The Insights and Perspectives of Nitric Oxide-mediated Biofilm Eradication

: Biofilms are among the most important causes of nosocomial and recurrent infections as biofilms confer antibiotic resistance to pathogenic bacteria and protect them from the host’s immune system. Thus, it is imperative to investigate effective therapeutic agents to counteract biofilms. As an important signaling molecule, nitric oxide (NO) plays a crucial role in various biological and pathological processes. NO could disperse biofilm and restore the drug sensitivity by reducing intracellular cyclic-diguanosine monophosphate (c-di-GMP) levels. This review highlights recent advances on antibacterial and antibiofilm effects of NO when NO was co-administered with other antimicrobial agents. A significant improvement in drug permeability and biofilm cell targeting and reduced cytotoxicity could be attained with this strategy. In this review, we briefly lay out challenges and propose future directions in this appealing avenue of research on NO-based therapy for biofilm eradication.

Mechanism of Nitrite Transporter NirC in Motility, Biofilm Formation, and Adhesion of Avian Pathogenic Escherichia Coli

The Escherichia coli (E. coli) nirC gene encodes a nitrite transporter, which involved in transporting toxic nitrite (NO2-) from the environment into the bacteria. Although the deletion of nirC gene could cause changes in motility, adhesion in the previous study, and the virulence involved in the specified mechanism for pathogenic E. coli remains to be known. In the present work, we aimed to evaluate the role of NirC in a serotype O2:K1:H7 avian pathogenic Escherichia coli (APEC) strain. For this purpose, we generated a NirC-deficient mutant of APEC XM strain and examined its biological characteristics. The nirC gene deletion mutant enhanced ability of motility, decreased in biofilm formation, and it markedly reduced ability to adhere mouse brain microvascular endothelial cell b.End3 cells. For understanding its mechanism, sequentially we detected and found the stress regulator rpoS and its downstream genes csrA were up-regulated in NirC-deficient mutant while diguanylate cyclase gene dgcT was down-regulated. By high-performance liquid chromatography (HPLC) experiment, we demonstrated the concentration of intracellular 3',5'-cyclic diguanosine monophosphate (c-di-GMP) significantly decrease in nirC gene deletion mutant. Taken data together, we may make a conclusion with a possible signal pathway clue, due to NirC mutation, environmental NO2- accumulation leads to nitrite stress and inactivates c-di-GMP synthesis by stimulating the stress regulator RpoS, resulting in changes of biological characteristics.

BIOPREPARATION FOR IN SITU ANTITUMOR VACCINATION

The present study is focused on the first attempt to use an enzymatically produced biological preparation of cyclic diguanosine monophosphate (cyclic di-GMP) for the therapy of animal cancer. Feline breast carcinoma was chosen as the test model. The preparation was administered intratumorally to induce the immunogenic death of a part of the cancer cells and thus carry out the so-called in situ antitumor vaccination. Preliminary results indicate good therapeutic prospects of studied biopreparation for animal cancer treatment. In conclusion, the expedience of further trials of cyclic di-GMP preparation for in situ antitumor vaccination was stated. The need to supplement this mono-preparation with another immunostimulating adjuvant characterized by a mechanism of action distinct from that exhibited by cyclic di-GMP was emphasized. DNA preparation comprising the so-called immunostimulating CpG motifs was provided as an example of such compound.

Insights into the ligand-free structure of cyclic diguanosine monophosphate I riboswitch of Vibrio cholerae using molecular dynamics simulation

Riboswitches are key cis regulatory elements present at 5’ UTRs of mRNAs. They play a critical role in gene expression regulation at transcriptional and translational level by binding selectively to specific ligands followed by conformational changes. Ligands bind to the aptamer of riboswitches and their complex structures have been solved, but ligand-free riboswitches structures are not available which is important to understand specific ligand binding mechanism. In this paper, an all atom 150 nano-second (ns) molecular dynamics (MD) simulations of cyclic diguanosine monophosphate (c-di-GMP I) riboswitch aptamer domain from Vibrio cholerae were carried out to study ligand-free c-di-GMP I riboswitch aptamer structure and the binding mechanism. The Principle component analysis, cross correlation dynamics analysis and trajectory analyses revealed that the ligand-free structure has stable conformation with folded P2, P3 and an open P1 helix which opens the ligand binding helix-join-helix while the ligand-bound structure shows less deviation and remains as closed structure compared to the ligand-free structure. The junction residues significantly showed anti-correlated motions with each other elucidating the open conformation of the ligand-free aptamer of riboswitch. The identified key residues involved in binding are A18, G20, C46, A47 and C92.HighlightsThe c-di-GMP I riboswitch regulates the essential genes involved in the virulence of human bacterial pathogen V. Cholera.A 150 ns molecular dynamics run was performed to find a ligand-free stable structure of c-di-GMP I riboswitch aptamer.The trajectory analysis resulted in stable conformation of ligand-free structure with folded P2, slightly open P3 and an unwind P1 helix.The atomic level analyses through cross correlation dynamics and RMSF values showed the opening of catalytic pocket and unwinding P1 helix.The identified key residues involved in binding are A18, G20, C46, A47 and C92 at the catalytic pocket.

The histidine kinase PdtaS is a cyclic di-GMP binding metabolic sensor that controls mycobacterial adaptation to nutrient deprivation

Cell signalling relies on second messengers to transduce signals from the sensory apparatus to downstream components of the signalling pathway. In bacteria, one of the most important and ubiquitous second messengers is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes is not completely understood. Here we present the first report of a c-di-GMP regulated sensor histidine kinase previously named PdtaS (Rv3220c), which binds to c-di-GMP at sub-micromolar concentrations, subsequently perturbing signalling of the PdtaS-PdtaR (Rv1626) two component system. Aided by biochemical analysis, molecular docking and structural modelling, we have characterized the binding site of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in M. smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. Perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multi-parametric systems approach of transcriptomics, unbiased metabolomics and lipid analyses.One-sentence summaryThe universal bacterial second messenger cyclic di-GMP controls the mycobacterial nutrient stress response

ON THE PROBLEM OF DEVELOPMENT OF THE UNIVERSAL IMMUNOTHERAPEUTIC ANTICANCER VACCINE

The authors of this paper theoretically substantiated the cancer treatment method, using in situ activation of dendritic cells with intratumoral injection of two molecular “danger signals” of bacterial origin – plasmid DNA containing unmethylated CpG-dinucleotides and cyclic diguanosine monophosphate (cyclo-diGMP). Based on literature data it might be presumed that this procedure is capable to release from the dying cancer cells a large number of tumor-associated mutant proteins, to recruit effector immune cells into the tumor bed, to activate dendritic cells and as a result to induce a potent anti-cancer T-cellular immune response leading to elimination of both primary solid tumors and possible metastases.

Burkholderia cenocepacia integrates cis-2-dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virulence

Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia, the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.

Degradation of cyclic diguanosine monophosphate by a hybrid two-component protein protects Azoarcus sp. strain CIB from toluene toxicity

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls diverse functions in bacteria, including transitions from planktonic to biofilm lifestyles, virulence, motility, and cell cycle. Here we describe TolR, a hybrid two-component system (HTCS), from the β-proteobacterium Azoarcus sp. strain CIB that degrades c-di-GMP in response to aromatic hydrocarbons, including toluene. This response protects cells from toluene toxicity during anaerobic growth. Whereas wild-type cells tolerated a sudden exposure to a toxic concentration of toluene, a tolR mutant strain or a strain overexpressing a diguanylate cyclase gene lost viability upon toluene shock. TolR comprises an N-terminal aromatic hydrocarbon-sensing Per–Arnt–Sim (PAS) domain, followed by an autokinase domain, a response regulator domain, and a C-terminal c-di-GMP phosphodiesterase (PDE) domain. Autophosphorylation of TolR in response to toluene exposure initiated an intramolecular phosphotransfer to the response regulator domain that resulted in c-di-GMP degradation. The TolR protein was engineered as a functional sensor histidine kinase (TolRSK) and an independent response regulator (TolRRR). This classic two-component system (CTCS) operated less efficiently than TolR, suggesting that TolR was evolved as a HTCS to optimize signal transduction. Our results suggest that TolR enables Azoarcus sp. CIB to adapt to toxic aromatic hydrocarbons under anaerobic conditions by modulating cellular levels of c-di-GMP. This is an additional role for c-di-GMP in bacterial physiology.

Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

Clostridium difficileis an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration inC. difficilewas recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes,pilA1(CD3513) andpilB1(CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions.pilA1is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-roundin vitrotranscription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation inC. difficile.

Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis

We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.