ABSTRACT: various strains of Pseudomonas plecoglossicida and Pseudomonas guariconensis, all containing the metallo-beta-lactamase gene blaVIM-5 Genome sequencing and assembly
Project description:Pseudomonas plecoglossicida is a facultative pathogen that is associated with diseases of multiple fish, mainly at 15-20 °C. Although fish disease caused by P. plecoglossicida has led to significant economic losses, the mechanisms of the temperature-dependent virulence are unclear. Here, we try to identify potential pathogenicity mechanisms and demonstrate the direct regulation of virulence factors by temperature with iTRAQ.
Project description:The <i>C. elegans</i> gene swip-10 encodes an orphan metallo beta-lactamase that genetic studies indicate is vital for limiting neuronal excitability and viability. Sequence analysis indicates that the mammalian gene Mblac1 is the likely ortholog of swip-10, with greatest sequence identity localized to the encoded protein's single metallo beta-lactamase domain. The substrate for the SWIP-10 protein remains unknown and to date no functional roles have been ascribed to MBLAC1, though we have shown that the protein binds the neuroprotective beta-lactam antibiotic, ceftriaxone. To gain insight into the functional role of MBLAC1 in vivo, we used CRISPR/Cas9 methods to disrupt N-terminal coding sequences of the mouse Mblac1 gene, resulting in a complete loss of protein expression in viable, homozygous knockout (KO) animals. Using serum from both WT and KO mice, we performed global, untargeted metabolomic analyses, resolving small molecules via hydrophilic interaction chromatography (HILIC) based ultra-performance liquid chromatography, coupled to mass spectrometry (UPLC-MS/MS). Unsupervised principal component analysis reliably segregated the metabolomes of MBLAC1 KO and WT mice, with 92 features subsequently nominated as significantly different by ANOVA, and for which we made tentative and putative metabolite assignments. Bioinformatic analyses of these molecules nominate validated pathways subserving bile acid biosynthesis and linoleate metabolism, networks known to be responsive to metabolic and oxidative stress. Our findings lead to hypotheses that can guide future targeted studies seeking to identify the substrate for MBLAC1 and how substrate hydrolysis supports the neuroprotective actions of ceftriaxone.<br>
