Patricia Anglade

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.

Low-pH Adaptation and the Acid Tolerance Response of Bifidobacterium longum Biotype longum

ABSTRACT Bifidobacteria are one of the main microbial inhabitants of the human colon. Usually administered in fermented dairy products as beneficial microorganisms, they have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in Bifidobacterium longum biotype longum NCIMB 8809 and its acid-pH-resistant mutant B. longum biotype longum 8809dpH were studied. Comparison of protein maps, and protein identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. These included general stress response chaperones and proteins involved in transcription and translation as well as in carbohydrate and nitrogen metabolism, among others. Significant differences in the levels of metabolic end products and in the redox status of the cells were also detected between the wild-type strain and its acid-pH-resistant mutant in response to, or as a result of, adaptation to acid. Remarkably, the results of this work indicated that adaptation and response to low pH in B. longum biotype longum involve changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm, likely coming from amino acid deamination, and a decrease of the bile salt hydrolase activity.

Adaptation and Response of Bifidobacterium animalis subsp. lactis to Bile: a Proteomic and Physiological Approach

ABSTRACT Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cells physiology.

Lactic acid bacteria and proteomics: current knowledge and perspectives.

Lactic acid bacteria (LAB) are widely used in the agro-food industry. Some of the LAB also participate in the natural flora in humans and animals. We review here proteomic studies concerning LAB. Two methods of research can be distinguished. In the first one, a systematic mapping of proteins is attempted, which will be useful for taxonomy and to function assignment of proteins. The second one focuses particularly on proteins whose synthesis is induced by various environmental situations or stresses. However, both approaches are complementary and will give new insights for the use of bacteria in industry, in human health and in the struggle against bacterial pathogens. Interest in LAB is growing, showing thus an increasing concern of their rational use and one can foresee in the near future an increasing use of proteomics as well as genomics.

Proteome Analyses of Heme-Dependent Respiration in Lactococcus lactis: Involvement of the Proteolytic System

Sugar fermentation was long considered the sole means of energy metabolism available to lactic acid bacteria. We recently showed that metabolism of Lactococcus lactis shifts progressively from fermentation to respiration during growth when oxygen and heme are available. To provide insights into this phenomenon, we compared the proteomic profiles of L. lactis under fermentative and respiratory growth conditions in rich medium. We identified 21 proteins whose levels differed significantly between these conditions. Two major groups of proteins were distinguished, one involved in carbon metabolism and the second in nitrogen metabolism. Unexpectedly, enzymes of the proteolytic system (PepO1 and PepC) which are repressed in rich medium in fermentation growth were induced under respiratory conditions despite the availability of free amino acids. A triple mutant (dtpT dtpP oppA) deficient in oligopeptide transport displayed normal respiration, showing that increased proteolytic activity is not an absolute requirement for respiratory metabolism. Transcriptional analysis confirmed that pepO1 is induced under respiration-permissive conditions. This induction was independent of CodY, the major regulator of proteolytic functions in L. lactis. We also observed that pepO1 induction is redox sensitive. In a codY mutant, pepO1 expression was increased twofold in aeration and eightfold in respiration-permissive conditions compared to static conditions. These observations suggest that new regulators activate proteolysis in L. lactis, which help to maintain the energetic needs of L. lactis during respiration.

Intraspecies Genomic Diversity and Natural Population Structure of the Meat-Borne Lactic Acid Bacterium Lactobacillus sakei

ABSTRACT Lactobacillus sakei is a food-borne bacterium naturally found in meat and fish products. A study was performed to examine the intraspecies diversity among 73 isolates sourced from laboratory collections in several different countries. Pulsed-field gel electrophoresis analysis demonstrated a 25% variation in genome size between isolates, ranging from 1,815 kb to 2,310 kb. The relatedness between isolates was then determined using a PCR-based method that detects the possession of 60 chromosomal genes belonging to the flexible gene pool. Ten different strain clusters were identified that had noticeable differences in their average genome size reflecting the natural population structure. The results show that many different genotypes may be isolated from similar types of meat products, suggesting a complex ecological habitat in which intraspecies diversity may be required for successful adaptation. Finally, proteomic analysis revealed a slight difference between the migration patterns of highly abundant GapA isoforms of the two prevailing L. sakei subspecies (sakei and carnosus). This analysis was used to affiliate the genotypic clusters with the corresponding subspecies. These findings reveal for the first time the extent of intraspecies genomic diversity in L. sakei. Consequently, identification of molecular subtypes may in the future prove valuable for a better understanding of microbial ecosystems in food products.

Identification of the binding sites of benzyl penicilloyl, the allergenic metabolite of penicillin, on the serum albumin molecule

Tryptic digests of fragment A299 585 of penicilloylated serum albumin obtained from two penicillin‐treated patients or prepared by in vitro conjugation, were analyzed by a tandem immunoaffinity reversed‐phase HLPC. Determinations of benzyl penicilloyl groups (BPO) were performed on the different fractions. Three BPO containing peptides were identified by their amino acid sequence and the bound BPO were located on lysines 432, 541 and 545. Six major BPO binding sites were thus identified on the whole albumin molecule. All of them are lysine residues and correspond to a limited number of definite structures in which lysine and serine residues appear to be closely associated.

A Preliminary Analysis of Bifidobacterium longum Exported Proteins by Two-Dimensional Electrophoresis

Extracellular proteins of Bifidobacterium longum may mediate important interactions with the host. Here, we report on a comprehensive analysis of such proteins by using protein-free culture conditions and two-dimensional gel electrophoresis followed by mass spectrometry for protein identification. Seventeen proteins were detected in the culture supernatant, and 14 of them could be identified. Among these were 3 hypothetical solute-binding proteins of ABC transporters, an invasion-associated protein homolog, putative enzymes catalyzing cell wall turnover, several polypeptides with similarity to bacterial conjugation proteins, and 3 proteins of unknown function. Surprisingly, aldolase, usually considered as a cytoplasmic protein, was found in the culture supernatant. All proteins, excluding aldolase, were predicted to contain a signal peptide and a signal peptide cleavage site in their immature form. Some of the excreted proteins are interesting targets for further genetic and physiological studies.

Binding of benzyl penicilloyl to human serum albumin Evidence for a highly reactive region at the junction of domains 1 and 2 of the albumin molecule

Tryptic digests of fragment C124–298 of penicilloylated serum albumin, obtained from a penicillin‐treated patient or prepared by in vitro conjugation, were analyzed by HPLC. Determinations of benzyl penicilloyl groups (BPO) were performed on the different fractions. Three BPO‐containing peptides were identified by their amino acid sequence and the bound BPO was located on lysines 190, 195 and 199 and serine 193. These four main BPO‐binding sites are all located on a very short region (10 amino acid residues) of the albumin molecule at the junction of domains 1 and 2.

Analysis and characterization of glutathione S-transferase subunits from wheat (Triticum aestivum L.)

Abstract The total subunit complement of glutathione S -transferases (GSTs) of wheat ( Triticum aestivum L.) was isolated by glutathione-agarose affinity chromatography. The proteins bound to the affinity column were analysed by SDS-PAGE, which separated six to seven protein bands. Reverse phase-HPLC analysis revealed six major and about 15 minor peaks. The amounts of three major components were increased upon treatment with the herbicide safener naphthalic anhydride. The six major components were isolated. Their relative molecular masses, determined by electrospray ionization mass spectrometry, were between 23 140±2 and 24 957±3 Da. The sequences of the 20 N-terminal amino acids were also determined, and revealed strong similarities with GST subunits from various monocotyledonous plants. Wheat GST subunits can be assigned to two groups: constitutive subunits, with identical N-termini and molecular masses around 23.2 kDa, and constitutive and inducible subunits, with some differences in N-terminal sequences, and molecular masses around 24.9 kDa.