Antimicrobial Activity of Trigonelline Hydrochloride Against Pseudomonas aeruginosa and Its Quorum-Sensing Regulated Molecular Mechanisms on Biofilm Formation and Virulence.

Pseudomonas aeruginosa, a vivid biofilm-producing bacterium, is considered a dreadful opportunistic pathogen, and thus, management of biofilm-associated infections due to multidrug resistant strains by traditional drugs currently is of great concern. This study was aimed to assess the impact of trigonelline hydrochloride, a pyridine alkaloid, on P. aeruginosa PAO1, in search of an alternative therapeutant. The effect of trigonelline on colony morphology and motility was studied along with its role on biofilm and expression virulence factors. Trigonelline influenced the colony structure, motility, biofilm architecture, and the production of virulence factors in a dose-dependent manner. Alterations in quorum sending (QS)-regulated gene expression after treatment and molecular docking analysis for certain regulator proteins confirmed its effect on the QS-system network by affecting Las, Rhl, and Pqs signaling pathways and as possible molecular targets. Thus, trigonelline might be considered as a potential chemical lead to manage biofilm-associated pathogenesis or to develop other analogues with enhanced pharmacokinetic actions.

Naringin sensitizes the antibiofilm effect of ciprofloxacin and tetracycline against Pseudomonas aeruginosa biofilm.

The study aims to explore the combinatorial effect of naringin with antibiotics, ciprofloxacin and tetracycline on Pseudomonas aeruginosa biofilms. The antibiofilm efficacy of selected treatment regimes against P. aeruginosa biofilm were quantified by crystal violet assay, MTT assay, Congo red binding assay, and were visualized by confocal laser scanning microscopy and scanning electron microscopy. All the assays reflected antibiofilm activities, however, combinatorial performances of naringin with antibiotics were found to be more significant. A significant reduction in swimming and swarming motilities along with pellicle formation and altered colony morphology were observed as a result of combinatorial effect. The cytotoxicity of naringin and its antibiotic combinations was assayed on murine macrophage cell line. The applicability of such combinations was tested for their relative eradication against pre-formed biofilm on urinary catheter surface. This finding indicated that naringin potentiates the efficacy of both ciprofloxacin and tetracycline on P. aeruginosa biofilm in comparison to their solo treatment. The finding would help to open hitherto unexplored possibilities of establishing naringin as a potential antibiofilm agent and suggest on the possibility of its use in drug-herb combinations for managing biofilm-associated bacterial infections.

Helicase Activity Plays a Crucial Role for RNase R Function in Vivo and for RNA Metabolism.

RNase R is a 3' to 5' hydrolytic exoribonuclease that has the unusual ability to digest highly structured RNA. The enzyme possesses an intrinsic, ATP-dependent RNA helicase activity that is essential in vitro for efficient nuclease activity against double-stranded RNA substrates, particularly at lower temperatures, with more stable RNA duplexes, and for duplexes with short 3' overhangs. Here, we inquired whether the helicase activity was also important for RNase R function in vivo and for RNA metabolism. We find that strains containing a helicase-deficient RNase R due to mutations in its ATP-binding Walker motifs exhibit growth defects at low temperatures. Most importantly, cells also lacking polynucleotide phosphorylase (PNPase), and dependent for growth on RNase R, grow extremely poorly at 34, 37, and 42 °C and do not grow at all at 31 °C. Northern analysis revealed that in these cells, fragments of 16S and 23S rRNA accumulate to high levels, leading to interference with ribosome maturation and ultimately to cell death. These findings indicate that the intrinsic helicase activity of RNase R is required for its proper functioning in vivo and for effective RNA metabolism.

How RNase R Degrades Structured RNA: ROLE OF THE HELICASE ACTIVITY AND THE S1 DOMAIN.

RNase R, a ubiquitous 3' exoribonuclease, plays an important role in many aspects of RNA metabolism. In contrast to other exoribonucleases, RNase R can efficiently degrade highly structured RNAs, but the mechanism by which this is accomplished has remained elusive. It is known that RNase R contains an unusual, intrinsic RNA helicase activity that facilitates degradation of duplex RNA, but how it stimulates the nuclease activity has also been unclear. Here, we have made use of specifically designed substrates to compare the nuclease and helicase activities of RNase R. We have also identified and mutated several residues in the S1 RNA-binding domain that are important for interacting with duplex RNA and have measured intrinsic tryptophan fluorescence to analyze the conformational changes that occur upon binding of structured RNA. Using these approaches, we have determined the relation of the RNA helicase, ATP binding, and nuclease activities of RNase R. This information has been combined with a structural analysis of RNase R, based on its homology to RNase II, whose structure has been determined, to develop a detailed model that explains how RNase R digests structured RNA and how this differs from its action on single-stranded RNA.

The Helicase Activity of Ribonuclease R Is Essential for Efficient Nuclease Activity.

RNase R, which belongs to the RNB family of enzymes, is a 3' to 5' hydrolytic exoribonuclease able to digest highly structured RNA. It was previously reported that RNase R possesses an intrinsic helicase activity that is independent of its ribonuclease activity. However, the properties of this helicase activity and its relationship to the ribonuclease activity were not clear. Here, we show that helicase activity is dependent on ATP and have identified ATP-binding Walker A and Walker B motifs that are present in Escherichia coli RNase R and in 88% of mesophilic bacterial genera analyzed, but absent from thermophilic bacteria. We also show by mutational analysis that both of these motifs are required for helicase activity. Interestingly, the Walker A motif is located in the C-terminal region of RNase R, whereas the Walker B motif is in its N-terminal region implying that the two parts of the protein must come together to generate a functional ATP-binding site. Direct measurement of ATP binding confirmed that ATP binds only when double-stranded RNA is present. Detailed analysis of the helicase activity revealed that ATP hydrolysis is not required because both adenosine 5'-O-(thiotriphosphate) and adenosine 5'-(ß,γ-imino)triphosphate can stimulate helicase activity, as can other nucleoside triphosphates. Although the nuclease activity of RNase R is not needed for its helicase activity, the helicase activity is important for effective nuclease activity against a dsRNA substrate, particularly at lower temperatures and with more stable duplexes. Moreover, competition experiments and mutational analysis revealed that the helicase activity utilizes the same catalytic channel as the nuclease activity. These findings indicate that the helicase activity plays an essential role in the catalytic efficiency of RNase R.

Brevibacillus sp. KUMAs2, a bacterial isolate for possible bioremediation of arsenic in rhizosphere.

Arsenic (As) contamination of soil and water has been considered as a major global environmental issue during last few decades. Among the various methods so far reported for reclamation of As contaminated rhizosphere soil, bioremediation using bacteria has been found to be most promising. An As resistant bacterial isolate Brevibacillus sp. KUMAs2 was obtained from As contaminated soil of Nadia, West Bengal, India, which could resist As(V) and As(III) a maximum of 265mM and 17mM, respectively. The strain could remove ~40 percent As under aerobic culture conditions. As resistant property in KUMAs2 was found to be plasmid-borne, which carried both As oxidizing and reducing genes. The strain could promote chilli plant growth under As contaminated soil environment by decreasing As accumulation in plant upon successful colonization in the rhizosphere, which suggests the possibility of using this isolate for successful bioremediation of As in the crop field.

Toxicity of cadmium sulfide (CdS) nanoparticles against Escherichia coli and HeLa cells.

The present study endeavours to assess the toxic effect of synthesized CdS nanoparticles (NPs) on Escherichia coli and HeLa cells. The CdS NPs were characterized by DLS, XRD, TEM and AFM studies and the average size of NPs was revealed as ∼3 nm. On CdS NPs exposure bacterial cells changed morphological features to filamentous form and damage of the cell surface was found by AFM study. The expression of two conserved cell division components namely ftsZ and ftsQ in E. coli was decreased both at transcriptional and translational levels upon CdS NPs exposure. CdS NPs inhibited proper cell septum formation without affecting the nucleoid segregation. Viability of HeLa cells declined with increasing concentration of CdS NPs and the IC50 value was found to be 4 µg/mL. NPs treated HeLa cells showed changed morphology with condensed and fragmented nuclei. Increased level of reactive oxygen species (ROS) was found both in E. coli and HeLa cells on CdS NPs exposure. The inverse correlation between declined cell viabilities and elevated ROS level suggested that oxidative stress seems to be the key event by which NPs induce toxicity both in E. coli and HeLa cells.

CdO nanoparticle toxicity on growth, morphology, and cell division in Escherichia coli.

This Article deals with the toxicological study of synthesized CdO nanoparticles (NPs) on Escherichia coli . Characterization of the CdO NPs was done by DLS, XRD, TEM, and AFM studies, and the average size of NPs was revealed as 22 ± 3 nm. The NPs showed bactericidal activity against E. coli. When NPs were added at midlog phase of growth, complete growth inhibitory concentration was found as 40 µg/mL. Bacterial cells changed morphological features to filamentous form with increasing CdO NPs exposure time, and thereafter resulted in filamentation-associated clumping. From AFM study, severe damage of the cell surface was found in CdO NPs-treated cells. CdO NPs were found to interfere with the expression level of two conserved cell division components, ftsZ and ftsQ, in E. coli at both transcriptional and translational levels. Interference of CdO NPs in proper septum formation without affecting the nucleoid segregation was also observed in confocal micrographs. The elevated intracellular oxidative stress due to CdO NPs exposure seems to be one of the reasons for the changes in cell morphology and expression of division proteins in E. coli.

Cadmium toxicity in Escherichia coli: Cell morphology, Z-ring formation and intracellular oxidative balance.

This article deals with toxicological study of cadmium (Cd) as CdCl(2) on the growth and cell morphology of Escherichia coli K-12 MG1655. The minimum inhibitory concentration of Cd was 15µM. When cadmium was added at mid-log phase, growth was completely inhibited at 0.6mM and 50% of the bacterial growth retardation was found at 0.3mM concentration. At sublethal dose of Cd (0.2mM), majority of the cells showed filamentous form, suggested the possible effect of Cd on cell division. AFM study of bacterial cell morphology revealed severe surface damage of the treated cells in comparison to untreated cells. The expression of FtsZ decreased both at transcriptional and translational levels with the time of Cd exposure, thus cell division was affected and as a result cells took filamentous form. Due to Cd exposure, the nucleoid segregation remained unaffected, but improper Z-ring formation was observed. Activities of peroxidase and superoxide dismutase significantly decreased in treated cells with exposure time, which might elevate intracellular ROS level, as a consequence metabolic dysfunction and toxic effect were resulted.