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Host cell response after infection with a virulent and a non virulent Acinetobacter baumannii IC2 OXA-23 strain

ABSTRACT: Acinetobacter baumannii is an emerging nosocomial pathogen that causes severe infections such as pneumonia or blood stream infections. As the incidence of multidrug-resistant A. baumannii infections in intensive care units increases, the pathogen is considered of greater clinical concern. Little is known about the molecular interaction of A. baumannii with its host yet. In order to study the host cell response upon A. baumannii infection, a complexome analysis was performed. For this, we identified a virulent ( A. baumannii 2778) and a non virulent (A. baumannii 1372) clinical isolate of genetic similarity > 95 % (both isolates from IC 2 harboring OXA 23). HUVECs were infected with each strain and enriched mitochondrial fraction was used for complexome profiling. Complexome analysis identified dramatic reduction of mitochondrial protein complexes in the strain of greater virulence.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Acinetobacter Baumannii Acicu Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Ilka Wittig

LAB HEAD: Stephan Göttig

PROVIDER: PXD035235 | Pride | 2023-04-10

REPOSITORIES: Pride

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Project description:Apoptosis plays a pivotal role in embryogenesis and postnatal cell homeostasis, involving DNA or subcellular fragmentation, and shedding of small membranous microvesicles termed apoptotic bodies (AB). Following DNA damage, hypoxia, or vascular injury, the chemokine CXCL12 has been implicated in the recruitment of progenitor cells for tissue regeneration through its receptor CXCR4 and in mechanisms counteracting apoptosis. Whether AB deliver alarm signals for regenerative responses to neighbouring cells beyond recruitment or eat-me signals for phagocytes and relevance to diseases with abundant apoptosis, eg atherosclerosis, remains unknown. Here we show that endothelial cell-derived AB are generated during diet-induced atherosclerosis and can be transferred to recipient endothelial or smooth muscle cells to induce functional expression of CXCL12. This is mediated through miRNA-126 enriched in AB, which acts by silencing RGS16 translation and unlocking CXCR4 to unleash an auto-regulatory feedback loop inducing CXCL12. Injection of AB promoted mobilization and incorporation of progenitor cells, reducing diet-induced atherosclerosis in apolipoprotein E-deficient mice, and local transfer of microRNA-126 inhibited collar-induced arterial plaque formation. This was associated with increased smooth muscle content but decreased macrophage and apoptotic cell content, all features of plaque stability. Our data identify a new mechanism, by which AB confer microRNA-126 as a paracrine alarm messenger to enhance CXCR4 signals and CXCL12 expression, thereby limiting or repairing vascular damage. This adds to the important functions of microRNAs in health and disease and may extend to progenitor cell recruitment during other forms of tissue repair or homeostasis. AB were isolated from supernatants of apoptotic, serum-starved human umbilical vein endothelial cells (HUVECs) by sequential centrifugation steps. Total RNA was isolated from AB or HUVECs and microRNA was purified using the mirVanaTM miRNA Isolation Kit (Ambion). microRNA obtained from 10 µg of total RNA was labeled using the mirVanaTM miRNA Labeling Kit (Ambion) and fluorescent Cy3 (Molecular Probes), and hybridized to the Ambion mirVanaTM miRNA Bioarray (1566 v.1). Hybridized mirVana miRNA Bioarrays were scanned and quantified by using ImaGene 5.5.4 (Bio Discovery). Resulted signal intensities were background corrected and then normalized using variance stabilization normalization. (Huber, 2002).

Project description:Background: In diabetes chronic hyperinsulinemia is responsible for the instability of the atherosclerotic plaque and stimulates cellular proliferation through the activation of the MAP kinases, which, in turn regulate cellular proliferation. So far, it is not known, however, whether insulin itself could increase the transcription of specific genes for cellular proliferation in the endothelium. Hence, the characterization of the global pattern of transcriptional modifications in endothelium is an important step for a better understanding of the mechanism of action of insulin and of its defects in situations of insulin resistance. Methodology and principal findings: The transcriptional response of endothelial cells in the eight hours following insulin stimulation was monitored using microarrays and compared to a control condition. About 1700 genes were selected as differentially expressed based on their treated minus control profile, thus allowing the detection of even small but systematic changes in gene expression. Genes were clustered in 7 groups according to their time expression profile and classified into 15 functional categories that can support the biological effects of insulin, based on Gene Ontology enrichment analysis. In terms of endothelial function, the most prominent processes affected were NADH dehydrogenase activity, N-terminal myristoylation domain binding, nitric-oxide synthase regulator activity and growth factor binding. Pathway based enrichment analysis revealed âElectron Transport Chainâ and âMatrix Metalloproteinasesâ significantly enriched. Results were validated on genes belonging to âElectron Transport Chainâ pathway, using quantitative RT-PCR. Conclusions: As far as we know, this is the first systematic study in the literature monitoring transcriptional response to insulin in endothelial cells, in a time series microarray experiment. Since chronic hyperinsulinemia is responsible for the instability of the atherosclerotic plaque and stimulates cellular proliferation, some of the genes identified in the present work are potential novel candidates in diabetes complications related to endothelial dysfunction. Experiments were carried on human umbilical venous endothelial cells (HUVEC) to elucidate the regulatory aspects of insulin from dynamic gene expression data. HUVEC incubated with or without insulin were collected at times 0', 40', 100', 200', 340' and 440'. RNA was extracted and quantified for high-density oligonucleotide microarrays Human Genome U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA).

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Host cell response after infection with a virulent and a non virulent Acinetobacter baumannii IC2 OXA-23 strain

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