Biomedical subjects

Abelardo Margolles

Publications and source records attributed to Abelardo Margolles.

18 recordsLinked to original sources

PubMed2006

Induction of alpha-L-arabinofuranosidase activity by monomeric carbohydrates in Bifidobacterium longum and ubiquity of encoding genes.

Bifidobacterium longum can be isolated from human faeces, some strains being considered probiotics. B. longum NIZO B667 produces an exo-acting alpha-L-arabinofuranosidase, AbfB, previously purified by us, that releases L-arabinose from arabinan and arabinoxylan. This activity was subjected to two-seven-fold induction by L-arabinose, D-xylose, L-arabitol and xylitol and to repression by glucose. Maximum activity was obtained at 48 h incubation except for D-xylose that was at 24 h. High concentrations (200 mM) of L-arabitol also caused repression of the arabinofuranosidase. A unique band of activity showing the same migration pattern as the purified AbfB was found in zymograms of cell free extracts, indicating that the activity was likely due to this sole enzyme. The assessment of the influence of inducers and repressors on the activity of AbfB and on the expression of the abfB gene by real time PCR indicated that regulation was transcriptional. DNA amplifications using a pair of degenerated primers flanking an internal fragment within alpha-L-arabinofuranosidase genes of the family 51 of glycoside hydrolases evidenced that these enzymes are widespread in Bifidobacterium. The aminoacidic sequences of bifidobacteria included a fragment of four to six residues in the position 136-141 that was absent in other microorganisms.

Arabinose↗

PubMed2006

Molecular analysis of tet(W) gene-mediated tetracycline resistance in dominant intestinal Bifidobacterium species from healthy humans.

tet(W) was found responsible for tetracycline resistance (MICs, 4 to > or =32 microg ml(-1)) in dominant bifidobacterial species from the gastrointestinal tracts of healthy humans. The gene from Bifidobacterium longum H66 proved to be identical over a 2.6-kbp region to the recently described tet(W) determinant of Butyrivibrio fibrisolvens.

Anti-Bacterial Agents↗

PubMed2006

Competitive exclusion of enteropathogens from human intestinal mucus by Bifidobacterium strains with acquired resistance to bile--a preliminary study.

The ability to inhibit the adhesion and to displace selected pathogens from human intestinal mucus of two Bifidobacterium strains with acquired resistance to bile, were assessed and compared with those of their bile sensitive original strains. A preliminary characterization of the macromolecules involved in the adhesion was also carried out. The inhibition of adhesion and the displacement of enteropathogens previously adhered were found to be specific, depending on the strains used. The cholate-resistant strain Bifidobacterium bifidum M6dCo, that adhered more to mucus than its original, was able to inhibit the adhesion and to displace pathogens from mucus significantly more than its original cholate-sensitive strain B. bifidum M6. Contrary to this, two strains showing similar adhesion levels, B. bifidum A1 and its bile resistant derivative B. bifidum A1dOx, did not display any differences. Different molecules appear to be involved in the adhesion of the strains B. bifidum M6 and B. bifidum M6dCo. These differences in the cellular surface may explain the differences in competitive exclusion observed between both strains.

Adaptation, Physiological↗

PubMed2006

Two membrane proteins from Bifidobacterium breve UCC2003 constitute an ABC-type multidrug transporter.

Intrinsic resistance to drugs is one of the main determining factors in bacterial survival in the intestinal ecosystem. This is mediated by, among others, multidrug resistance (MDR) transporters, membrane proteins which extrude noxious compounds with very different chemical structures and cellular targets. Two genes from Bifidobacterium breve encoding hypothetical membrane proteins with a high homology with members of the ATP-binding cassette (ABC) family of multidrug efflux transporters, were expressed separately and jointly in Lactococcus lactis. Cells co-expressing both proteins exhibited enhanced resistance levels to the antimicrobials nisin and polymyxin B. Furthermore, the drug extrusion activity in membrane vesicles was increased when both proteins were co-expressed, compared to membranes in which the proteins were produced independently. Both proteins were co-purified from the membrane as a stable complex in a 1:1 ratio. This is believed to be the first study of a functional ABC-type multidrug transporter in Bifidobacterium and contributes to our understanding of the molecular mechanisms underlying the capacity of intestinal bacteria to tolerate cytotoxic compounds.

ATP-Binding Cassette Transporters↗

PubMed2006

The F1F0-ATPase of Bifidobacterium animalis is involved in bile tolerance.

Adaptation and tolerance to bile stress are important factors for the survival of bifidobacteria in the intestinal tract. Bifidobacterium animalis is a probiotic microorganism which has been largely applied in fermented dairy foods due to its technological properties and its health-promoting effects for humans. The effect of the presence of bile on the activity and expression of F(1)F(0)-ATPase, the pool of ATP and the intracellular pH of B. animalis IPLA 4549 and its mutant with acquired resistance to bile B. animalis 4549dOx was determined. The bile-resistant mutant tolerated the acid pH better than its parent strain. Bile induced the expression of the F(1)F(0)-ATPase and increased the membrane-bound H(+)-ATPase activity, in both parent and mutant strains. In acidic conditions (pH 5.0), the expression and the activity of this enzyme were higher in the mutant than in the parent strain when cells were grown in the absence of bile. Total ATP content was higher for the mutant in the absence of bile, whereas the presence of bile induced a decrease of intracellular ATP levels, which was much more pronounced for the parent strain. At pH 4.0, and independently on the presence or absence of bile, the mutant showed a higher intracellular pH than its parent strain. These findings suggest that the bile-adapted B. animalis strain is able to tolerate bile by increasing the intracellular ATP reserve, and by inducing proton pumping by the F(1)F(0)-ATPase, therefore tightly regulating the internal pH, and provide a link between the physiological state of the cell and the response to bile.

Adenosine Triphosphate↗

PubMed2006

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis strains isolated between 1936 and 1995.

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strains.

Bacterial Proteins↗

PubMed2006

Fluorescence spectroscopy: a rapid tool for assessing tetracycline resistance in Bifidobacterium longum.

The tetracycline uptake kinetics of 35 Bifidobacterium longum strains isolated from the human gastrointestinal tract were examined by fluorescence spectroscopy, and the suitability of the technique as a screening tool of tetracycline resistance or susceptibility was determined. The strains were first grouped into three classes based on their corresponding minimum inhibitory concentrations (MICs) of tetracycline, as established by the microdilution method: susceptible (MICs or=32 microg mL(-1)). The kinetics of tetracycline uptake for the strains in each resistance group were then analyzed over a 20 min period by fluorescence spectroscopy (absorbance wavelength 524 nm, excitation wavelength 400 nm) in a buffer system containing 100 microg mL(-1) tetracycline. Principal component analysis and factorial discriminant analysis of the results showed excellent distinction among susceptible, semi-resistant, and resistant strains. The proposed method provides a powerful and convenient means of rapidly screening tetracycline resistance in B. longum.

Bifidobacterium↗

PubMed2006

Exopolysaccharides produced by probiotic strains modify the adhesion of probiotics and enteropathogens to human intestinal mucus.

Exopolysaccharides (EPSs) are exocellular polymers present in the surface of many bacteria, including Lactobacillus and Bifidobacterium. The genome sequence of several strains revealed the presence of EPS-encoding genes. However, the physiological role that EPSs play in the bacterial ecology still remains uncertain. In this study, we have assessed the effect of EPSs produced by Lactobacillus rhamnosus GG, Bifidobacterium longum NB667, and Bifidobacterium animalis IPLA-R1 on the adhesion of probiotic and enteropathogen strains to human intestinal mucus. The EPS fraction GG had no significant effect on the adhesion of L. rhamnosus GG and B. animalis IPLA-R1. However, the EPS fractions NB667 and IPLA-R1 significantly reduced the adherence of both probiotic strains. In contrast, the three EPS fractions increased the adhesion of Enterobacter sakazakii ATCC 29544 and Escherichia coli NCTC 8603. Higher adherence of Salmonella enterica serovar Typhimurium ATCC 29631 and Clostridium difficile ATCC 9689 was detected in the presence of the EPS fractions GG and NB667. In general, these effects were obtained at EPS concentrations of up to 5 mg/ml, and they were EPS dose dependent. The competitive exclusion of probiotics in the presence of EPS could suggest the involvement of these biopolymers in the adhesion to mucus. The increase in the adherence of enteropathogens could be explained if components of the pathogen surface are able to bind to specific EPSs and the bound EPSs are able to adhere to mucus. To the best of our knowledge, this is the first work reporting the effect of EPSs from probiotics on bacterial adhesion properties.

Antibiosis↗

PubMed2005

Ability of Bifidobacterium strains with acquired resistance to bile to adhere to human intestinal mucus.

The hydrophobicity and capacity to adhere to human intestinal mucus of Bifidobacterium strains with acquired resistance to bile were assessed and compared with those of their more sensitive original strains. The resistant variants used were previously obtained [Int. J. Food Microbiol. 82 (2003) 191; Int. J. Food Microbiol. 94 (2004) 79] by progressive adaptation of originally more sensitive strains to gradually increasing concentrations of bile. In five out of the seven groups of original and bile-resistant variants tested the resistant strains showed higher adhesion levels to human mucus (range between 1.4- and 4-fold) than their corresponding original strains. However, in the presence of physiologic concentrations of bile (0.3%, w/v) the adhesion level of all Bifidobacterium strains dropped between 7% and 74%, depending on the strain. In spite of this, the adhesion capability of three bile-resistant variants remained higher than that of their originals. Hydrophobicity evidenced considerable variability; in four out of the seven bile-resistant strains it was higher than in the original strains, although no direct correlation between adhesion and hydrophobicity could be established. It was concluded that the acquisition of bile resistance by our Bifidobacterium strains promoted changes in hydrophobicity and in the adhesion of these microorganisms to human intestinal mucus.

Adaptation, Physiological↗

PubMed2005

Macrolide resistance mediated by a Bifidobacterium breve membrane protein.

A gene coding for a hypothetical membrane protein from Bifidobacterium breve was expressed in Lactococcus lactis. Immunoblotting demonstrated that this protein is located in the membrane. Phenotypical changes in sensitivity towards 21 antibiotics were determined. The membrane protein-expressing cells showed higher levels of resistance to several macrolides.

Anti-Bacterial Agents↗

PubMed2005

A bile salt-resistant derivative of Bifidobacterium animalis has an altered fermentation pattern when grown on glucose and maltose.

The growth of Bifidobacterium animalis subsp. lactis IPLA 4549 and its derivative with acquired resistance to bile, B. animalis subsp. lactis 4549dOx, was evaluated in batch cultures with glucose or the glucose disaccharide maltose as the main carbon source. The acquisition of bile salt resistance caused a change in growth pattern for both sugars, which mainly resulted in a preferential use of maltose compared to glucose, whereas the mother strain used both carbohydrates in a similar way. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses were performed to determine the amounts of glucose consumption and organic acid and ethanol formation from glucose by buffered resting cells taken at different points during growth. Resting cells of the bile-adapted strain generally consumed less glucose than those of the nonadapted one but showed an enhanced production of ethanol and higher acetic acid-to-lactic acid as well as formic acid-to-lactic acid ratios. These findings suggest a shift in the catabolism of carbohydrates promoted by the acquisition of bile resistance that may cause changes in the redox potential and improvements in the cellular ATP yield.

Bifidobacterium↗

PubMed2005

Proteomic analysis of global changes in protein expression during bile salt exposure of Bifidobacterium longum NCIMB 8809.

Adaptation to and tolerance of bile stress are among the main limiting factors to ensure survival of bifidobacteria in the intestinal environment of humans. The effect of bile salts on protein expression patterns of Bifidobacterium longum was examined. Protein pattern comparison of strains grown with or without bile extract allowed us to identify 34 different proteins whose expression was regulated. The majority of these proteins were induced after both a minor (0.6 g liter(-1)) and a major (1.2 g liter(-1)) exposure to bile. These include general stress response chaperones, proteins involved in transcription and translation and in the metabolism of amino acids and nucleotides, and several enzymes of glycolysis and pyruvate catabolism. Remarkably, xylulose 5-phosphate/fructose 6-phosphate phosphoketolase, the key enzyme of the so-called bifidobacterial shunt, was found to be upregulated, and the activity on fructose 6-phosphate was significantly higher for protein extracts of cells grown in the presence of bile. Changes in the levels of metabolic end products (acetate and lactate) were also detected. These results suggest that bile salts, to which bifidobacteria are naturally exposed, induce a complex physiological response rather than a single event in which proteins from many different functional categories take part. This study has extended our understanding of the molecular mechanism underlying the capacity of intestinal bifidobacteria to tolerate bile.

Aldehyde-Lyases↗

PubMed2005

Acquisition of bile salt resistance promotes antibiotic susceptibility changes in bifidobacterium.

The effect of acquired resistance to bile on the antimicrobial sensitivity of two Bifidobacterium strains (Bifidobacterium animalis IPLA4549 and Bifidobacterium longum NIZO B667) was studied. The MICs of 23 different antibiotics belonging to the most clinically important groups were determined by using the Etest method, which comprises nonporous plastic strips calibrated with a predefined gradient of antibiotic concentrations covering 15 twofold dilutions. The strains were sensitive to most antibiotics assayed, although they tolerated relatively high concentrations of gentamicin, kanamycin, streptomycin, polymyxin B, and ciprofloxacin (from 32 to more than 1,024 microg/ml). One of the bile-adapted strains was more strongly resistant to ceftazidime than was its parent bile-sensitive strain, and the other bile-adapted strain had increased resistance to tetracyclines. Therefore, to test the possibility that the acquisition of stable resistance to bile could be associated with a general increase in resistance to some antibiotics, we analyzed the sensitivities of four additional pairs of parent strains and their bile-adapted derivatives to ceftazidime and three tetracyclines (doxycycline, minocycline, and tetracycline). Three of the bile-resistant derivatives had increased resistance to ceftazidime (more than 256-fold) compared with their parents, and two had enhanced resistance to tetracyclines (at least 12-fold). Thus, the acquisition of bile salts resistance in Bifidobacterium induced modifications of the antibiotic resistance patterns. These results suggest that adaptation of probiotics to bile could also change their potential impact on intestinal microbiota, and this possibility deserves further attention.

Adaptation, Physiological↗

PubMed2004

Effect of the adaptation to high bile salts concentrations on glycosidic activity, survival at low PH and cross-resistance to bile salts in Bifidobacterium.

Six derivatives with increased resistance to ox gall (MIC: > or = 1% w/v) and one derivative resistant to sodium cholate (MIC: 0.8% w/v) were obtained from more sensitive original Bifidobacterium strains. These microorganisms, and two additional cholate resistant derivatives obtained in a previous study (Int. J. Food Microbiol. 82 (2003) 191), were partially characterised in this study. Acquisition of resistance against a given bile salt, also conferred cross-resistance to other bile salts, and promoted an increase in the survival of these microorganisms at low pH. Bile resistance levels of derivatives were dependent on the external pH so that the resistance was lower at neutral pH values than in acidic environments. In addition, the acquisition of bile resistance induced changes on glycoside-hydrolysing activities of derivatives obtained from five out of eight original strains, with certain activities such as beta-glucosidase showing more than tenfold increases in some of these microorganisms. These data suggest that the exposure to high bile salts concentrations may have induced a synergic response on Bifidobacterium for the adaptation to the conditions of the gastrointestinal tract. This could have improved the survival at low pH in these microorganisms, the resistance to high bile salts concentrations, and the assimilation of non-digestible carbohydrates by the enhancement of some glycoside-hydrolysing activities.

Adaptation, Physiological↗

PubMed2004

Acquired resistance to bile increases fructose-6-phosphate phosphoketolase activity in Bifidobacterium.

Two Bifidobacterium strains with acquired resistance to bile were used in this study. Significant differences on membrane-associated protein profiles were found between the bile resistant derivatives and their corresponding original strains. One of the major species detected in one of the resistant derivatives had an apparent denatured molecular mass of approximately 90 kDa, and was identified as xylulose-5-phosphate/fructose-6-phosphate phosphoketolase, the key enzyme of Bifidobacterium carbohydrate catabolism. Phosphoketolase activity was considerably higher in membrane preparations and cell-free extracts of the two bile resistant derivatives. This correlated to a greater consumption rate of glucose in resistant strains. Fructose-6-phosphate phosphoketolase activity in the strain Bifidobacterium bifidum CECT4549 and its resistant derivative was found to be partially associated with the cytoplasmic membrane through weak interactions.

Adaptation, Physiological↗

PubMed2003

Characterisation of a Bifidobacterium strain with acquired resistance to cholate--a preliminary study.

The susceptibility levels against bile salts (ox gall) and sodium cholate of 19 Bifidobacterium strains from different origins ranged between 0.125% and 2.0% and 0.05% and 0.4%, respectively. Seven of these strains were subsequently selected for the isolation of sodium cholate resistant derivatives by exposure to gradually increasing concentrations of this compound. Derivatives resistant to at least 1.2% of cholate were obtained from Bifidobacterium bifidum CECT 4549 and B. bifidum M6. Further analysis of the CECT 4549 derivative indicated that the phenotype induced was stable; the enzymatic activities analysed remained unchanged and no major rearrangements of chromosomal DNA were produced. However, the cells of the resistant derivative were smaller and more regular in average size than those of the original strain. In addition, the derivative had lost the ability to use lactose, fructose and galactose, and showed reduced expression levels of two membrane proteins of 78 and 114 kDa. Finally, the resistant strain displayed higher survival and lower growth inhibition in the presence of cholate than the original strain.

Bifidobacterium↗

PubMed2003

Purification and functional characterization of a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667.

The gene encoding a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667, abfB, was cloned and sequenced. The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with alpha-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism. A histidine tag was introduced at the C terminus of AbfB, and the recombinant protein was overexpressed in Lactococcus lactis under control of the tightly regulated, nisin-inducible nisA promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure. AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70 degrees C), and it had an optimum pH of 6.0 and an optimum temperature of 45 degrees C. The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55 degrees C. Chelating and reducing agents did not have any effect on its activity, but the presence of Cu(2+), Hg(2+), and Zn(2+) markedly reduced enzymatic activity. The protein exhibited a high level of activity with p-nitrophenyl alpha-L-arabinofuranoside, with apparent K(m) and V(max) values of 0.295 mM and 417 U/mg, respectively. AbfB released L-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. No endoarabinanase activity was detected. These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of L-arabinose-containing polysaccharides.

Base Sequence↗

PubMed2002

A new experimental approach to detect long-range conformational changes transmitted between the membrane and cytosolic domains of LmrA, a bacterial multidrug transporter.

LmrA confers multidrug resistance to Lactococcus lactis by mediating the extrusion of antibiotics, out of the bacterial membrane, using the energy derived from ATP hydrolysis. Cooperation between the cytosolic and membrane-embedded domains plays a crucial role in regulating the transport ATPase cycle of this protein. In order to demonstrate the existence of a structural coupling required for the cross-talk between drug transport and ATP hydrolysis, we studied specifically the dynamic changes occurring in the membrane-embedded and cytosolic domains of LmrA by combining infrared linear dichroic spectrum measurements in the course of H/D exchange with Trp fluorescence quenching by a water-soluble attenuator. This new experimental approach, which is of general interest in the study of membrane proteins, detects long-range conformational changes, transmitted between the membrane-embedded and cytosolic regions of LmrA. On the one hand, nucleotide binding and hydrolysis in the cytosolic nucleotide binding domain cause a repacking of the transmembrane helices. On the other hand, drug binding to the transmembrane helices affects both the structure of the cytosolic regions and the ATPase activity of the nucleotide binding domain.

Adenosine Triphosphate↗