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Methods in Molecular Biology (Clifton,... 2024One of the major plant stress level indicators is reactive oxygen species (ROS). They have been known to play a central role in regulating plant responses to various...
One of the major plant stress level indicators is reactive oxygen species (ROS). They have been known to play a central role in regulating plant responses to various environmental stresses. This book chapter specifically covers abiotic stress induced by a drought hormone abscisic acid and biotic stress induced by Pseudomonas syringe DC3000 on single cell-type guard cells. We describe in detail the measurement of ROS production starting from sample preparation to data analysis by fluorescence intensity acquisition using ImageJ software. We discussed the problems faced while performing the experiment and addressed how to overcome them by providing specific guidelines to ensure high quality repeatable data.
Topics: Reactive Oxygen Species; Arabidopsis; Stress, Physiological; Abscisic Acid; Pseudomonas syringae
PubMed: 38869797
DOI: 10.1007/978-1-0716-3973-3_14 -
World Journal of Microbiology &... Jun 2024It was known that UVc irradiation increases the reactive oxygen species' (ROS) levels in bacteria hence the intervention of antioxidant enzymes and causes also changes...
It was known that UVc irradiation increases the reactive oxygen species' (ROS) levels in bacteria hence the intervention of antioxidant enzymes and causes also changes in fatty acids (FAs) composition enabling bacteria to face antibiotics. Here, we intended to elucidate an interrelationship between SOD and susceptibility to antibiotics by studying FA membrane composition of UVc-treated P. aeruginosa PAO1 and its isogenic mutants (sodM, sodB and sod MB) membrane, after treatment with antibiotics. Swarmer mutants defective in genes encoding superoxide dismutase were pre-exposed to UVc radiations and then tested by disk diffusion method for their contribution to antibiotic tolerance in comparison with the P. aeruginosa wild type (WT). Moreover, fatty acid composition of untreated and UVc-treated WT and sod mutants was examined by Gaz chromatography and correlated to antibiotic resistance. Firstly, it has been demonstrated that after UVc exposure, swarmer WT strain, sodM and sodB mutants remain resistant to polymixin B, a membrane target antibiotic, through membrane unsaturation supported by the intervention of Mn-SOD after short UVc exposure and cyclopropanation of unsaturated FAs supported by the action of Fe-SOD after longer UVc exposure. However, resistance for ciprofloxacin is correlated with increase in saturated FAs. This correlation has been confirmed by a molecular docking approach showing that biotin carboxylase, involved in the initial stage of FA biosynthesis, exhibits a high affinity for ciprofloxacin. This investigation has explored the correlation of antibiotic resistance with FA content of swarmer P.aeruginosa pre-exposed to UVc radiations, confirmed to be antibiotic target dependant.
Topics: Anti-Bacterial Agents; Pseudomonas aeruginosa; Ultraviolet Rays; Superoxide Dismutase; Mutation; Cyclopropanes; Drug Resistance, Bacterial; Fatty Acids; Ciprofloxacin; Microbial Sensitivity Tests; Bacterial Proteins; Cell Membrane; Computer Simulation; Polymyxin B
PubMed: 38869625
DOI: 10.1007/s11274-024-04033-8 -
Frontiers in Immunology 2024Zinc finger Asp-His-His-Cys motif-containing (zDHHC) proteins, known for their palmitoyltransferase (PAT) activity, play crucial roles in diverse cellular processes,...
Zinc finger Asp-His-His-Cys motif-containing (zDHHC) proteins, known for their palmitoyltransferase (PAT) activity, play crucial roles in diverse cellular processes, including immune regulation. However, their non-palmitoyltransferase immunomodulatory functions and involvement in teleost immune responses remain underexplored. In this study, we systematically characterized the zDHHC family in the large yellow croaker (), identifying 22 members. Phylogenetic analysis unveiled that each of the 22 zDHHCs formed distinct clusters with their orthologues from other teleost species. Furthermore, all zDHHCs exhibited a highly conserved DHHC domain, as confirmed by tertiary structure prediction. Notably, zDHHC23 exhibited the most pronounced upregulation following () infection of macrophage/monocyte cells (MO/MΦ). Silencing zDHHC23 led to heightened pro-inflammatory cytokine expression and diminished anti-inflammatory cytokine levels in MO/MΦ during infection, indicating its anti-inflammatory role. Functionally, zDHHC23 facilitated M2-type macrophage polarization, as evidenced by a significant skewing of MO/MΦ towards the pro-inflammatory M1 phenotype upon zDHHC23 knockdown, along with the inhibition of MO/MΦ necroptosis induced by infection. These findings highlight the non-PAT immunomodulatory function of zDHHC23 in teleost immune regulation, broadening our understanding of zDHHC proteins in host-pathogen interactions, suggesting zDHHC23 as a potential therapeutic target for immune modulation in aquatic species.
Topics: Animals; Perciformes; Macrophages; Fish Proteins; Necroptosis; Phylogeny; Macrophage Activation; Fish Diseases; Acyltransferases; Pseudomonas; Cytokines
PubMed: 38868779
DOI: 10.3389/fimmu.2024.1401626 -
Microbial Cell Factories Jun 2024The objectives of the current study were to extract pyocyanin from Pseudomonas aeruginosa clinical isolates, characterize its chemical nature, and assess its biological...
Transforming microbial pigment into therapeutic revelation: extraction and characterization of pyocyanin from Pseudomonas aeruginosa and its therapeutic potential as an antibacterial and anticancer agent.
BACKGROUND
The objectives of the current study were to extract pyocyanin from Pseudomonas aeruginosa clinical isolates, characterize its chemical nature, and assess its biological activity against different bacteria and cancer cells. Due to its diverse bioactive properties, pyocyanin, being one of the virulence factors of P. aeruginosa, holds a promising, safe, and available therapeutic potential.
METHODS
30 clinical P. aeruginosa isolates were collected from different sources of infections and identified by routine methods, the VITEK 2 compact system, and 16 S rRNA. The phenazine-modifying genes (phzM, phzS) were identified using polymerase chain reaction (PCR). Pyocyanin chemical characterization included UV-Vis spectrophotometry, Fourier Transform Infra-Red spectroscopy (FTIR), Gas Chromatography-Mass Spectrometry (GC-MS), and Liquid Chromatography-Mass Spectrometry (LC-MS). The biological activity of pyocyanin was explored by determining the MIC values against different clinical bacterial strains and assessing its anticancer activity against A549, MDA-MB-231, and Caco-2 cancer cell lines using cytotoxicity, wound healing and colony forming assays.
RESULTS
All identified isolates harboured at least one of the phzM or phzS genes. The co-presence of both genes was demonstrated in 13 isolates. The UV-VIS absorbance peaks were maxima at 215, 265, 385, and 520 nm. FTIR could identify the characteristic pyocyanin functional groups, whereas both GC-MS and LC-MS elucidated the chemical formula CHNO, with a molecular weight 210. The quadri-technical analytical approaches confirmed the chemical nature of the extracted pyocyanin. The extract showed broad-spectrum antibacterial activity, with the greatest activity against Bacillus, Staphylococcus, and Streptococcus species (MICs 31.25-125 µg/mL), followed by E. coli isolates (MICs 250-1000 µg/mL). Regarding the anticancer activity, the pyocyanin extract showed IC values against A549, MDA-MB-231, and Caco-2 cancer cell lines of 130, 105, and 187.9 µg/mL, respectively. Furthermore, pyocyanin has markedly suppressed colony formation and migratory abilities in these cells.
CONCLUSIONS
The extracted pyocyanin has demonstrated to be a potentially effective candidate against various bacterial infections and cancers. Hence, the current findings could contribute to producing this natural compound easily through an affordable method. Nonetheless, future studies are required to investigate pyocyanin's effects in vivo and analyse the results of combining it with other traditional antibiotics or anticancer drugs.
Topics: Pyocyanine; Pseudomonas aeruginosa; Anti-Bacterial Agents; Humans; Antineoplastic Agents; Microbial Sensitivity Tests; Cell Line, Tumor; Caco-2 Cells
PubMed: 38867319
DOI: 10.1186/s12934-024-02438-6 -
Scientific Reports Jun 2024Rhizoctonia solani, the causal agent of banded leaf and sheath blight (BL&SB), poses a significant threat to maize and various crops globally. The increasing concerns...
Rhizoctonia solani, the causal agent of banded leaf and sheath blight (BL&SB), poses a significant threat to maize and various crops globally. The increasing concerns surrounding the environmental and health impacts of chemical fungicides have encouraged intensified concern in the development of biological control agents (BCAs) as eco-friendly alternatives. In this study, we explored the potential of 22 rhizobacteria strains (AS1-AS22) isolates, recovered from the grasslands of the Pithoragarh region in the Central Himalayas, as effective BCAs against BL&SB disease. Among these strains, two Pseudomonas isolates, AS19 and AS21, exhibited pronounced inhibition of fungal mycelium growth in vitro, with respective inhibition rates of 57.04% and 54.15% in cell cultures and 66.56% and 65.60% in cell-free culture filtrates. Additionally, both strains demonstrated effective suppression of sclerotium growth. The strains AS19 and AS21 were identified as Pseudomonas sp. by 16S rDNA phylogeny and deposited under accession numbers NAIMCC-B-02303 and NAIMCC-B-02304, respectively. Further investigations revealed the mechanisms of action of AS19 and AS21, demonstrating their ability to induce systemic resistance (ISR) and exhibit broad-spectrum antifungal activity against Alternaria triticina, Bipolaris sorokiniana, Rhizoctonia maydis, and Fusarium oxysporum f. sp. lentis. Pot trials demonstrated significant reductions in BL&SB disease incidence (DI) following foliar applications of AS19 and AS21, with reductions ranging from 25 to 38.33% compared to control treatments. Scanning electron microscopy revealed substantial degradation of fungal mycelium by the strains, accompanied by the production of hydrolytic enzymes. These findings suggest the potential of Pseudomonas strains AS19 and AS21 as promising BCAs against BL&SB and other fungal pathogens. However, further field trials are warranted to validate their efficacy under natural conditions and elucidate the specific bacterial metabolites responsible for inducing systemic resistance. This study contributes to the advancement of sustainable disease management strategies and emphasizes the potential of Pseudomonas strains AS19 and AS21 in combating BL&SB and other fungal diseases affecting agricultural crops.
Topics: Plant Diseases; Zea mays; Pseudomonas; Rhizoctonia; Plant Leaves; Biological Control Agents; Pest Control, Biological; Antibiosis; Phylogeny
PubMed: 38866928
DOI: 10.1038/s41598-024-64028-1 -
PloS One 2024Evaluate the effects of five disinfection methods on bacterial concentrations in hospital sink drains, focusing on three opportunistic pathogens (OPs): Serratia...
OBJECTIVE
Evaluate the effects of five disinfection methods on bacterial concentrations in hospital sink drains, focusing on three opportunistic pathogens (OPs): Serratia marcescens, Pseudomonas aeruginosa and Stenotrophomonas maltophilia.
DESIGN
Over two years, three sampling campaigns were conducted in a neonatal intensive care unit (NICU). Samples from 19 sink drains were taken at three time points: before, during, and after disinfection. Bacterial concentration was measured using culture-based and flow cytometry methods. High-throughput short sequence typing was performed to identify the three OPs and assess S. marcescens persistence after disinfection at the genotypic level.
SETTING
This study was conducted in a pediatric hospitals NICU in Montréal, Canada, which is divided in an intensive and intermediate care side, with individual rooms equipped with a sink.
INTERVENTIONS
Five treatments were compared: self-disinfecting drains, chlorine disinfection, boiling water disinfection, hot tap water flushing, and steam disinfection.
RESULTS
This study highlights significant differences in the effectiveness of disinfection methods. Chlorine treatment proved ineffective in reducing bacterial concentration, including the three OPs. In contrast, all other drain interventions resulted in an immediate reduction in culturable bacteria (4-8 log) and intact cells (2-3 log). Thermal methods, particularly boiling water and steam treatments, exhibited superior effectiveness in reducing bacterial loads, including OPs. However, in drains with well-established bacterial biofilms, clonal strains of S. marcescens recolonized the drains after heat treatments.
CONCLUSIONS
Our study supports thermal disinfection (>80°C) for pathogen reduction in drains but highlights the need for additional trials and the implementation of specific measures to limit biofilm formation.
Topics: Serratia marcescens; Intensive Care Units, Neonatal; Disinfection; Humans; Pseudomonas aeruginosa; Infant, Newborn; Stenotrophomonas maltophilia; Serratia Infections; Cross Infection
PubMed: 38865328
DOI: 10.1371/journal.pone.0304378 -
Proceedings of the National Academy of... Jun 2024Nitrous oxide (NO), a potent greenhouse gas, can be generated by multiple biological and abiotic processes in diverse contexts. Accurately tracking the dominant sources...
Nitrous oxide (NO), a potent greenhouse gas, can be generated by multiple biological and abiotic processes in diverse contexts. Accurately tracking the dominant sources of NO has the potential to improve our understanding of NO fluxes from soils as well as inform the diagnosis of human infections. Isotopic "Site Preference" (SP) values have been used toward this end, as bacterial and fungal nitric oxide reductases (NORs) produce NO with different isotopic fingerprints, spanning a large range. Here, we show that flavohemoglobin (Fhp), a hitherto biogeochemically neglected yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto NO under anoxic conditions (~+10‰) that correlates with typical environmental NO SP measurements. Using as a model organism, we generated strains that only contained Fhp or the dissimilatory NOR, finding that in vivo NO SP values imparted by these enzymes differ by over 10‰. Depending on the cellular physiological state, the ratio of Fhp:NOR varies significantly in wild-type cells and controls the net NO SP biosignature: When cells grow anaerobically under denitrifying conditions, NOR dominates; when cells experience rapid, increased nitric oxide concentrations under anoxic conditions but are not growing, Fhp dominates. Other bacteria that only make Fhp generate similar NO SP biosignatures to those measured from our Fhp-only strain. Fhp homologs in sequenced bacterial genomes currently exceed NOR homologs by nearly a factor of four. Accordingly, we suggest a different framework to guide the attribution of NO biological sources in nature and disease.
Topics: Nitrous Oxide; Oxidoreductases; Pseudomonas aeruginosa; Anaerobiosis; Bacterial Proteins; Nitric Oxide
PubMed: 38865268
DOI: 10.1073/pnas.2319960121 -
Protein Science : a Publication of the... Jul 2024Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent...
Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (K < 0.5 μM) include PBPs (PBP1a, K = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, K = 0.09 μM; RlpA, K = 0.4 μM); a type VI secretion system effector (Tse5, K = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, K < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.
Topics: Pseudomonas aeruginosa; Bacterial Proteins; Periplasm; Periplasmic Proteins; Glycosyltransferases; Peptidoglycan
PubMed: 38864725
DOI: 10.1002/pro.5038 -
Microbial Biotechnology Jun 2024Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major...
Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major pathogen of cherry (Prunus avium) causing bacterial canker of the stem, leaf and fruit, impacting productivity and leading to a loss of trees. In an attempt to find a treatment for this disease, naturally occurring bacteriophage (phage) that specifically target Pss is being investigated as a biocontrol strategy. However, before using them as a biocontrol treatment, it is important to both understand their efficacy in reducing the bacterial population and determine if the bacterial pathogens can evolve resistance to evade phage infection. To investigate this, killing curve assays of five MR phages targeting Pss showed that phage resistance rapidly emerges in vitro, even when using a cocktail of the five phages together. To gain insight to the changes occurring, Pss colonies were collected three times during a 66-h killing curve assay and separately, Pss and phage were also coevolved over 10 generations, enabling the measurement of genomic and fitness changes in bacterial populations. Pss evolved resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways. Bacterial fitness (growth) and virulence were affected in only a few mutants. Deletion of LPS-associated genes suggested that LPS was the main target receptor for all five MR phages. Later generations of coevolved phages from the coevolution experiment were more potent at reducing the bacterial density and when used with wild-type phages could reduce the emergence of phage-resistant mutants. This study shows that understanding the genetic mechanisms of bacterial pathogen resistance to phages is important for helping to design a more effective approach to kill the bacteria while minimizing the opportunity for phage resistance to manifest.
Topics: Pseudomonas syringae; Plant Diseases; Pseudomonas Phages; Bacteriophages
PubMed: 38864499
DOI: 10.1111/1751-7915.14489 -
Microbial Biotechnology Jun 2024Antimicrobial resistance (AMR) is an escalating global health crisis, driven by the overuse and misuse of antibiotics. Multidrug-resistant Gram-negative bacteria, such...
Antimicrobial resistance (AMR) is an escalating global health crisis, driven by the overuse and misuse of antibiotics. Multidrug-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, are particularly concerning due to their high morbidity and mortality rates. In this context, endolysins, derived from bacteriophages, offer a promising alternative to traditional antibiotics. This study introduces LysJEP8, a novel endolysin derived from Escherichia phage JEP8, which exhibits remarkable antimicrobial activity against key Gram-negative members of the ESKAPE group. Comparative assessments highlight LysJEP8's superior performance in reducing bacterial survival rates compared to previously described endolysins, with the most significant impact observed against P. aeruginosa, and notable effects on A. baumannii and K. pneumoniae. The study found that LysJEP8, as predicted by in silico analysis, worked best at lower pH values but lost its effectiveness at salt concentrations close to physiological levels. Importantly, LysJEP8 exhibited remarkable efficacy in the disruption of P. aeruginosa biofilms. This research underscores the potential of LysJEP8 as a valuable candidate for the development of innovative antibacterial agents, particularly against Gram-negative pathogens, and highlights opportunities for further engineering and optimization to address AMR effectively.
Topics: Endopeptidases; Drug Resistance, Multiple, Bacterial; Anti-Bacterial Agents; Gram-Negative Bacteria; Pseudomonas aeruginosa; Biofilms; Microbial Sensitivity Tests; Bacteriophages; Klebsiella pneumoniae; Hydrogen-Ion Concentration; Acinetobacter baumannii; Microbial Viability
PubMed: 38864495
DOI: 10.1111/1751-7915.14483