role in the response of the bacterium to oxidative stress. The protein was cloned, expressed, purified to homogeneity and crystallized. The protein was crystallized from 2 M ammonium sulfate, 0.1 M Na HEPES pH 7, 2%(v/v) PEG 400. A complete diffraction data set was collected to 2.3 angstrom resolution using a Rigaku MicroMax HF007 Cu K alpha X-ray generator and a Rigaku R-AXIS IV(+)(+) detector. The crystals belonged to space group P2(1), with unit-cell parameters a = 43.50, b = 149.35, c = 73.73 angstrom, beta = 104.4 degrees, and contained four molecules in the asymmetric unit.
Project description:Tn551 insertional inactivation of femC is known to reduce methicillin resistance levels in methicillin-resistant and -susceptible Staphylococcus aureus. By use of cotransductional crosses, femC was mapped close to thrB on the SmaI-A fragment of the S. aureus NCTC 8325 chromosome. The Tn551 insertion femC::omega 2005 was found to interrupt an open reading frame coding for a putative protein of 121 amino acids which is highly similar to the glutamine synthetase repressors (GlnR) of Bacillus spp. Downstream of femC, an open reading frame highly similar to Bacillus sp. glutamine synthetases (GlnA) was found. Northern (RNA) blots probed with putative glnR or glnA fragments revealed that 1.7- and 1.9-kb transcripts characteristic of wild-type cells were replaced by less abundant 7.0- and 7.2-kb transcripts in the femC::omega 2005 mutant. Total glutamine synthetase activity was also decreased in the mutant strain; the addition of glutamine to defined media restored the wild-type methicillin resistance phenotype of the femC mutant. This result suggests that the omega 2005 insertion in glnR has a polar effect on glnA and that glnR and glnA are transcribed together as an operon. These results suggest that the loss of wild-type levels of glutamine synthetase and the consequent decrease in glutamine availability cause a decreased level of methicillin resistance.