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Microbiology Spectrum Aug 2023The bacterium Riemerella anatipestifer requires iron for growth, but the mechanism of iron uptake is not fully understood. In this study, we disrupted the Feo system and...
The bacterium Riemerella anatipestifer requires iron for growth, but the mechanism of iron uptake is not fully understood. In this study, we disrupted the Feo system and characterized its function in iron import in R. anatipestifer ATCC 11845. Compared to the parent strain, the growth of the Δ Δ and Δ strains was affected under Fe-limited conditions, since the absence of the system led to less intracellular iron than in the parent strain. In parallel, the Δ strain was shown to be less sensitive to streptonigrin, an antibiotic that requires free iron to function. The sensitivity of the Δ strain to hydrogen peroxide was also observed to be diminished compared with that of the parent strain, which could be related to the reduced intracellular iron content in the Δ strain. Further research revealed that and were directly regulated by iron through the Fur regulator and that the transcript levels of and were significantly increased in medium supplemented with 1 mM MnCl, 400 μM ZnSO, and 200 μM CuCl. Finally, it was shown that the Δ strain of R. anatipestifer ATCC 11845 was significantly impaired in its ability to colonize the blood, liver, and brain of ducklings. Taken together, these results demonstrated that FeoAB supports ferrous iron acquisition in R. anatipestifer and plays an important role in R. anatipestifer colonization. In Gram-negative bacteria, the Feo system is an important ferrous iron transport system. R. anatipestifer encodes an Feo system, but its function unknown. As iron uptake may be required for oxidative stress protection and virulence, understanding the contribution of iron transporters to these processes is crucial. This study showed that the Δ strain is debilitated in its ability to import iron and that its intracellular iron content was constitutively low, which enhanced the resistance of the deficient strain to HO. We were surprised to find that, in addition to responding to iron, the Feo system may play an important role in sensing manganese, zinc, and copper stress. The reduced colonization ability of the Δ strain also sheds light on the role of iron transporters in host-pathogen interactions. This study is important for understanding the cross talk between iron and other metal transport pathways, as well as the pathogenic mechanism in R. anatipestifer.
Topics: Virulence; Bacterial Proteins; Hydrogen Peroxide; Iron; Membrane Transport Proteins
PubMed: 37272830
DOI: 10.1128/spectrum.01373-23 -
Journal of Biomolecular Structure &... 2022Antibiotic resistance is a global concern. Two members of the bacterial genus namely, and have raised much concern in recent years because of their resistance to...
Antibiotic resistance is a global concern. Two members of the bacterial genus namely, and have raised much concern in recent years because of their resistance to multiple commonly used antibiotics. Identification of multidrug resistant and pan-drug resistant bacteria has propelled the search for new antibiotics that can act on unconventional targets. Researches are going on to find out the possibility of using bacterial ribonucleotide reductases as a novel target for antibiotic development. Through evaluations, this study aims for characterization and functional annotation of ribonucleotide reductase enzymes of and . Binding affinities with these enzymes of the compounds that have shown promising results in inhibiting growth by acting on its ribonucleotide reductase were also assessed by molecular docking and dynamics simulations. Insights from this study will help in battling these infections in the near future. Communicated by Ramaswamy H. Sarma.
Topics: Humans; Ribonucleotide Reductases; Molecular Docking Simulation; Streptonigrin; Flavobacteriaceae Infections; Genome, Bacterial; Phylogeny; Anti-Bacterial Agents
PubMed: 34048660
DOI: 10.1080/07391102.2021.1930166 -
Journal of Microbiology, Immunology,... Dec 2022Klebsiella pneumoniae is a gram-negative opportunistic pathogen that causes diseases mostly in immunocompromised individuals. Recently, hypervirulent K. pneumoniae...
BACKGROUND
Klebsiella pneumoniae is a gram-negative opportunistic pathogen that causes diseases mostly in immunocompromised individuals. Recently, hypervirulent K. pneumoniae strains also cause severe disease in healthy individuals. Capsular polysaccharide (CPS) is the major virulence determinant in hypervirulent K. pneumoniae and protects the cell against the bactericidal activity of the immune system. Gallic acid (GA), a natural phenolic compound, is known to exhibit wide spectrum antibacterial activity; however, its effect on hypervirulent K. pneumoniae remains largely unresolved. We aimed to identify the effects of GA on CPS biosynthesis in hypervirulent K. pneumoniae.
METHODS
Antibacterial activity of GA was evaluated by counting colonies. CPS amount was determined by glucuronic acid content. The transcriptions of cps gene cluster were measured by quantitative real time PCR (qRT-PCR) and the β-galactosidase activity. The effect of GA on the resistance of K. pneumoniae to streptonigrin (SNG), an iron-activated antibiotic, was evaluated. The effect of GA on the resistance of K. pneumoniae to serum killing and phagocytosis by macrophages was observed.
RESULTS
GA inhibited the growth and CPS biosynthesis in K. pneumoniae. GA may affect the iron availability in K. pneumoniae, thus possibly repressing the cps transcription. In addition, GA reduced the resistance of K. pneumoniae to serum killing and enhanced its susceptibility to phagocytosis.
CONCLUSION
GA possesses bactericidal activity and inhibits the CPS biosynthesis in hypervirulent K. pneumoniae, thereby facilitating pathogen clearance by the host immune system. Therefore, GA may represent a promising strategy for the prevention or treatment of patients with hypervirulent K. pneumoniae infections.
Topics: Humans; Klebsiella pneumoniae; Gallic Acid; Virulence Factors; Anti-Bacterial Agents; Iron; Klebsiella Infections
PubMed: 34326026
DOI: 10.1016/j.jmii.2021.07.002 -
Archives of Biochemistry and Biophysics Sep 2022Several compounds have been found capable of diverting the electron flow in Escherichia coli and thus causing increased intracellular production of O and HO. One...
Several compounds have been found capable of diverting the electron flow in Escherichia coli and thus causing increased intracellular production of O and HO. One indication of this electron-shunting action was increased cyanide-resistant respiration and one cellular response was increased biosynthesis of the manganese-containing superoxide dismutase and of catalase. Blocking cytochrome oxidase with cyanide or azide increased the electron flow available for reduction of paraquat and presumably of the other exogenous compounds tested and thus increased their biological effects. Paraquat, pyocyanine, phenazine methosulfate, streptonigrin, juglone, menadione, plumbagin, methylene blue, and azure C were all effective in elevating intracellular production of O and HO. The effect of alloxan appeared paradoxical in that it increased cyanide-resistant respiration without significantly increasing the cell content of the manganese-superoxide dismutase and with only a small effect on the level of catalase. The alloxan effect on cyanide-resistant respiration was artifactual and was due to an oxygen-consuming reaction between alloxan and cyanide, rather than to a diversion of the intracellular electron flow. With paraquat as a representative electron-shunting compound, the increase in biosynthesis of the manganese-superoxide dismutase was prevented by inhibitors of transcription or of translation, but not by an inhibitor of replication. The increase in this enzyme activity, caused by paraquat and presumably by the other compounds, was thus due to de novo enzyme synthesis activated or derepressed at the level of transcription.
Topics: Alloxan; Catalase; Cyanides; Escherichia coli; Hydrogen Peroxide; Manganese; Oxidation-Reduction; Oxygen; Paraquat; Superoxide Dismutase; Superoxides
PubMed: 35660299
DOI: 10.1016/j.abb.2022.109229 -
Cells Jan 2021Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the...
Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.
Topics: Animals; Biomarkers; Colitis; DNA; DNA, Mitochondrial; Deoxyribonucleases; Dextran Sulfate; Endoscopy; Extracellular Space; Extracellular Traps; Inflammation; Intestinal Mucosa; Intestines; Mice, Inbred C57BL; Ornithine; Protein-Arginine Deiminase Type 4; Severity of Illness Index; Streptonigrin; Mice
PubMed: 33418977
DOI: 10.3390/cells10010081 -
Food Microbiology Oct 2023Salmonella is known to survive in raw/pasteurized milk and cause foodborne outbreaks. Lactoferrin, present in milk from all animal sources, is an iron-binding...
Salmonella is known to survive in raw/pasteurized milk and cause foodborne outbreaks. Lactoferrin, present in milk from all animal sources, is an iron-binding glycoprotein that limits the availability of iron to pathogenic bacteria. Despite the presence of lactoferrins, Salmonella can grow in milk obtained from different animal sources. However, the mechanism by which Salmonella overcomes iron scarcity induced by lactoferrin in milk is not evaluated yet. Salmonella employs the DNA binding transcriptional regulator Fur (ferric update regulator) to mediate iron uptake during survival in iron deplete conditions. To understand the importance of Fur in Salmonella milk growth, we profiled the growth of Salmonella Typhimurium Δfur (ST4/74Δfur) in both bovine and camel milk. ST4/74Δfur was highly inhibited in milk compared to wild-type ST4/74, confirming the importance of Fur mediated regulation of iron metabolism in Salmonella milk growth. We further studied the biology of ST4/74Δfur to understand the importance of iron metabolism in Salmonella milk survival. Using increasing concentrations of FeCl, and the antibiotic streptonigrin we show that iron accumulates in the cytoplasm of ST4/74Δfur. We hypothesized that the accumulated iron could activate oxidative stress via Fenton's reaction leading to growth inhibition. However, the inhibition of ST4/74Δfur in milk was not due to Fenton's reaction, but due to the 'iron scarce' conditions of milk and microaerophilic incubation conditions which made the presence of the fur gene indispensable for Salmonella milk growth. Subsequently, survival studies of 14 other transcriptional mutants of ST4/74 in milk confirmed that RpoE-mediated response to extracytoplasmic stress is also important for the survival of Salmonella in milk. Though we have data only for fur and rpoE, many other Salmonella transcriptional factors could play important roles in the growth of Salmonella in milk, a theme for future research on Salmonella milk biology. Nevertheless, our data provide early insights into the biology of milk-associated Salmonella.
Topics: Animals; Cattle; Salmonella typhimurium; Lactoferrin; Repressor Proteins; Iron; Milk; Bacterial Proteins; Gene Expression Regulation, Bacterial
PubMed: 37567619
DOI: 10.1016/j.fm.2023.104326 -
Microorganisms May 2024is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within , which...
is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within , which may be a key factor in its pathogenesis. This novel pathway is encoded in a gene cluster spanning MAB_0284c to 0305 and is related to pathways, producing the secondary metabolites streptonigrin and nybomycin. We constructed an in-frame deletion of the MAB_0295 () gene and tested it in our animal model. We have previously shown that tadpoles, which have functional lungs and T cells, can serve as a reliable comparative model for persistent infection and pathogenesis. Here, we report that tadpoles intraperitoneally infected with the ∆ mutant exhibit early decreased bacterial loads and significantly increased survival compared with those infected with WT . ∆ mutant also induced lower transcript levels of several pro-inflammatory cytokines (, , , ) than those of WT in the liver and lungs. In addition, there was impaired macrophage recruitment and decreased macrophage infection in tadpoles infected with the ∆ mutant, by tail wound inoculation, compared to those infected with the WT bacteria, as assayed by intravital confocal microscopy. These data underline the relevance and usefulness of tadpoles as a novel comparative animal model to identify genetic determinants of immunopathogenesis, suggesting a role for this novel and uncharacterized pathway in pathogenesis and macrophage recruitment.
PubMed: 38930501
DOI: 10.3390/microorganisms12061120 -
Molecules (Basel, Switzerland) Sep 2022Hybrids 1,4-quinone with quinoline were obtained by connecting two active structures through an oxygen atom. This strategy allows to obtain new compounds with a high...
Hybrids 1,4-quinone with quinoline were obtained by connecting two active structures through an oxygen atom. This strategy allows to obtain new compounds with a high biological activity and suitable bioavailability. Newly synthesized compounds were characterized by various spectroscopic methods. The enzymatic assay used showed that these compounds were a suitable DT-diaphorase (NQO1) substrates as evidenced by increasing enzymatic conversion rates relative to that of streptonigrin. Hybrids were tested in vitro against a panel of human cell lines including melanoma, breast, and lung cancers. They showed also a high cytotoxic activity depending on the type of 1,4-quinone moiety and the applied tumor cell lines. It was found that cytotoxic activity of the studied hybrids was increasing against the cell lines with higher NQO1 protein level, such as breast (MCF-7 and T47D) and lung (A549) cancers. Selected hybrids were tested for the transcriptional activity of the gene encoding a proliferation marker (H3 histone), cell cycle regulators (p53 and p21) and the apoptosis pathway (BCL-2 and BAX). The molecular docking was used to examine the probable interaction between the hybrids and NQO1 protein.
Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Cell Line, Tumor; Drug Screening Assays, Antitumor; Histones; Humans; Hydroxyquinolines; Molecular Docking Simulation; NAD(P)H Dehydrogenase (Quinone); Oxygen; Quinolines; Quinones; Streptonigrin; Tumor Suppressor Protein p53; bcl-2-Associated X Protein
PubMed: 36234741
DOI: 10.3390/molecules27196206 -
Bioorganic Chemistry Jan 2021In this research, betulin derivatives were bonded to the 1,4-quinone fragment by triazole linker. Furthermore, the enzymatic assay used has shown that these compounds...
In this research, betulin derivatives were bonded to the 1,4-quinone fragment by triazole linker. Furthermore, the enzymatic assay used has shown that these compounds are a good DT-diaphorase (NQO1) substrates as evidenced by increasing enzymatic conversion rates relative to that of streptonigrin. The anticancer activities of the hybrids were tested against a panel of human cell lines, like: melanoma, ovarian, breast, colon, and lung cancers. The structure-activity relationship showed that the activity depends on the type of 1,4-quinone moiety and the tumor cell lines used. It was also found that the anticancer effects were increasing against the cell line with higher NQO1 protein level, like: breast (T47D, MCF-7), colon (Caco-2), and lung (A549) cancers. The transcriptional activity of the gene encoding a proliferation marker (H3 histone), cell cycle regulators (p53 and p21) and apoptosis pathway (BCL-2 and BAX) for selected compounds were determined. The molecular docking study was carried out to examine the interaction between the hybrids and NQO1 enzyme. The computational simulation showed that the type of the 1,4-quinone moiety influences location of the compound in the active site of the enzyme. It is worth noting that the study of new hybrids of betulin as substrate for NQO1 protein may lead to new medical therapeutic applications in the future.
Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Humans; Molecular Docking Simulation; Molecular Structure; NAD(P)H Dehydrogenase (Quinone); Quinones; Structure-Activity Relationship; Substrate Specificity; Triterpenes
PubMed: 33272711
DOI: 10.1016/j.bioorg.2020.104478 -
Scientific Reports Jun 2020Elevated levels of plasma alpha1-antitrypsin (AAT) correlate with a poor prognosis of various cancers. Herein, we investigated effects of exogenous AAT on non-small lung...
Elevated levels of plasma alpha1-antitrypsin (AAT) correlate with a poor prognosis of various cancers. Herein, we investigated effects of exogenous AAT on non-small lung cancer cell lines with high (H1975) and very low (H661) baseline expression of SERPINA1 gene encoding AAT protein. Comparison of cells grown for 3 weeks in a regular medium versus medium supplemented with 2 mg/ml of AAT revealed that in the presence of AAT cells acquire better proliferative properties, resistance to staurosporine (STS)-induced apoptosis, and show higher expression of CLU, a pro-tumorigenic gene coding clusterin protein. Similarly, the co-administration of STS with AAT or addition of AAT to the cells pre-treated with STS abrogated effects of STS in both cell lines. Following experiments with H1975 cells have shown that AAT blocks critical steps in STS-induced cell death: inhibition of AKT/MAPK pathways, and activation of caspase 3 and autophagy. AAT does not inhibit apoptosis-triggered by chloroquine (inhibitor of autophagy) or streptonigrin (inducer of p53 pathway). The anti-apoptotic effects of AAT were unaffected by lipopolysaccharide (LPS). However, AAT induced TLR4 levels and enhanced LPS effects on the production of IL-6, a tumor-promoting cytokine. Our data provide further evidence that AAT plays a significant role in the tumorigenesis.
Topics: Apoptosis; Autophagy; Cell Movement; Cell Proliferation; Cytokines; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Lipopolysaccharides; Lung Neoplasms; Protective Agents; Serine Proteinase Inhibitors; Staurosporine; Tumor Cells, Cultured; alpha 1-Antitrypsin
PubMed: 32533048
DOI: 10.1038/s41598-020-66825-w