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1.
Food Chem Toxicol ; 140: 111241, 2020 Jun.
Article in English | MEDLINE | ID: mdl-32194137

ABSTRACT

Ingestion of deoxynivalenol (DON), one of the most common mycotoxin contaminants of cereals, leads to adverse effects for animal and human health. Bacterial biotransformation is a strategy to mitigate the toxicity of this mycotoxin. The present study aims to evaluate the toxicity of two bacterial biotranformation products of DON: 3-epi-deoxynivalenol (3-epi-DON) and de-epoxy-deoxynivalenol (DOM-1) through zootechnical, hematological, histological and immunological assays. Twenty-four 4-weeks-old piglets received a control diet or a diet contaminated with 3 mg kg-1 DON, DOM-1, or 3-epi-DON for 7 days. Sample tissues were collected for histomorphometrical analysis, expression of cytokines and cell protein junctions. The zootechnical and hematological parameters were not modulated by any treatment. Ingestion of DON induced histological alterations in the intestine, liver and lymphoid organs, as well as an overexpression of pro-inflammatory cytokines, E-cadherin and occludin. These changes were not observed in piglets receiving the DOM-1 and 3-epi-DON contaminated diets. Pigs fed 3-epi-DON contaminated diet showed an increase in IgM levels in comparison with other diets, while no change was observed in IgA and IgG levels among the diets. Our results indicate that DOM-1 and 3-epi-DON are not toxic for piglets; thus bacterial biotransformation seems to be a sustainable alternative to reduce mycotoxin toxicity.


Subject(s)
Trichothecenes/toxicity , Animal Feed/analysis , Animals , Biotransformation , Cytokines/metabolism , Food Contamination/analysis , Immunoglobulins/blood , Intestine, Small/drug effects , Intestine, Small/metabolism , Intestine, Small/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Lymphoid Tissue/drug effects , Lymphoid Tissue/metabolism , Lymphoid Tissue/pathology , Male , Organ Size/drug effects , Swine , Trichothecenes/chemistry , Trichothecenes/pharmacokinetics , Weight Gain/drug effects
2.
Environ Int ; 132: 105082, 2019 11.
Article in English | MEDLINE | ID: mdl-31400600

ABSTRACT

Deoxynivalenol (DON), one of the most widespread mycotoxins in Europe, and cadmium (Cd), a widespread environmental pollutant, are common food contaminants. They exert adverse effects on different organs including kidney, liver, and intestine. The intestine is a common target of DON and Cd when they are ingested. Most studies have focused on their individual effects whereas their combined toxicity has rarely been studied. The aim of this study was thus to evaluate their individual and combined effects on the intestinal barrier function in vitro and in vivo. In vitro, Caco-2 cells were treated with increasing concentrations of DON and Cd (1-30 µM). In vivo, Wistar rats were used as controls or exposed to DON contaminated feed (8.2 mg/kg feed), Cd-contaminated water (5 mg/l) or both for four weeks. In Caco-2 cells, DON, Cd and the DON+Cd mixture reduced transepithelial electrical resistance (TEER) and increased paracellular permeability in a dose-dependent manner. Impairment of the barrier function was associated with a decrease in the amount of E-cadherin and occludin after exposure to the two contaminants alone or combined. A decrease in E-cadherin expression was observed in rats exposed to the two contaminants alone or combined, whereas occludin expression only decreased in animals exposed to DON and DON+Cd. Jejunal crypt depth was reduced in rats exposed to DON or Cd, whereas villi height was not affected. In vitro and in vivo results showed that the effects of exposure to combined DON and Cd on the intestinal barrier function in the jejunum of Wistar rats and in the colorectal cancer cell line (Caco-2) was similar to the effects of each individual contaminant. This suggests that regulations for each individual contaminant are sufficiently protective for consumers.


Subject(s)
Cadmium/toxicity , Environmental Pollutants/toxicity , Intestinal Absorption/drug effects , Intestinal Mucosa/drug effects , Trichothecenes/toxicity , Aged , Animals , Caco-2 Cells , Electric Impedance , Europe , Food Contamination , Humans , Male , Permeability , Rats , Rats, Wistar
3.
Arch Toxicol ; 92(11): 3381-3389, 2018 11.
Article in English | MEDLINE | ID: mdl-30171291

ABSTRACT

Deoxynivalenol (DON) is the most abundant trichothecene in food and feed. It causes both acute and chronic disorders of the human and animal intestine, liver and the immune system. The structural basis for the toxicity of DON has not been fully elucidated. Using the pig as a target and a model species for human, the toxicity of DON and its deepoxy-metabolite (DOM-1) was compared. Animals were exposed by gavage to 1 and 0.5 nmol toxin/kg b.w./day for 2 and 3 weeks respectively. Whatever the dose/duration, DOM-1 was less toxic than DON in terms of weight gain and emesis. In the 3-week experiment, animals were vaccinated with ovalbumin, and their immune response was analyzed in addition to tissue morphology, biochemistry and hematology. DON impaired the morphology of the jejunum and the ileum, reduced villi height, decreased E-cadherin expression and modified the intestinal expression of cytokines. Similarly, DON induced hepatotoxicity as indicated by the lesion score and the blood biochemistry. By contrast, DOM-1 only induced minimal intestinal toxicity and did not trigger hepatotoxicity. As far as the immune response was concerned, the effects of ingesting DOM-1 were similar to those caused by DON, as measured by histopathology of lymphoid organs, PCNA expression and the specific antibody response. Taken together, these data demonstrated that DOM-1, a microbial detoxification product of DON, was not toxic in the sensitive pig model but retained some immune-modulatory properties of DON, especially its ability to stimulate a specific antibody response during a vaccination protocol.


Subject(s)
Immune System/drug effects , Trichothecenes/toxicity , Animals , Intestine, Small/drug effects , Intestine, Small/immunology , Liver/drug effects , Male , Swine , Trichothecenes/pharmacology , Weight Gain/drug effects
4.
mBio ; 8(2)2017 03 14.
Article in English | MEDLINE | ID: mdl-28292979

ABSTRACT

An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food-especially cereal products-in Europe and North America. This study investigates the effect of the food contaminant DON on the genotoxicity of the E. coli strains producing colibactin. In vitro, intestinal epithelial cells were coexposed to DON and E. coli producing colibactin. In vivo, newborn rats colonized at birth with E. coli producing colibactin were fed a DON-contaminated diet. Intestinal DNA damage was estimated by the phosphorylation of histone H2AX. DON exacerbates the genotoxicity of the E. coli producing colibactin in a time- and dose-dependent manner in vitro Although DON had no effect on the composition of the gut microbiota, and especially on the number of E. coli, a significant increase in DNA damage was observed in intestinal epithelial cells of animals colonized by E. coli strains producing colibactin and coexposed to DON compared to animals colonized with E. coli strains unable to produce colibactin or animals exposed only to DON. In conclusion, our data demonstrate that the genotoxicity of E. coli strains producing colibactin, increasingly present in the microbiota of asymptomatic human beings, is modulated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis.IMPORTANCE An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food-especially cereal products-in Europe and North America. Our in vitro and in vivo results demonstrate that the intestinal DNA damage induced by colibactin-producing E. coli strains was exacerbated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis.


Subject(s)
DNA Damage/drug effects , Escherichia coli/drug effects , Escherichia coli/metabolism , Gastrointestinal Tract/microbiology , Mutagens/toxicity , Peptides/toxicity , Polyketides/toxicity , Trichothecenes/metabolism , Animals , Coculture Techniques , Epithelial Cells/drug effects , Escherichia coli/growth & development , Histones/analysis , Rats
5.
Sci Rep ; 6: 29105, 2016 07 06.
Article in English | MEDLINE | ID: mdl-27381510

ABSTRACT

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 µM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.


Subject(s)
Bacteria/metabolism , Biotransformation , Mitogen-Activated Protein Kinases/genetics , Trichothecenes/toxicity , Animals , Bacteria/drug effects , Bacteria/genetics , Caco-2 Cells , Epithelial Cells/drug effects , Gene Expression Regulation/drug effects , Humans , Intestines/chemistry , Intestines/drug effects , Oxygen Consumption/genetics , Ribosomes/drug effects , Ribosomes/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Swine , Transcriptome/drug effects , Transcriptome/genetics , Trichothecenes/chemistry
6.
Arch Toxicol ; 90(8): 2037-46, 2016 Aug.
Article in English | MEDLINE | ID: mdl-26404761

ABSTRACT

Natural food contaminants such as mycotoxins are an important problem for human health. Deoxynivalenol (DON) is one of the most common mycotoxins detected in cereals and grains. Its toxicological effects mainly concern the immune system and the gastrointestinal tract. This toxin is a potent ribotoxic stressor leading to MAP kinase activation and inflammatory response. DON frequently co-occurs with its glucosylated form, the masked mycotoxin deoxynivalenol-3-ß-D-glucoside (D3G). The toxicity of this later compound remains unknown in mammals. This study aimed to assess the ability of D3G to elicit a ribotoxic stress and to induce intestinal toxicity. The toxicity of D3G and DON (0-10 µM) was studied in vitro, on the human intestinal Caco-2 cell line, and ex vivo, on porcine jejunal explants. First, an in silico analysis revealed that D3G, contrary to DON, was unable to bind to the A-site of the ribosome peptidyl transferase center, the main targets for DON toxicity. Accordingly, D3G did not activate JNK and P38 MAPKs in treated Caco-2 cells and did not alter viability and barrier function on cells, as measured by the trans-epithelial electrical resistance. Treatment of intestinal explants for 4 h with 10 µM DON induced morphological lesions and up-regulated the expression of pro-inflammatory cytokines as measured by qPCR and pan-genomic microarray analysis. By contrast, expression profile of D3G-treated explants was similar to that of controls, and these explants did not show histomorphology alteration. In conclusion, our data demonstrated that glucosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity.


Subject(s)
Food Contamination/analysis , Glucosides/toxicity , Jejunum/drug effects , Trichothecenes/toxicity , Animals , Caco-2 Cells , Cell Culture Techniques , Cell Survival/drug effects , Cytokines/genetics , Humans , Jejunum/metabolism , Jejunum/pathology , MAP Kinase Signaling System/drug effects , Peptidyl Transferases/metabolism , Protein Binding , Ribosomes/drug effects , Ribosomes/enzymology , Swine , Transcriptome/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism
7.
Toxins (Basel) ; 7(6): 1945-61, 2015 May 29.
Article in English | MEDLINE | ID: mdl-26035490

ABSTRACT

The mycotoxins deoxynivalenol (DON) and nivalenol (NIV), worldwide cereal contaminants, raise concerns for animal and human gut health, following contaminated food or feed ingestion. The impact of DON and NIV on intestinal mucosa was investigated after acute exposure, in vitro and in vivo. The histological changes induced by DON and NIV were analyzed after four-hour exposure on pig jejunum explants and loops, two alternative models. On explants, dose-dependent increases in the histological changes were induced by DON and NIV, with a two-fold increase in lesion severity at 10 µM NIV. On loops, NIV had a greater impact on the mucosa than DON. The overall proliferative cells showed 30% and 13% decrease after NIV and DON exposure, respectively, and NIV increased the proliferative index of crypt enterocytes. NIV also increased apoptosis at the top of villi and reduced by almost half the proliferative/apoptotic cell ratio. Lamina propria cells (mainly immune cells) were more sensitive than enterocytes (epithelial cells) to apoptosis induced by NIV. Our results demonstrate a greater impact of NIV than DON on the intestinal mucosa, both in vitro and in vivo, and highlight the need of a specific hazard characterization for NIV risk assessment.


Subject(s)
Intestinal Mucosa/drug effects , Jejunum/drug effects , Trichothecenes/toxicity , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Enterocytes/drug effects , Enterocytes/pathology , Female , In Vitro Techniques , Intestinal Mucosa/pathology , Jejunum/pathology , Reproducibility of Results , Swine
8.
Epidemiol Infect ; 143(14): 3114-7, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25703810

ABSTRACT

Dengue virus, commonly transmitted by mosquitoes, causes a human disease of significant social impact and presents a serious public health problem in Brazil. This report describes the unusual emergence of DENV-4 in northern Brazil after a nearly 30-year-long absence. DENV-4 genotype I is of Asian origin and was identified in the serum of patients receiving treatment at a hospital serving the Salvador area (Brazilian state of Bahia). The identification of dengue virus serotypes through molecular and phylogenetic analysis is essential for predicting disease severity or fatal illness, principally in endemic countries such as Brazil.


Subject(s)
Dengue Virus/classification , Dengue Virus/genetics , Dengue/virology , Brazil/epidemiology , Dengue/epidemiology , Dengue Virus/isolation & purification , Genotype , Humans , Molecular Epidemiology
9.
Exp Toxicol Pathol ; 67(2): 89-98, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25467749

ABSTRACT

Deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEA) are mycotoxins commonly produced by Fusarium species. The purpose of the present study was to investigate the effects of DON alone and in combination with NIV and ZEA on several parameters including weight gain and histological aspects of pigs submitted to chronic intoxication. Twenty, 5-week-old piglets received for 28 days one of the following diets: a control diet, a diet mono- contaminated with DON (1.5mg/kg), a diet multi-contaminated with DON (2mg/kg)+NIV (1.3mg/kg)+ZEA (1.5mg/kg) or a diet contaminated with DON (3mg/kg)+NIV (1.3mg/kg)+ZEA (1.5mg/kg). Animals fed the multi-contaminated diets presented a significant decrease in weight gain over the total period. The chronic ingestion of the contaminated diets induced a significant increase on histological changes on the intestine, liver and lymphoid organs. In addition, a significant increase on lymphocyte apoptosis was observed in lymph nodes and spleen in the animals receiving the contaminated diets. These data provide a better understanding of the possible effects of Fusarium toxins, alone or in combinations on the morphology of the intestine and lymphoid organs, which would contribute to the risk assessment of these toxins.


Subject(s)
Intestines/drug effects , Liver/drug effects , Trichothecenes/toxicity , Zearalenone/toxicity , Animal Feed , Animals , Cell Proliferation/drug effects , Drug Interactions , Food Contamination , Intestines/pathology , Liver/pathology , Male , Swine/growth & development , Weight Gain/drug effects
10.
J Agric Food Chem ; 61(27): 6711-9, 2013 Jul 10.
Article in English | MEDLINE | ID: mdl-23758213

ABSTRACT

Mycotoxin mitigation is of major interest as ingestion of mycotoxins results in poor animal health, decreased productivity, as well as substantial economic losses. A feed additive (FA) consisting of a combination of bacteria (Eubacterium BBSH797) and enzyme (fumonisin esterase FumD) was tested in pigs for its ability to neutralize the effects of mono- and co-contaminated diets with deoxynivalenol (DON) and fumonisins (FB) on hematology, biochemistry, tissue morphology, and immune response. Forty-eight animals, allocated into eight groups, received one of eight diets for 35 days: a control diet, a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg), or both toxins, and the same four diets with FA. Inclusion of FA restored the circulating number of neutrophils of piglets fed the FB and DON + FB diets. Similarly, FA counteracted the minor changes observed on plasma concentrations of albumin and creatinine. In lung, the lesions induced by the ingestion of FB in mono- and co-contaminated diets were no longer observed after addition of FA in these diets. Lesions recorded in the liver of pigs fed either of the contaminated diets with FA were partly reduced, and the increased hepatocyte proliferation was totally neutralized when FA was present in the co-contaminated diet. After 35 days of exposure, the development of the vaccinal response was significantly improved in animals fed diets supplemented with FA, as shown by results of lymphocyte proliferation, cytokine expression in spleen, and the production of specific Ig. Similarly, in jejunum of animals fed diets with FA, occurrence of lesions and upregulation of pro-inflammatory cytokines were much less obvious. The ameliorative effects provided by FA suggest that this approach would be suitable in the control of DON and FB that commonly co-occur in feed.


Subject(s)
Animal Feed/microbiology , Dietary Supplements/analysis , Eubacterium/metabolism , Fusariosis/microbiology , Fusarium/metabolism , Mycotoxins/metabolism , Swine Diseases/microbiology , Swine/microbiology , Animal Feed/analysis , Animals , Biotransformation , Dietary Supplements/microbiology , Fusariosis/pathology , Lung/pathology , Mycotoxins/blood , Mycotoxins/toxicity , Swine/blood , Swine Diseases/pathology
11.
Eur Phys J E Soft Matter ; 36(4): 9856, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23612746

ABSTRACT

In this work we focus on the surface charging properties of core shell ferrite nanoparticles dispersed in water, namely magnetic nanocolloids. This structural charge results from the Brönsted acid-base behavior of the particles surface sites and is achieved through hydrolysis reactions. It can be modeled by considering identical charged sites behaving as weak diprotic acids. Then, electrochemical techniques could be implemented to study the acid-base equilibrium between the particle surface and the colloid bulk solution. Simultaneous potentio-conductimetric titrations are therefore performed to determine the thermodynamical constants of the p H-dependent reactions and to obtain the p H variations of the surface charge density. The results reveal that the saturation value of the structural charge strongly depends on the nanoparticle mean size. For large particles, the surface tends to be fully ionized whereas for smaller particles the saturated structural charge decreases drastically. This surface charge reduction is attributed to the existence in smaller particles of metallic surface sites, which cannot be accessible to the proton charge. The existence of such dead sites would be related to hydroxo-bonded sites with very low acidity combined with a quantum size effect, which would affect the charging/discharging process at the surface of the semiconductor ferrite quantum dot.

12.
Biochem Pharmacol ; 83(10): 1465-73, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22366513

ABSTRACT

Fumonisins are mycotoxins frequently found as natural contaminants in maize, where they are produced by the plant pathogen Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisin B1 (FB1) is the predominant fumonisin in this family. FB1 is converted to its hydrolyzed analogs HFB1, by alkaline cooking (nixtamalization) or through enzymatic degradation. The toxicity of HFB1 is poorly documented especially at the intestinal level. The objectives of this study were to compare the toxicity of HFB1 and FB1 and to assess the ability of these toxins to disrupt sphingolipids biosynthesis. HFB1 was obtained by a deesterification of FB1 with a carboxylesterase. Piglets, animals highly sensitive to FB1, were exposed by gavage for 2 weeks to 2.8 µmol FB1 or HFB1/kg body weight/day. FB1 induced hepatotoxicity as indicated by the lesion score, the level of several biochemical analytes and the expression of inflammatory cytokines. Similarly, FB1 impaired the morphology of the different segments of the small intestine, reduced villi height and modified intestinal cytokine expression. By contrast, HFB1 did not trigger hepatotoxicity, did not impair intestinal morphology and slightly modified the intestinal immune response. This low toxicity of HFB1 correlates with a weak alteration of the sphinganine/sphingosine ratio in the liver and in the plasma. Taken together, these data demonstrate that HFB1 does not cause intestinal or hepatic toxicity in the sensitive pig model and only slightly disrupts sphingolipids metabolism. This finding suggests that conversion to HFB1 could be a good strategy to reduce FB1 exposure.


Subject(s)
Fumonisins/toxicity , Intestines/drug effects , Liver/drug effects , Sphingolipids/metabolism , Animals , Base Sequence , DNA Primers , Female , Hydrolysis , Real-Time Polymerase Chain Reaction , Swine
13.
Br J Nutr ; 107(12): 1776-86, 2012 Jun.
Article in English | MEDLINE | ID: mdl-21936967

ABSTRACT

Deoxynivalenol (DON) and fumonisins (FB) are mycotoxins produced by Fusarium species, which naturally co-occur in animal diets. The gastrointestinal tract represents the first barrier met by exogenous food/feed compounds. The purpose of the present study was to investigate the effects of DON and FB, alone and in combination, on some intestinal parameters, including morphology, histology, expression of cytokines and junction proteins. A total of twenty-four 5-week-old piglets were randomly assigned to four different groups, receiving separate diets for 5 weeks: a control diet; a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg); or both toxins. Chronic ingestion of these contaminated diets induced morphological and histological changes, as shown by the atrophy and fusion of villi, the decreased villi height and cell proliferation in the jejunum, and by the reduced number of goblet cells and lymphocytes. At the end of the experiment, the expression levels of several cytokines were measured by RT-PCR and some of them (TNF-α, IL-1ß, IFN-γ, IL-6 and IL-10) were significantly up-regulated in the ileum or the jejunum. In addition, the ingestion of contaminated diets reduced the expression of the adherent junction protein E-cadherin and the tight junction protein occludin in the intestine. When animals were fed with a co-contaminated diet (DON+FB), several types of interactions were observed depending on the parameters and segments assessed: synergistic (immune cells); additive (cytokines and junction protein expression); less than additive (histological lesions and cytokine expression); antagonistic (immune cells and cytokine expression). Taken together, the present data provide strong evidence that chronic ingestion of low doses of mycotoxins alters the intestine, and thus may predispose animals to infections by enteric pathogens.


Subject(s)
Diet , Food Contamination , Fumonisins/adverse effects , Fusarium/chemistry , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Trichothecenes/adverse effects , Animals , Cadherins/metabolism , Cytokines/metabolism , Goblet Cells/drug effects , Infections/etiology , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Intestine, Small/immunology , Intestine, Small/metabolism , Intestine, Small/pathology , Lymphocytes/drug effects , Male , Membrane Proteins/metabolism , Occludin , Random Allocation , Reverse Transcriptase Polymerase Chain Reaction , Swine , Up-Regulation
14.
Arch Microbiol ; 188(3): 243-50, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17492271

ABSTRACT

Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.


Subject(s)
Bacterial Proteins/metabolism , Flagella/physiology , Xanthomonas axonopodis/physiology , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Two-Hybrid System Techniques , Xanthomonas axonopodis/genetics , Xanthomonas axonopodis/metabolism
15.
Protein Expr Purif ; 53(2): 363-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17350859

ABSTRACT

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker in plantations around the world and is of particular significance in Brazil where its incidence has risen exponentially over the past decade. Approximately one third of the predicted Xac open reading frames show no homology, or homology with very low score with that of known sequences. It is believed that Xac utilizes secretion systems to transfer virulence proteins into susceptible eukaryotic cells. This process is assisted by secretion chaperones that maintain virulence proteins partly or completely unfolded during translocation. We have cloned three of these hypothetical secretion chaperones: XAC0419 and XAC1346 from type III secretion system (TTSS) and XACb0033 from type IV secretion system (TFSS). All proteins were cloned in a pET23a vector (Novagen), expressed at 37 degrees C using a BL21(DE3)pLysS Escherichia coli strain and purified by ion exchange and gel-filtration chromatographic methods. Pure proteins were characterized using spectroscopic measurements: circular dichroism, and both static and lifetime emission fluorescence in the case of XACb0033. The analyzed proteins are stable at elevated temperatures (up to 65 degrees C) and exhibit alpha-helix content from approximately 30% (XACb003) to approximately 87% (XAC1346). XACb0033 exhibits lifetimes in the fluorescence experiments that indicate different neighborhoods for its tryptophan residues. These chaperones have the characteristics of TTSS and TFSS: all are small, with a high alpha-helix content, and without ATP-binding or ATP-hydrolyzing activity.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Molecular Chaperones/genetics , Molecular Chaperones/isolation & purification , Xanthomonas axonopodis/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Circular Dichroism , Citrus/microbiology , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Genes, Bacterial , Molecular Chaperones/chemistry , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Plant Diseases/microbiology , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Spectrometry, Fluorescence , Virulence , Xanthomonas axonopodis/pathogenicity
16.
J Clin Microbiol ; 44(8): 2767-72, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16891490

ABSTRACT

In Brazil, Salmonella enterica serovar Infantis resistant to various antimicrobials, including cephalosporins, has been identified as an etiological agent of severe gastroenteritis in hospitalized children since 1994. In this study, 35 serovar Infantis strains, isolated from children admitted to four different Rio de Janeiro, Brazil, hospitals between 1996 and 2001, were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing in order to determine their genetic relatedness and antimicrobial resistance profiles. Thirty-four serovar Infantis strains were resistant to at least two antibiotic classes, and all 35 strains were susceptible to fluoroquinolones, cephamycin, and carbapenem. Extended-spectrum beta-lactamase (ESBL) screening by double-disk diffusion indicated that 32 serovar Infantis strains (91.4%) produced beta-lactamases that were inhibited by clavulanic acid. Antimicrobial resistance gene profiles were determined by PCR for a subset of 11 multidrug-resistant serovar Infantis strains, and putative ESBLs were detected by isoelectric focusing. Ten serovar Infantis strains carried bla(TEM), catI, ant(3")Ia and/or ant(3")Ib, sulI and/or sulII, and tet(D) genes as well as an integron-associated aac(6')-Iq cassette. Eight strains possessed at least four different beta-lactamases with pI profiles that confirmed the presence of both ESBLs and non-ESBLs. Our PFGE profiles indicated that 33 serovar Infantis strains isolated from Rio de Janeiro hospitals came from the same genetic lineage.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Gastroenteritis/microbiology , Salmonella Infections/microbiology , Salmonella enterica/drug effects , Salmonella enterica/genetics , Brazil , Clavulanic Acid/pharmacology , Cluster Analysis , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Enzyme Inhibitors/pharmacology , Genes, Bacterial , Humans , Infant , Infant, Newborn , Inpatients , Microbial Sensitivity Tests , Molecular Epidemiology , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Salmonella enterica/classification , Salmonella enterica/isolation & purification , beta-Lactamase Inhibitors , beta-Lactamases/analysis
17.
Arch Biochem Biophys ; 427(2): 135-42, 2004 Jul 15.
Article in English | MEDLINE | ID: mdl-15196987

ABSTRACT

We report here on the stability and folding of the 91 residue alpha-helical F29W N-terminal domain of chicken skeletal muscle troponin C (TnC(1-91)F29W), the thin filament calcium-binding component. Unfolding was monitored by differential scanning calorimetry, circular dichroism, and intrinsic fluorescence spectroscopy using urea, pH, and temperature as denaturants, in the absence and in the presence of calcium. The unfolding of TnC(1-91)F29W was reversible and did not follow a two-state transition, suggesting that an intermediate may be present during this reaction. Our results support the hypothesis that intermediates are likely to occur during the folding of small proteins and domains. The physiological significance of the presence of an intermediate in the folding pathway of troponin C is discussed.


Subject(s)
Calcium/chemistry , Troponin C/chemistry , Urea/chemistry , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Protein Conformation , Protein Denaturation , Protein Folding , Protein Structure, Tertiary , Temperature
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