RESUMO
We previously reported that Vibrio cholerae in a viable but non-culturable (VBNC) state can be converted to a culturable state by treatment with catalase. This finding enabled us to develop an assay system to observe the time course of the conversion from VBNC to culturable in V. cholerae. VBNC cells began to convert to culturable cells as early as 2 h after catalase supplementation. Gene expression in VBNC cells during catalase treatment was analyzed using RNA microarray. Many ribosomal DNA genes were stimulated 6 h post catalase exposure, suggesting that the conversion-driving signal started prior to 6 h. Focusing on the period prior to cell proliferation, we found that 16 genes might be involved in the conversion mechanism in V. cholerae, and they showed enhanced expression at 2 h and 4 h after catalase addition. These upregulated genes included phage shock proteins (pspA, B, and C), alternative sigma factor (rpoE) and its negative regulator (rseA), cobW C terminal domain-containing protein, damage-inducible helicase (dinG), cholerae toxin secretion protein epsM, HTH-type transcription regulator (iscR), mechanosensitive ion channel family protein, anthranilate synthase component I, fructose-specific IIBC component, molybdenum import ATP-binding protein (modC), LysE family translocator, putative organic hydroperoxide resistance protein, and a hypothetical protein. This study identified genes involved in the catalase-induced conversion of V. cholerae VBNC cells to a culturable state and provided valuable insights into the mechanisms involved in the conversion process.
Assuntos
Vibrio cholerae , Vibrio cholerae/genética , Catalase/genética , Perfilação da Expressão GênicaRESUMO
In addition to catalyzing coupled transport and phosphorylation of carbohydrates, the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulates various physiological processes in most bacteria. Therefore, the transcription of genes encoding the PTS is precisely regulated by transcriptional regulators depending on substrate availability. As the distribution of the mannose-specific PTS (PTSMan) is limited to animal-associated bacteria, it has been suggested to play an important role in host-bacteria interactions. In Vibrio cholerae, mannose is known to inhibit biofilm formation. During host infection, the transcription level of the V. cholerae gene encoding the putative PTSMan (hereafter referred to as manP) significantly increases, and mutations in this gene increase host survival rate. Herein, we show that an AraC-type transcriptional regulator (hereafter referred to as ManR) acts as a transcriptional activator of the mannose operon and is responsible for V. cholerae growth and biofilm inhibition on a mannose or fructose-supplemented medium. ManR activates mannose operon transcription by facilitating RNA polymerase binding to the promoter in response to mannose 6-phosphate and, to a lesser extent, to fructose 1-phosphate. When manP or manR is impaired, the mannose-induced inhibition of biofilm formation was reversed and intestinal colonization was significantly reduced in a Drosophila melanogaster infection model. Our results show that ManR recognizes mannose and fructose in the environment and facilitates V. cholerae survival in the host.
Assuntos
Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato , Vibrio cholerae , Animais , Citarabina , Drosophila melanogaster/metabolismo , Frutose , Regulação Bacteriana da Expressão Gênica , Humanos , Manose/metabolismo , Fosfatos/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMO
Antimicrobial resistance (AMR) has become a serious public and economic threat. The rate of bacteria acquiring AMR surpasses the rate of new antibiotics discovery, projecting more deadly AMR infections in the future. The Pathogen Box is an open-source library of drug-like compounds that can be screened for antibiotic activity. We have screened molecules of the Pathogen Box against Vibrio cholerae, the cholera-causing pathogen, and successfully identified two compounds, MMV687807 and MMV675968, that inhibit growth. RNA-seq analyses of V. cholerae after incubation with each compound revealed that both compounds affect cellular functions on multiple levels including carbon metabolism, iron homeostasis, and biofilm formation. In addition, whole-genome sequencing analysis of spontaneous resistance mutants identified an efflux system that confers resistance to MMV687807. We also identified that the dihydrofolate reductase is the likely target of MMV675968 suggesting it acts as an analog of trimethoprim but with a MIC 14-fold lower than trimethoprim in molar concentration. In summary, these two compounds that effectively inhibit V. cholerae and other bacteria may lead to the development of new antibiotics for better treatment of the cholera disease. IMPORTANCE Cholera is a serious infectious disease in tropical regions causing millions of infections annually. Vibrio cholerae, the causative agent of cholera, has gained multi-antibiotic resistance over the years, posing greater threat to public health and current treatment strategies. Here we report two compounds that effectively target the growth of V. cholerae and have the potential to control cholera infection.
Assuntos
Antibacterianos/farmacologia , Cólera/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Antagonistas do Ácido Fólico/farmacologia , Vibrio cholerae/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/análogos & derivados , Trimetoprima/farmacologia , Vibrio cholerae/genética , Vibrio cholerae/crescimento & desenvolvimento , Sequenciamento Completo do GenomaRESUMO
Cholera is a severe small intestine bacterial disease caused by consumption of water and food contaminated with Vibrio cholera. The disease causes watery diarrhea leading to severe dehydration and even death if left untreated. In the past few decades, V. cholerae has emerged as multidrug-resistant enteric pathogen due to its rapid ability to adapt in detrimental environmental conditions. This research study aimed to design inhibitors of a master virulence gene expression regulator, HapR. HapR is critical in regulating the expression of several set of V. cholera virulence genes, quorum-sensing circuits and biofilm formation. A blind docking strategy was employed to infer the natural binding tendency of diverse phytochemicals extracted from medicinal plants by exposing the whole HapR structure to the screening library. Scoring function criteria was applied to prioritize molecules with strong binding affinity (binding energy < -11 kcal/mol) and as such two compounds: Strychnogucine A and Galluflavanone were filtered. Both the compounds were found favourably binding to the conserved dimerization interface of HapR. One rare binding conformation of Strychnogucine A was noticed docked at the elongated cavity formed by α1, α4 and α6 (binding energy of -12.5 kcal/mol). The binding stability of both top leads at dimer interface and elongated cavity was further estimated using long run of molecular dynamics simulations, followed by MMGB/PBSA binding free energy calculations to define the dominance of different binding energies. In a nutshell, this study presents computational evidence on antibacterial potential of phytochemicals capable of directly targeting bacterial virulence and highlight their great capacity to be utilized in the future experimental studies to stop the evolution of antibiotic resistance evolution.
Assuntos
Vibrio cholerae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Compostos Fitoquímicos , Percepção de Quorum , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMO
Diarrheal illness is a major cause of morbidity and mortality among children in Haiti, and the impact of diarrheal illness was compounded by a cholera outbreak between 2010 and 2019. Our understanding of risk factors for diarrhea among children during this outbreak is limited. We conducted a secondary analysis of data collected as part of a cholera vaccine effectiveness study to identify factors associated with medically attended diarrhea among children in central Haiti from October of 2012 through November of 2016. We identified 47 children aged one to five years old who presented to medical clinics with acute, watery diarrhea, and 166 matched controls who did not have diarrhea, and we performed conditional logistic regression to identify factors associated with diarrhea. Discontinuing exclusive breastfeeding within one month of birth was associated with increased risk of diarrhea (RR 6.9, 95% CI 1.46-32.64), and diarrhea was inversely associated with reported history of supplementation with vitamin A (RR 0.05, 95% CI 0.004-0.56) and zinc (reported among 0% of cases vs. 17% of controls). Because of the concordance in supplementation patterns, it was not possible to attribute the association to vitamin A or zinc independently. While having a respondent who correctly identified ≥3 means of avoiding cholera was associated with reduced risk of diarrhea (RR 0.43, 95% CI 0.19-1.01), reported household sanitation practices and knowledge of cholera were not consistently associated with risk of diarrhea. These findings support ongoing efforts to reduce barriers to breastfeeding and promote pediatric supplementation with vitamin A and zinc in Haiti. Given the reduced efficacy of current oral cholera vaccines (OCV) among children, the results reinforce the importance of breastfeeding and micronutrient supplementation in preventing all-cause pediatric diarrheal illness generally and during cholera outbreaks.
Assuntos
Vacinas contra Cólera/administração & dosagem , Cólera/prevenção & controle , Diarreia/prevenção & controle , Estudos de Casos e Controles , Pré-Escolar , Cólera/epidemiologia , Cólera/microbiologia , Diarreia/epidemiologia , Diarreia/microbiologia , Epidemias , Feminino , Haiti/epidemiologia , Humanos , Lactente , Masculino , População Rural/estatística & dados numéricos , Eficácia de Vacinas , Vibrio cholerae/genética , Vibrio cholerae/imunologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (L.) Urb or Indian pennywort is a plant of ethnopharmacological relevance, commonly called as Brahmi in South India known for its antimicrobial property in gut and for the treatment of other gut ailments. Natural anti-virulence drugs that disarm pathogens by directly targeting virulence factors or the cell viability and are thus preferred over antibiotics as these drugs impose limited selection pressure for resistance development. In this regard, an in-vitro experimental study was conducted to know the effect of extract of Centella asiatica(L.) Urb. on cholera toxin, gene expression and its vibriocidal effect on five standard strains of Vibrio cholerae; IDH03097 (El Tor variant), N16961 (El Tor), O395 (Classical) as well as five clinical strains (Haitian variant). AIM OF THE STUDY: To study the effect of extract of Centella asiatica on Vibrio cholerae. MATERIALS AND METHODS: Crude extract was prepared from the leaves and stem part of the plant. The vibriocidal concentration was tested at different concentrations of the extract. The amount of cholera toxin released from the strains before and after exposure to the extract of Centella asiatica to Vibrio cholerae was measured using Bead ELISA. ctxA gene expression in the strains before and after exposure to extract of Centella asiatica was measured using quantitative real time PCR. All the above assays were performed with commercially obtained asiaticoside as well. RESULTS: The vibriocidal activity was tested at the different concentration of the extract, where 1g/mL of crude extract and 12.5mg/mL of asiaticoside was found to be vibriocidal. The amount of cholera toxin released before and after the exposure to extract of C. asiatica was measured using Bead ELISA, showing a reduction of 70%, 89% and 93% toxin produced by classical, El Tor and variant respectively. ctxA gene expression before and after exposure to extract of Centella asiatica as well as asiaticoside was measured using qRT-PCR. We found a decrease in expression of ctxA gene transcription by 6.19 fold in classical strain, 4.29 fold in El Tor, 1.133 fold in variant strains and about 10.13-10.20 fold for the clinical strains of V. cholerae using the extract of C.asiatica while, the reduction with the exposure to the asiaticoside were 2.762 fold in classical strain, 4.809 in El Tor, 24.1 in variant strain and 34.77 - 34.8 for the clinical strains. CONCLUSION: Centella asiatica extract inhibited the CT production in Vibrio cholerae as well as decreased the transcription of ctxA gene expression.
Assuntos
Toxina da Cólera/biossíntese , Genes Bacterianos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Triterpenos/farmacologia , Vibrio cholerae/efeitos dos fármacos , Antibacterianos/administração & dosagem , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Centella , Relação Dose-Resposta a Droga , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Triterpenos/administração & dosagem , Triterpenos/isolamento & purificação , Vibrio cholerae/genéticaRESUMO
Vibrio cholera is a Gram-negative pathogen that causes diarrheal disease. The B subunit of Chlora toxin (CtxB) is one of the most important antigens of Vibrio cholera in which mediates the attachment of the bacteria to target cells. The aim of this study was to prepare chitosan nanoparticles containing CtxB and evaluate the effect of the antigen entrapment on the immunogenicity of this antigen. For this, the pET28a vector was induced using IPTG. Recombinant CtxB protein was expressed and purified using Ni-NTA column and finally was confirmed by western blotting. Following the confirmation of the protein entrapment onto the chitosan nanoparticles, the formulation was prescribed to BALB/c mice in three groups, including oral, oral-injection and injection groups. Serum and fecal IgA and IgG were evaluated by ELISA test. Finally, challenge of immunized mice was performed using Ctx toxin and rabbit ileal loop test. Using SDS-PAGE and western blotting, the 17.5â¯kDa recombinant CtxB was confirmed. Size electrical charge and of nanoparticles were determined and approved by Zetasizer. Nanoparticles prescription showed 1/102400 IgG endpoint titers for injection groups and 1/1600, 1/6400 for oral, oral-injection groups respectively and Serum and fecal IgA endpoint titers showed above 1/160 in all groups. Furthermore, immunized mice were able to neutralize Ctx toxin by ileal loop test. The CtxB is a suitable immunogen of V. cholera to be incorporated in both protective and preventive vaccines. Chitosan nanoparticles improve the immune responses and it may be used as a carrier for vaccine delivery.
Assuntos
Antígenos de Bactérias/imunologia , Toxina da Cólera/imunologia , Cólera/prevenção & controle , Nanopartículas/química , Vibrio cholerae/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Quitosana/administração & dosagem , Quitosana/química , Cólera/imunologia , Cólera/microbiologia , Toxina da Cólera/administração & dosagem , Toxina da Cólera/química , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Coelhos , Vibrio cholerae/química , Vibrio cholerae/genéticaRESUMO
Peptides that are synthesized independently of the ribosome in plants, fungi, and bacteria can have clinically relevant anticancer, antihemochromatosis, and antiviral activities, among many other. Despite their natural origin, discovering new natural products is challenging, and there is a need to expand the chemical diversity that is accessible. In this work, we created a novel, compressed synthetic pathway for the heterologous expression and diversification of nonribosomal peptides (NRPs) based on homologs of siderophore pathways from Escherichia coli and Vibrio cholerae To enhance the likelihood of successful molecule production, we established a selective pressure via the iron-chelating properties of siderophores. By supplementing cells containing our synthetic pathway with different precursors that are incorporated into the pathway independently of NRP enzymes, we generated over 20 predesigned, novel, and structurally diverse NRPs. This engineering approach, where phylogenetically related genes from different organisms are integrated and supplemented with novel precursors, should enable heterologous expression and molecular diversification of NRPs.IMPORTANCE Nonribosomal peptides (NRPs) constitute a source of bioactive molecules with potential therapeutic applications. However, discovering novel NRPs by rational engineering of biosynthetic pathways remains challenging. Here, we show that a synthetic compressed pathway in which we replaced biosynthetic genes with their ancestral homologs and orthologs enabled successful heterologous NRP expression. Polyamines added exogenously were incorporated into nascent NRPs, and molecular production was pressured by growing the host under conditions that make such NRPs beneficial for survival. This multilayered approach resulted in the assembly of over 20 distinct and novel molecules. We envision this strategy being used to enable the production of NRPs from heterologous pathways.
Assuntos
Produtos Biológicos/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeos/metabolismo , Produtos Biológicos/química , Vias Biossintéticas , Escherichia coli/genética , Fungos/genética , Fungos/metabolismo , Expressão Gênica , Peptídeos/química , Peptídeos/genética , Sideróforos/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMO
UNLABELLED: Manganese plays an important role in the cellular physiology and metabolism of bacterial species, including the human pathogen Vibrio cholerae The intracellular level of manganese ions is controlled through coordinated regulation of the import and export of this element. We have identified a putative manganese exporter (VC0022), named mneA (manganese exporter A), which is highly conserved among Vibrio spp. An mneA mutant exhibited sensitivity to manganese but not to other cations. Under high-manganese conditions, the mneA mutant showed an almost 50-fold increase in intracellular manganese levels and reduced intracellular iron relative to those of its wild-type parent, suggesting that the mutant's manganese sensitivity is due to the accumulation of toxic levels of manganese and reduced iron. Expression of mneA suppressed the manganese-sensitive phenotype of an Escherichia coli strain carrying a mutation in the nonhomologous manganese export gene, mntP, further supporting a manganese export function for V. cholerae MneA. The level of mneA mRNA was induced approximately 2.5-fold after addition of manganese to the medium, indicating regulation of this gene by manganese. This study offers the first insights into understanding manganese homeostasis in this important pathogen. IMPORTANCE: Bacterial cells control intracellular metal concentrations by coordinating acquisition in metal-limited environments with export in metal-excess environments. We identified a putative manganese export protein, MneA, in Vibrio cholerae An mneA mutant was sensitive to manganese, and this effect was specific to manganese. The mneA mutant accumulated high levels of intracellular manganese with a concomitant decrease in intracellular iron levels when grown in manganese-supplemented medium. Expression of mneA in trans suppressed the manganese sensitivity of an E. coli mntP mutant. This study is the first to investigate manganese export in V. cholerae.
Assuntos
Proteínas de Bactérias/metabolismo , Manganês/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Proteínas de Membrana Transportadoras/genética , Mutação , Vibrio cholerae/genéticaRESUMO
UNLABELLED: Recently, our group along with others reported that the Vibrio FadR regulatory protein is unusual in that, unlike the prototypical fadR product of Escherichia coli, which has only one ligand-binding site, Vibrio FadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of the vc2105 gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of the vc2105 gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates the vc2105 promoter from the FadR regulator. The expression level of the Vibrio cholerae vc2105 gene was elevated 2- to 3-fold in a fadR null mutant strain, validating that FadR is a repressor for the vc2105 gene. The ß-galactosidase activity of a vc2105-lacZ transcriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike the fadD gene, a member of the Vibrio fad regulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression of ctxAB (cholera toxin A/B) and tcpA (toxin coregulated pilus A). Given that the transcriptional regulation of vc2105 fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of the Vibrio fad regulon. IMPORTANCE: The Vibrio FadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterized in vitro and in vivo. An auxiliary fad gene (vc2105) from Vibrio was proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Further in vitro and in vivo experiments revealed that the Vibrio FadR is a repressor for the vc2105 gene. Unlike fadD, a member of the Vibrio fad regulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxAB and tcpA). Therefore, since transcriptional regulation of vc2105 fits the criteria for fad genes, it seems likely that vc2105 acts as a new auxiliary member of the Vibrio fad regulon.
Assuntos
Proteínas de Bactérias/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Vibrio cholerae/genética , Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , Proteínas de Fímbrias/genética , Regiões Promotoras Genéticas , Ligação Proteica , Regulon , Ressonância de Plasmônio de Superfície/métodos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Vibrio cholerae/metabolismo , beta-Galactosidase/metabolismoRESUMO
A newly emerged Vibrio cholerae O1 El Tor variant strain with multidrug resistance is considered a threat to public health. Recent strategies to suppress virulence factors production instead of bacterial growth may lead to less selective pressure for the emergence of resistant strains. The use of spices and their active constituents as the inhibitory agents against cholera toxin (CT) production in V. cholerae may be an alternative approach to treat cholera. In this study, we examined the potential of sweet fennel seed (Foeniculum vulgare Miller var. dulce) methanol extract to inhibit CT production in V. cholerae without affecting viability. The methanol extract of sweet fennel seeds significantly inhibited CT production in various V. cholerae strains, regardless of serogroup or biotype. Interestingly, trans-anethole and 4-allylanisole, essential oil components of sweet fennel seeds, also demonstrated similar effects. Here, we report that sub-bactericidal concentrations of sweet fennel seed methanol extract and its major components can drastically inhibit CT production in various V. cholerae strains.
Assuntos
Antibacterianos/metabolismo , Toxina da Cólera/antagonistas & inibidores , Toxina da Cólera/biossíntese , Foeniculum/química , Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/metabolismo , Vibrio cholerae/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Metanol , Viabilidade Microbiana/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Sementes/química , Solventes , Vibrio cholerae/genéticaRESUMO
BACKGROUND: Singapore's diarrhoeal notification system is based on specific pathogens. Official data may thus be skewed towards notifiable diseases. Limited information is available on the profiles of aetiological agents responsible for acute gastroenteritis (AGE) cases, especially among the adult population. To understand the frequency and distribution of potential causative agents of diarrheal disease in Singapore, we screened adults' stool samples collected from a large public hospital. METHODS: The stool samples were screened for 18 diarrheagenic pathogens using a combination of commercial multiplex polymerase chain reaction (PCR), in-house singleplex PCR and immunochromatographic assays. One hundred adult faecal samples that were collected from October 2013 to January 2014 for routine diagnostic purposes and submitted for culture at Tan Tock Seng Hospital, Singapore were used. RESULTS: Pathogens were detected in 32% of the samples. The predominant organisms encountered were norovirus genogroup II (11%), Aeromonas spp. (9%) and Campylobacter spp. (5%). One sample was positive for both verocytotoxigenic E. coli (VTEC) and E. coli O157:H7. Two other samples were positive for VTEC only, and one other sample was positive for E. coli O157:H7 only. Astrovirus, C. perfringens, Shigella spp. and toxigenic C. difficile were each detected in 2% of the samples. Cryptosporidium parvum, Giardia lamblia, group A rotavirus, Salmonella spp. and Vibrio spp. were each detected in 1% of the samples. No L. monocytogenes, Y. enterocolitica, enteric adenovirus, or norovirus genogroup I were detected. CONCLUSION: Our preliminary findings suggest that pathogens causing non-notifiable diseases might have contributed considerably to the adult hospitalised AGE cases. However, as the samples were from an adult hospital, the data obtained may not be representative of the whole community. Thus, a larger study to collect clinical samples and risk exposure data from primary healthcare clinics and children hospital is planned for, to gain a more holistic perspective on the epidemiology of AGE in Singapore. A larger study may also offer valuable insights for improving the approach of microbiological surveillance of food, as well as strategizing inspection efforts along the food supply chain by public health authorities.
Assuntos
Diarreia/microbiologia , Gastroenterite/microbiologia , Doença Aguda , Adulto , Campylobacter/genética , Campylobacter/isolamento & purificação , Cromatografia de Afinidade , DNA Bacteriano/análise , Diarreia/epidemiologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Gastroenterite/epidemiologia , Hospitais , Humanos , Reação em Cadeia da Polimerase Multiplex , Norovirus/genética , Norovirus/isolamento & purificação , RNA Viral/análise , Rotavirus/genética , Rotavirus/isolamento & purificação , Salmonella/genética , Salmonella/isolamento & purificação , Singapura/epidemiologia , Vibrio cholerae/genética , Vibrio cholerae/isolamento & purificaçãoRESUMO
Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/µL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/µL and a regression coefficient (R(2)) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera.
Assuntos
Técnicas Biossensoriais , Cólera/diagnóstico , DNA Bacteriano/isolamento & purificação , Óxido de Magnésio/química , Nanoestruturas/química , Vibrio cholerae/isolamento & purificação , Cólera/microbiologia , Cólera/patologia , Sondas de DNA/síntese química , Sondas de DNA/química , DNA Bacteriano/genética , DNA Complementar/química , DNA Complementar/genética , Humanos , Limite de Detecção , Medições Luminescentes , Nanoestruturas/ultraestrutura , Hibridização de Ácido Nucleico/métodos , Processos Fotoquímicos , Espectroscopia de Infravermelho com Transformada de Fourier , Vibrio cholerae/genética , Vibrio cholerae/patogenicidadeRESUMO
The antimicrobial effects of aqueous extracts of blueberry, raspberry, and strawberry on 13 pathogenic bacteria were evaluated. The minimum inhibitory concentrations and minimum bactericidal concentrations of the extracts were determined before and after neutralization to pH 7.03 ± 0.15. Both Gram-positive and Gram-negative pathogenic bacteria were selectively inhibited by the non-neutralized berries. Blueberry was the best inhibitor, and Vibrio and Listeria were the most sensitive bacteria. After neutralization, blueberry affected only Vibrio and Listeria, whereas the antimicrobial activities of raspberry and strawberry were abolished. The total contents of phenolics, flavonoids, and proanthocyanidins in the extracts were measured with colorimetric methods and were highest in strawberry, followed by raspberry, and then blueberry. We also studied the effects of sub-bactericidal concentrations of the three berry extracts on virulence gene expression in Vibrio cholerae. Real-time quantitative reverse transcription-polymerase chain reaction revealed that the three berry extracts effectively repressed the transcription of the tcpA gene. Raspberry also repressed the transcription of the ctxA gene, whereas blueberry and strawberry did not. However, the three berry extracts did not affect the transcription of toxT. These results suggest that the three berry extracts exert potent antimicrobial effects and inhibit the expression of the virulence factors of V. cholerae.
Assuntos
Antibacterianos/farmacologia , Mirtilos Azuis (Planta) , Fragaria , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Rubus , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Mirtilos Azuis (Planta)/química , Flavonoides/farmacologia , Frutas/química , Expressão Gênica/efeitos dos fármacos , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Virulência/genéticaRESUMO
Advances in high-throughput DNA sequencing allow for a comprehensive analysis of bacterial genes that contribute to virulence in a specific infectious setting. Such information can yield new insights that affect decisions on how to best manage major public health issues such as the threat posed by increasing antimicrobial drug resistance. Much of the focus has been on the consequences of the selective advantage conferred on drug-resistant strains during antibiotic therapy. It is thought that the genetic and phenotypic changes that confer resistance also result in concomitant reductions in in vivo fitness, virulence, and transmission. However, experimental validation of this accepted paradigm is modest. Using a saturated transposon library of Pseudomonas aeruginosa, we identified genes across many functional categories and operons that contributed to maximal in vivo fitness during lung infections in animal models. Genes that bestowed both intrinsic and acquired antibiotic resistance provided a positive in vivo fitness advantage to P. aeruginosa during infection. We confirmed these findings in the pathogenic bacteria Acinetobacter baumannii and Vibrio cholerae using murine and rabbit infection models, respectively. Our results show that efforts to confront the worldwide increase in antibiotic resistance might be exacerbated by fitness advantages that enhance virulence in drug-resistant microbes.
Assuntos
Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Efeitos Psicossociais da Doença , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/fisiologia , Animais , Antibacterianos/farmacologia , Contagem de Colônia Microbiana , Elementos de DNA Transponíveis/genética , Modelos Animais de Doenças , Resistência Microbiana a Medicamentos/genética , Trato Gastrointestinal/patologia , Genes Bacterianos , Pulmão/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Mutagênese Insercional/genética , Mutação/genética , Óperon/genética , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Coelhos , Análise de Sequência de DNA , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/fisiologiaRESUMO
Glycine riboswitches contain two aptamers and turn on the expression of downstream genes in bacteria. Although full-length glycine riboswitches were shown to exhibit no glycine-binding cooperativity, the truncated glycine riboswitches were confirmed to bind two glycine molecules cooperatively. Thorough understanding of the ligand-binding cooperativity may shed light on the molecular basis of the cooperativity and help design novel intricate biosensing genetic circuits for application in synthetic biology. A previously proposed sequential model does not readily provide explanation for published data showing a deleterious mutation in the first aptamer inhibiting the glycine binding of the second one. Using the glycine riboswitch from Vibrio cholerae as a model system, we have identified a region in the first aptamer that modulates the second aptamer function especially in the shortened glycine riboswitch. Importantly, this modulation can be rescued by the addition of a complementary oligodeoxynucleotide, demonstrating the feasibility of developing this system into novel genetic circuits that sense both glycine and a DNA signal.
Assuntos
Aptâmeros de Nucleotídeos/genética , Glicina/metabolismo , Riboswitch/genética , Vibrio cholerae/genética , Regulação Alostérica , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , DNA/metabolismo , Glicina/genética , Ligantes , Conformação de Ácido Nucleico , RNA Mensageiro/genéticaRESUMO
We cloned, overexpressed and purified Vibrio cholerae FtsZ protein for the first time. We used several complementary techniques to probe and compare the comparative assembly properties of recombinant Vibrio cholerae FtsZ (VcFtsZ) and Escherichia coli FtsZ (EcFtsZ). We observed that VcFtsZ polymerized at a slower rate than EcFtsZ and interestingly its polymerization was highly dependent on the presence of Ca(2+) ion. Furthermore, DMSO specifically modulated the polymerization of VcFtsZ, promoted polymer bundling and increased the stability of the VcFtsZ protofilaments. Whereas DMSO showed no significant stimulatory effect on the assembly and bundling of EcFtsZ. Transmission electron microscopy experiments demonstrated that in presence of 8% DMSO the average thickness of the VcFtsZ polymers were increased significantly. DMSO specifically stabilized the VcFtsZ polymers against dilution induced disassembly and it reduced the GTPase activity of VcFtsZ. These results collectively suggested that despite lot of sequence homology, the assembly of VcFtsZ and EcFtsZ are differently regulated processes. We expect to use this knowledge of assembly properties of VcFtsZ for screening of small molecules against VcFtsZ for development of anti-cholera agent.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cólera/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Vibrio cholerae/genética , Proteínas de Bactérias/química , Cálcio/metabolismo , Cólera/tratamento farmacológico , Cólera/microbiologia , Clonagem Molecular , Proteínas do Citoesqueleto/química , Dimetil Sulfóxido/química , Escherichia coli , GTP Fosfo-Hidrolases/metabolismo , Humanos , Estabilidade Proteica , Vibrio cholerae/químicaRESUMO
UNLABELLED: Vibrio cholerae, a Gram-negative bacterium, infects humans and causes cholera, a severe disease characterized by vomiting and diarrhea. These symptoms are primarily caused by cholera toxin (CT), whose production by V. cholerae is tightly regulated by the virulence cascade. In this study, we designed and carried out a high-throughput chemical genetic screen to identify inhibitors of the virulence cascade. We identified three compounds, which we named toxtazin A and toxtazin B and B', representing two novel classes of toxT transcription inhibitors. All three compounds reduce production of both CT and the toxin-coregulated pilus (TCP), an important colonization factor. We present evidence that toxtazin A works at the level of the toxT promoter and that toxtazins B and B' work at the level of the tcpP promoter. Treatment with toxtazin B results in a 100-fold reduction in colonization in an infant mouse model of infection, though toxtazin A did not reduce colonization at the concentrations tested. These results add to the growing body of literature indicating that small-molecule inhibitors of virulence genes could be developed to treat infections, as alternatives to antibiotics become increasingly needed. IMPORTANCE: V. cholerae caused more than 580,000 infections worldwide in 2011 alone (WHO, Wkly. Epidemiol. Rec. 87:289-304, 2012). Cholera is treated with an oral rehydration therapy consisting of water, glucose, and electrolytes. However, as V. cholerae is transmitted via contaminated water, treatment can be difficult for communities whose water source is contaminated. In this study, we address the need for new therapeutic approaches by targeting the production of the main virulence factor, cholera toxin (CT). The high-throughput screen presented here led to the identification of two novel classes of inhibitors of the virulence cascade in V. cholerae, toxtazin A and toxtazins B and B'. We demonstrate that (i) small-molecule inhibitors of virulence gene production can be identified in a high-throughput screen, (ii) targeting virulence gene production is an effective therapeutic strategy, and (iii) small-molecule inhibitors can uncover unknown layers of gene regulation, even in well-studied regulatory cascades.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Vibrio cholerae/efeitos dos fármacos , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/uso terapêutico , Cólera/microbiologia , Cólera/prevenção & controle , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Camundongos , Transcrição Gênica/efeitos dos fármacos , Resultado do Tratamento , Vibrio cholerae/genéticaRESUMO
The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in Vibrio cholerae, the causative agent of cholera. In this study, activation of the genes edd and eda, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in V. cholerae. Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in V. cholerae and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes (ctxA and tcpA) and their regulator (toxT). Further, lowering of these transcript levels and cholera toxin production in vitro by an ED pathway-defective mutant (strain N16961 with a Δedd mutation [Δedd(N16961) strain]) suggested the importance of this pathway in regulating V. cholerae virulence. The in vivo relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in V. cholerae was shown to inhibit biofilm formation in vitro that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported in vivo expressed genes. All of these results suggest the importance of the ED pathway in V. cholerae pathogenesis.
Assuntos
Aldeído Liases/metabolismo , Cólera/microbiologia , Regulação Bacteriana da Expressão Gênica , Gluconatos/metabolismo , Hidroliases/metabolismo , Vibrio cholerae/patogenicidade , Aldeído Liases/genética , Animais , Animais Lactentes , Meios de Cultura , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Inativação Gênica , Hidroliases/genética , Intestinos/microbiologia , Camundongos , Coelhos , Vibrio cholerae/genética , Vibrio cholerae/crescimento & desenvolvimento , Vibrio cholerae/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Regulatory 5' untranslated regions (r5'UTRs) of mRNAs such as riboswitches modulate the expression of genes involved in varied biological processes in both bacteria and eukaryotes. New high-throughput sequencing technologies could provide powerful tools for discovery of novel r5'UTRs, but the size and complexity of the datasets generated by these technologies makes it difficult to differentiate r5'UTRs from the multitude of other types of RNAs detected. Here, we developed and implemented a bioinformatic approach to identify putative r5'UTRs from within large datasets of RNAs recently identified by pyrosequencing of the Vibrio cholerae small transcriptome. This screen yielded only approximately 1% of all non-overlapping RNAs along with 75% of previously annotated r5'UTRs and 69 candidate V. cholerae r5'UTRs. These candidates include several putative functional homologues of diverse r5'UTRs characterized in other species as well as numerous candidates upstream of genes involved in pathways not known to be regulated by r5'UTRs, such as fatty acid oxidation and peptidoglycan catabolism. Two of these novel r5'UTRs were experimentally validated using a GFP reporter-based approach. Our findings suggest that the number and diversity of pathways regulated by r5'UTRs has been underestimated and that deep sequencing-based transcriptomics will be extremely valuable in the search for novel r5'UTRs.