Diguanylate Cyclases and Phosphodiesterases Required for Basal-Level c-di-GMP in Pseudomonas aeruginosa as Revealed by Systematic Phylogenetic and Transcriptomic Analyses.

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. aeruginosaIMPORTANCEPseudomonas 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.

Mitochondrial Bioenergetics of Functional Wound Closure is Dependent on Macrophage-Keratinocyte Exosomal Crosstalk.

Tissue nanotransfection (TNT)-based fluorescent labeling of cell-specific exosomes has shown that exosomes play a central role in physiological keratinocyte-macrophage (mϕ) crosstalk at the wound-site. Here, we report that during the early phase of wound reepithelialization, macrophage-derived exosomes (Exomϕ), enriched with the outer mitochondrial membrane protein TOMM70, are localized in leading-edge keratinocytes. TOMM70 is a 70 kDa adaptor protein anchored in the mitochondrial outer membrane and plays a critical role in maintaining mitochondrial function and quality. TOMM70 selectively recognizes cytosolic chaperones by its tetratricopeptide repeat (TPR) domain and facilitates the import of preproteins lacking a positively charged mitochondrial targeted sequence. Exosomal packaging of TOMM70 in mϕ was independent of mitochondrial fission. TOMM70-enriched Exomϕ compensated for the hypoxia-induced depletion of epidermal TOMM70, thereby rescuing mitochondrial metabolism in leading-edge keratinocytes. Thus, macrophage-derived TOMM70 is responsible for the glycolytic ATP supply to power keratinocyte migration. Blockade of exosomal uptake from keratinocytes impaired wound closure with the persistence of proinflammatory mϕ in the wound microenvironment, pointing toward a bidirectional crosstalk between these two cell types. The significance of such bidirectional crosstalk was established by the observation that in patients with nonhealing diabetic foot ulcers, TOMM70 is deficient in keratinocytes of wound-edge tissues.

Swine Model of Biofilm Infection and Invisible Wounds.

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the involvement of the host immune system. Iterative changes in the host and pathogen during the formation of such clinically relevant biofilm can only occur in vivo. The swine wound model is recognized for its advantages as a powerful pre-clinical model. There are several reported approaches for studying wound biofilms. In vitro and ex vivo systems are deficient in terms of the host immune response. Short-term in vivo studies involve acute responses and, thus, do not allow for biofilm maturation, as is known to occur clinically. The first long-term swine wound biofilm study was reported in 2014. The study recognized that biofilm-infected wounds may close as determined by planimetry, but the skin barrier function of the affected site may fail to be restored. Later, this observation was validated clinically. The concept of functional wound closure was thus born. Wounds closed but deficient in skin barrier function may be viewed as invisible wounds. In this work, we seek to report the methodological details necessary to reproduce the long-term swine model of biofilm-infected severe burn injury, which is clinically relevant and has translational value. This protocol provides detailed guidance on establishing an 8 week wound biofilm infection using P. aeruginosa (PA01). Eight full-thickness burn wounds were created symmetrically on the dorsum of domestic white pigs, which were inoculated with (PA01) at day 3 post-burn; subsequently, noninvasive assessments of the wound healing were conducted at different time points using laser speckle imaging (LSI), high-resolution ultrasound (HUSD), and transepidermal water loss (TEWL). The inoculated burn wounds were covered with a four-layer dressing. Biofilms, as established and confirmed structurally by SEM at day 7 post-inoculation, compromised the functional wound closure. Such an adverse outcome is subject to reversal in response to appropriate interventions.

Dual functions: A coumarin-chalcone conjugate inhibits cyclic-di-GMP and quorum-sensing signaling to reduce biofilm formation and virulence of pathogens.

Antibiotic resistance or tolerance of pathogens is one of the most serious global public health threats. Bacteria in biofilms show extreme tolerance to almost all antibiotic classes. Thus, use of antibiofilm drugs without bacterial-killing effects is one of the strategies to combat antibiotic tolerance. In this study, we discovered a coumarin-chalcone conjugate C9, which can inhibit the biofilm formation of three common pathogens that cause nosocomial infections, namely, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, with the best antibiofilm activity against P. aeruginosa. Further investigations indicate that C9 decreases the synthesis of the key biofilm matrix exopolysaccharide Psl and bacterial second messenger cyclic-di-GMP. Meanwhile, C9 can interfere with the regulation of the quorum sensing (QS) system to reduce the virulence of P. aeruginosa. C9 treatment enhances the sensitivity of biofilm to several antibiotics and reduces the survival rate of P. aeruginosa under starvation or oxidative stress conditions, indicating its excellent potential for use as an antibiofilm-forming and anti-QS drug.

Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair.

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.

Chinese medicinal herb extract inhibits PQS-mediated quorum sensing system in Pseudomonas aeruginosa.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese medicinal herbs have long been recognized as important resources that can be used for the struggle against diseases and a significant component of health care system for thousands of years. AIM OF THE STUDY: In order to understand their roles in the treatment against bacterial infections, we examined the underlying mechanisms of one of the medicinal herb extracts (MHE) (Artemisiae argyi Folium, the root bark of Cortex dictamni and the root of Solanum melongena) on the human opportunistic pathogen Pseudomonas aeruginosa. MATERIALS AND METHODS: We combined phenotypic assays, transcriptional analysis and chemical investigations to identify the mechanisms underlying MHE inhibition. The standard sample was prepared and transcriptional reporters for quorum sensing systems were constructed. Electrophoretic mobility shift assays were used to clarify the mechanism. GC-MS and molecular docking were used to identify the chemicals in MHE and potential binding agents. RESULTS: We found that co-culturing of MHE with bacterial cells did not change the growth rate but substantially attenuate the production of virulence factors such as phenazine pyocyanin, siderophore pyoverdine and biofilm formation. Transcriptional responses of three major quorum sensing (QS) systems of P. aeruginosa to MHE showed that Pseudomonas quinolone signaling (PQS) system was completely repressed, rhlR/rhlI QS system was moderately inhibited, while lasR/lasI QS system was only slightly affected, suggesting that MHE might selectively target the PQS system to inhibit bacterial virulence. Furthermore, electrophoretic mobility shift assays (EMSA) showed that MHE inhibited the binding of MvfR the corresponding pqsA promoter region, suggesting that MHE serves as a competitive agent to quench the QS functionality in P. aeruginosa. CONCLUSION: We prove that MHE functions as an effective countermeasure against bacterial infections.

Evaluation and characterization of the predicted diguanylate cyclase-encoding genes in Pseudomonas aeruginosa.

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.

Anti-biofilm activity of plant derived extracts against infectious pathogen-Pseudomonas aeruginosa PAO1.

BACKGROUND: Biofilm forming ability of Pseudomonas aeruginosa make them vulnerable, because it makes them recalcitrant against various antibiotics. Quorum sensing (QS) is cell density based signaling that helps in bacterial cell-cell communication, which regulated various virulence factors such as pigment and biofilm formation that contribute in the establishment of chronic infections. The interruption of QS is one of the effective approach to control various virulence factors. Present study was intended with the aim to authenticate antibiofilm potential in different solvents based extracts of selected medicinal plant species viz. Berginia ciliata, Clematis grata and Clematis viticella traditionally used by the inhabitants of Himalayan region of Pakistan to treat various pathogenic diseases. P. aeruginosa PAO1, an opportunistic pathogen and involves in various life-threatening infections specifically in immune deficient patients was used as a model pathogen. METHODS: Plants were extracted in various organic (ethanol, methanol, acetone, ethyl acetate, hexane, chloroform) as well as in aqueous solvents and their ability to inhibit biofilm was measured. Biofilm of PAO1 was grown in Jensen's medium while growing at 30°C and crystal violet assay was performed to assess the biofilm inhibiting activity of plant extracts. RESULTS: Solvents play a vital role in extraction of plant components and it was found that the plants in various solvents exhibit different activity against the PAO1 biofilm. Comparatively, 1% methanolic extract of B. ciliata (rhizome with skin), showed more than 80% inhibition of biofilm formation without effecting on the growth of the bacterium. Significant correlation between flavonoids content and antibiofilm activity in methanolic extract revealed the contribution of secondary metabolites in P. aeruginosa (PAO1) biofilm inhibition. CONCLUSION: Our study revealed that plants under investigation more specifically B. ciliata could be a potential candidate for drug discovery to treat P. aeruginosa PAO1, induced infectious diseases especially for its biofilm treatment.

In silico characterization of putative drug targets in Staphylococcus saprophyticus, causing bovine mastitis.

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.