Cyclic diguanosine monophosphate (c-di-GMP) is an important second messenger involved in bacterial switching from motile to sessile lifestyles. In the opportunistic pathogen Pseudomonas aeruginosa, at least 40 genes are predicted to encode proteins for the making and breaking of this signal molecule. However, there is still paucity of information concerning the systemic expression pattern of these genes and the functions of uncharacterized genes. In this study, we analyzed the phylogenetic distribution of genes from P. aeruginosa that were predicted to have a GGDEF domain and found five genes (PA5487, PA0285, PA0290, PA4367, and PA5017) with highly conserved distribution across 52 public complete pseudomonad genomes. PA5487 was further characterized as a typical diguanylate cyclase (DGC) and was named dgcH. A systemic analysis of the gene expression data revealed that the expression of dgcH is highly invariable and that dgcH probably functions as a conserved gene to maintain the basal level of c-di-GMP, as reinforced by gene expression analyses. The other four conserved genes also had an expression pattern similar to that of dgcH. The functional analysis suggested that PA0290 encoded a DGC, while the others functioned as phosphodiesterases (PDEs). Our data revealed that there are five DGC and PDE genes that maintain the basal level of c-di-GMP in P. aeruginosa.
IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in animals, humans, and plants. The formation of biofilms by P. aeruginosa is the central mode of action to persist in hosts and evade immune and antibiotic attacks. Cyclic-di-GMP (c-di-GMP) is an important second messenger involved in the regulation of biofilm formation. In P. aeruginosa PAO1 strain, there are around 40 genes that encode enzymes for making and breaking this dinucleotide. A major missing piece of information in this field is the phylogeny and expression profile of those genes. Here, we took a systemic approach to investigate this mystery. We found that among 40 c-di-GMP metabolizing genes, 5 have well-conserved phylogenetic distribution and invariable expression profiles, suggesting that there are enzymes required for the basal level of c-di-GMP in P. aeruginosa. This study thus provides putative therapeutic targets against P. aeruginosa infections.
Opportunistic pathogen Pseudomonas aeruginosa can cause acute and chronic infections in humans. It is notorious for its resistance to antibiotics due to the formation of biofilms. Cyclic‐di‐GMP is a bacterial second messenger that plays important roles during biofilm development. There are 40 genes in P. aeruginosa predicted to participate in c‐di‐GMP biosynthesis or degradation. It is time‐consuming for the functional characterization of these genes. Here, we cloned 16 genes from P. aeruginosa PAO1 that are predicted to encode diguanylate cyclases (DGCs, responsible for c‐di‐GMP biosynthesis) and constructed their corresponding in‐frame deletion mutants. We evaluated the methods to measure the intracellular c‐di‐GMP concentration by using deletion mutants and PAO1 strains containing a plasmid expressing one of the 16 genes, respectively. Functional outputs of all PAO1‐derived stains were also detected and evaluated, including biofilm formation, production of exopolysaccharide, swimming and swarming motilities. Our data showed that measuring the c‐di‐GMP level only characterized a few DGC by using either pCdrA::gfp as a reporter or LC/MS/MS. Functional output results indicated that overexpression of a DGC gave more pronounced phenotypes than the corresponding deletion mutant and suggested that the swimming motility assay could be a quick way to briefly estimate a predicted DGC for further studies. The overall evaluation suggested 15 out of 16 predicted DGCs were functional DGCs, wherein six were characterized to encode DGCs previously. Altogether, we have provided not only a cloning library of 16 DGC‐encoding genes and their corresponding in‐frame deletion mutants but also paved ways to briefly characterize a predicted DGC.
Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.
The bovine mastitis caused by coagulase negative staphylococci (CNS) has increased in many herds of urban and rural areas of
India. Emergence of multi drug resistant bacteria has further made its management more complex and serious. Therefore,
innovation of novel specific drug for the treatment of disease caused by particular organism remained to be a challenge. Hence, in
the present study a bacterium was isolated from milk of the cow with bovine mastitis and was identified as S. saprophyticus, 44
pathways of S. saprophyticus retrieved (KEGG) from web server were found to be non homologous to the host Bos taurus, out of
which 39 pathways were found to be in cytoplasm, 2 in cell wall and 3 in the cell membrane. The knowledge of the present study
could make the drug discovery easier which have high affinity to the target site of the causative organism.
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