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Comparison of Clinical Staphylococcus aureus Strains Demonstrating Differing Susceptibilities to the Biocide Ethanol

ABSTRACT: 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.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE17391 | GEO | 2009/08/17

SECONDARY ACCESSION(S): PRJNA118829

REPOSITORIES: GEO

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Project description:Background: Growth in the global population and industrial activities has increased world energy consumption. Bioethanol is considered as an alternative renewable energy source. Among various ethanol-producing microbes, Zymomonas mobilis ZM4 has received special attention due to its higher ethanol yield and tolerance. Advances in genetic engineering are particularly important for developing microorganisms with improved ethanol production. However, the variety of factors involved in the response to high concentrations of ethanol makes it difficult to devise genetic engineering strategies to generate alcohol tolerant strains. For a better understanding of the ethanol tolerance phenomenon, we obtained and characterized two Z. mobilis ZM4 mutants (ER79ap and ER79ag) with increased ethanol tolerance. Results: Mutants were obtained using a strain adaptive evolution method in mini-fermentors and sequential transfers to higher ethanol concentrations. Mutations were identified by Illumina genomic sequencing. Strain ER79ap possesses three point mutations in the following genes: SpoT/RelA, which synthesizes and degrades the alarmone ppGpp; clpB, encoding a disgregase; and clpP, a component of the Clp protease. In contrast, strain ER79ag has four mutations in the subsequent genes: spoT/relA; rimO; clpP; and in a gene encoding a hypothetical protein with a CBS domain. Transcript profiles of ZM4 and ER79ap were obtained with microarray analysis and identified 126 genes in ZM4 and 148 genes in ER79ap that were differentially expressed in response to ethanol. Conclusions: Both mutants carry mutations in clpP and relA/spoT genes, suggesting that these genes are responsible of their enhanced ethanol tolerance. Transcript profile analysis of the ZM4 and ER79ap showed that they share a set of forty genes that are differentially expressed under ethanol stress and this set may correspond to those that are crucial for the ethanol response. The expression profiles indicate that ethanol induces a major reprograming of transcription that involves changes in the cell membrane, protein synthesis, and in some metabolic pathways, especially those involved in the amino acid metabolism. Our data suggest that clpP and in particular the relA/spoT gene can be targets for bioengineering ethanol tolerance.

Project description:The collaborative cross (CC) founder strains include five classical inbred laboratory strains (129S1/SvlmJ, A/J, C57BL/6J (B6), NOD/ShiLtJ, and NZO/HILtJ) and three wild-derived strains (CAST/EiJ, PWK/PhJ (PWK) and WSB/EiJ) The strains encompass 89% of the genetic diversity available in Mus musculus and about 10 to 20 times more genetic diversity than found in Homo sapiens. For more than 60 years the B6 strain has been used as the model strain for high ethanol preference/high consumption. However, another one of the CC founder strains, PWK, was identified as a high ethanol preference/high consumption strain. The current study determined how the transcriptomes of the B6 and PWK strains differed from the 6 low preference CC strains across 3 nodes of the brain addiction circuit. RNA-Seq data were collected from the central nucleus of the amygdala, the nucleus accumbens core and the prelimbic cortex. Differential expression (DE) analysis was performed in each of these brain regions for all 28 possible pairwise comparisons of the CC founder strains. Unique genes for each strain were identified by selecting for genes that differed significantly (false discovery rate (FDR) < 0.05) from all other strains in the same direction. B6 was identified as the most distinct inbred laboratory strain, having the highest number of total differently expressed genes (DEGs) and DEGs with high log fold change, and unique genes compared to other CC strains. Less than 50 unique DEGs were identified in common between B6 and PWK within all three brain regions, indicating the strains potentially represent two distinct genetic signatures for risk for high ethanol-preference. 238 DEGs were found to be commonly different between B6, PWK and the average expression of the remaining CC strains within all three regions. The commonly different up-expressed genes were significantly enriched (FDR < 0.001) among genes associated with neuroimmune function. These data compliment the observation that neuroimmune signaling is key to understanding alcohol use disorder and and support use of these 8 strains and the highly heterogeneous mouse populations derived from them to identify alcohol-related brain mechanisms and treatment targets.

Project description:Polymorphonuclear leukocytes (PMN) from patients with chronic granulomatous disease (CGD) fail to produce microbicidal concentrations of reactive oxygen species due to mutations in NOX2. Patients with CGD suffer from severe, life-threatening infections and inflammatory complications. Granulibacter bethesdensis is an emerging Gram-negative pathogen in CGD that resists killing by CGD PMN and inhibits PMN apoptosis through unknown mechanisms. Microarray analysis was used to study mRNA expression in normal and CGD PMN during incubation with G. bethesdensis and, simultaneously, in G. bethesdensis with normal and CGD PMN. We detected upregulation of anti-apoptotic genes (e.g., XIAP, GADD45B) and downregulation of pro-apoptotic genes (e.g., CASP8, APAF1) in infected PMN. Transcript and protein levels of inflammation and immunity-related genes were also altered. Upon interaction with PMN, G. bethesdensis altered expression of ROS-resistance genes in the presence of normal but not CGD PMN. Bacterial stress response genes, including ClpB, increased during phagocytosis by both normal and CGD PMN demonstrating responses to oxygen-independent PMN antimicrobial systems. Antisense knock down demonstrated that ClpB is dispensable for extracellular growth but is essential for bacterial resistance to both normal and CGD PMN. Metabolic adaptation of Granulibacter growth in PMN included upregulation of pyruvate dehydrogenase. Pharmacologic inhibition of pyruvate dehydrogenase by triphenylbismuthdichloride was lethal to Granulibacter. This study expands knowledge of microbial pathogenesis by Granulibacter in cells from permissive (CGD) and non-permissive (normal) hosts and identifies potentially druggable microbial factors, such as pyruvate dehydrogenase and ClpB, to help combat this antibiotic-resistant pathogen. pathogen time series 0-1-4-24 in CGD subjects and normals : host time series 0-1-4-24 in CGD subjects and normals non-infected and infected with Gb

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Comparison of Clinical Staphylococcus aureus Strains Demonstrating Differing Susceptibilities to the Biocide Ethanol

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