Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq based transcriptome analysis of a highly virulent and multidrug resistant MRSA isolate for the understanding of virulence and resistance mechanisms

ABSTRACT: Staphylococcus aureus can infect a wide range of animals and pose as a serious threat to public health by transferring via animals or animal-derived food stuff. Even more importantly, multiple drug resistance development in the bacteria has resulted in therapeutic failure of a number of antibiotics. Therefore by realizing the need of time, this study was designed to investigate the underlying mechanisms of virulence and resistance in S. aureus. After screening through in vivo and in vitro virulence assays and susceptibility test, a highly virulent and multidrug resistant MRSA strain was selected for differential analysis by RNA-seq technology and gene expression results were verified by RT-qPCR. Up-regulation of crucial regulators like sarA and KdpDE seemed to play role in decreased expression of many exotoxin genes while enhanced the adhesion and cell wall protein expression, leading to strong biofilm production in the presence of inactivated agr system. In addition to resistance genes like blaZ, ermC and femA, up-regulation of vraS and multidrug ABC transporter genes contributed to the multidrug resistance in MRSA. Fluoroquinolone resistance was attributed to mutational changes in gyrA and parC genes. Our findings suggested that many virulence and resistance determinants in S. aureus are controlled by complex network of various regulators, and sarA is the most important of those as it adds to pathogenicity of the bacteria and ensures its survival in diverse environment. Further investigations are required to unveil these mechanisms in S. aureus. Four samples were analysed including 2 MRSA1679a test strain and 2 reference strain ATCC1 samples with two replicates of each.

ORGANISM(S): Staphylococcus aureus

SUBMITTER: Hanqi Yin

PROVIDER: E-GEOD-78764 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Approximatively 75% of the genome of S. aureus (“core” genome) are highly conserved between strains, whereas the remaining 25% (“accessory” genome) are composed of variable regions that are mostly composed of mobile genetic elements (MGE), containing virulence and resistance genes. We have developed a composite DNA-microarray (StaphVar Array) which selectively targets 403 genes located on the accessory or core variable genome. Target genes encode antimicrobial resistance factors (35 %), virulence factors (28 %) and adhesins (31%). This microarray was validated with reference strains and used to characterize the genomic DNA of 13 community-acquired methicillin resistant S. aureus (CA-MRSA) strains representative of all the Multilocus Sequence Types (STs) described to date in Belgium. Analysis of gene content of 8 reference strains by the StaphVar Array matched 96 to 99% of the theoretical results. Analysis of CA-MRSA strains showed that 54.4 % of the genes tested were strain-dependent. Strains presented specific exotoxin, enterotoxin, cytolysin and adhesin gene profile by multi-locus sequence typing (MLST) lineage. One exception to these “lineage-specific” gene profile was the variable presence of the Arginin Catabolic Mobile Element (ACME, characteristic of the USA300 clone) within ST8 strains. In conclusion, this novel StaphVar array enables characterization of more than 400 variable resistance and virulence determinants in S. aureus strains. CA-MRSA strains from Belgium displayed extensive diversity in virulence and resistance profile. The presence of the USA 300 clone in our country was confirmed. Although mainly located on MGE, association of virulence genes were highly conserved within strains of the same MLST lineage. CGH microarray was performed on 13 epidemiologically distinct clinical isolates of methicillin resistant S. aureus. S. aureus labeled genomic DNA were hybridized to StaphVar arrays containing 1326 60mer oligonucleotide probes (Eurogentec, Belgium).

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of gene regulon. The goal of this study is to investigate the genes regulated by Rsp in MRSA BD02-25 Methods: mRNA profiles of wild-type (WT) and Rsp knockout (â³Rsp) MRSA at mid-logarithmic growth phase (4h) were generated by deep sequencing, respectively in duplicate samples, using the Hiseq2000 (Illumina, CA) sequencer. The sequence reads that passed quality filters were aligned to S. aureus COL (RefSeq accession number NC_002951.2) using the Burrows-Wheeler Alignment tool (BWA) followed by ANOVA (ANOVA). Only the consistent data between the two WT or mutant samples were reserved for further analysis. qRTâPCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, RNA-seq analyses revealed that 328 genes were up-regulated and 176 genes were down-regulated in â³rsp compared with wild type. In order to explain the major relevant changes in â³rsp compare to wild-type, the differential expressed genes obtained in RNA-seq with three biological replicates were used to construct a regulation network. 108 up-regulated genes and 33 down-regulated genes in RNA-seq data had gathered in the protein-protein interaction network. 15 modules of biological processes were responsible, including ribosome, metabolism, biofilm formation, cell wall degradation, cytolysis, two-component system and so on. Biological processes related to biofilm formation, such as cell wall degradation and metabolism were up regulated, indicating that the defense systems were activated. However, the genes that responsible for the two-component system and cytolysis were down regulated, suggesting a possible pathway how Rsp controls the virulence. Conclusions: Our data provide new information to the virulence regulatory network in S. aureus. mRNA profiles of wild-type (WT) and Rsp knockout (â³Rsp) MRSA were generated by deep sequencing, in duplicate, using the Hiseq2000 (Illumina, CA) sequencer.

Project description:The staphylococcal accessory regulator A (sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. To assess the relative impact of these two functions, we previously used a proteomics approach that measures protein abundance as a function of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assess the impact of this on the accumulation of S. aureus exoproteins1. While this approach confirmed that protease-mediated degradation has a significant impact on the S. aureus exoproteome, it was potentially limited in that it did not take into account the possibility that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, we present an expanded proteomics approach that utilizes a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels. Specifically, proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases (sarA/protease) were resolved using one-dimensional gel electrophoresis. Using methods that focus on total proteoforms vs. methods that focus specifically on full-length proteins, quantitative proteomic comparisons of sarA vs sarA/protease mutants identified proteins that were degraded in a protease dependent manner owing to mutation of sarA, while comparisons of a sarA/protease mutant vs the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This approach provided for a more comprehensive and robust analysis of the impact of mutating sarA and protease-mediated degradation on the S. aureus exoproteome.

Project description:Methicillin resistant Staphylococcus aureus (MRSA) infection is becoming refractory to existing antibiotic therapy owing to the inherent ability of S. aureus to develop rapid resistance and is considered a major threat to public health. We found that a natural isolate of Bacillus pumilus from the Columbia River Estuary produces a strong anti-MRSA compound, amicoumacin A. As amicoumacin A has been reported to exhibit anti-microbial, anti-inflammatory, and anti-ulcer activities, we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in the expression of genes involved in several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which has been shown to be induced in response to a collapse of membrane potential. In order to gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by serial passage method was carried out. Single point mutations resulting in codon substitutions were uncovered in several distinct genes: ksgA, RNA dimethyltranferase; fusA, elongation factor G; dnaG, primase, ; lacD, tagatose 1,6-bisphosphate aldolase, ; and SACOL0611, encoding a putative glycosyl transferase gene. Based on these results, a candidate approach was undertaken to recreate the same amino acid substitution individually in FusA and KsgA, each of which resulted in two-fold resistance towards amicoumacin A. The fusA gene is known as the site for fusidic acid- resistant mutations; however the codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance occur in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability. Amicoumacin A, isolated from Bacillus pumilus, was added to exponentially growing cultures (OD600 =0.5) of Staphylococcus aureus COL at concentrations leading to around 12% and 20% reduction of OD600 after 10 min and 40 min, respectively. Total RNA was isolated from three biological replicates. Labeled cDNA from treated and control cultures (Cy5) was hybridized against a common reference cDNA pool (Cy3). The reference pool was prepared from a mixture of equal amounts of total RNA isolated from all stress and control samples in the experiment.

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RNA-seq based transcriptome analysis of a highly virulent and multidrug resistant MRSA isolate for the understanding of virulence and resistance mechanisms

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