A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese

<p>Abstract</p> <p>Background</p> <p>Lipolysis is an important process of cheese ripening that contributes to the formation of flavour.<it> Propionibacterium freudenreichii </it>is the main agent of lipolysis in Emmental cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of <it>P. freudenreichii </it>type strain CIP103027^Twas recently obtained in our laboratory.</p> <p>The aim of this study was to identify as exhaustively as possible the potential esterases in <it>P. freudenreichii </it>that could be involved in the hydrolysis of acylglycerols in Emmental cheese. The proteins identified were produced in a soluble and active form by heterologous expression in <it>Escherichia coli </it>for further study of their activity and specificity of hydrolysed substrates.</p> <p>Results</p> <p>The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among <it>P. freudenreichii </it>CIP103027^Tpredicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in <it>E. coli </it>and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method.</p> <p>Conclusion</p> <p>We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a <it>P. freudenreichii </it>strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of <it>P. freudenreichii </it>involved in the lipolysis in Emmental cheese.</p

A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening.

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered

Mycobacterium canettii Infection of Adipose Tissues

Adipose tissues were shown to host Mycobacterium tuberculosis which is persisting inside mature adipocytes. It remains unknown whether this holds true for Mycobacterium canettii, a rare representative of the M. tuberculosis complex responsible for lymphatic and pulmonary tuberculosis. Here, we infected primary murine white and brown pre-adipocytes and murine 3T3-L1 pre-adipocytes and mature adipocytes with M. canettii and M. tuberculosis as a positive control. Both mycobacteria were able to infect 18–22% of challenged primary murine pre-adipocytes; and to replicate within these cells during a 7-day experiment with the intracellular inoculums being significantly higher in brown than in white pre-adipocytes for M. canettii (p = 0.02) and M. tuberculosis (p = 0.03). Further in-vitro infection of 3T3-L1 mature adipocytes yielded 9% of infected cells by M. canettii and 17% of infected cells by M. tuberculosis (p = 0.001). Interestingly, M. canettii replicated and accumulated intra-cytosolic lipid inclusions within mature adipocytes over a 12-day experiment; while M. tuberculosis stopped replicating at day 3 post-infection. These results indicate that brown pre-adipocytes could be one of the potential targets for M. tuberculosis complex mycobacteria; and illustrate differential outcome of M. tuberculosis complex mycobacteria into adipose tissues. While white adipose tissue is an unlikely sanctuary for M. canettii, it is still an open question whether M. canettii and M. tuberculosis could persist in brown adipose tissues

Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

BACKGROUND: New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. METHODS: Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. RESULTS: A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. CONCLUSION: These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB

Etude biochimique, structurale et physiologique d enzymes lipolytiques chez les Mycobactéries

Les enzymes lipolytiques chez Mycobacterium tuberculosis, l'agent étiologique de la tuberculose, restent peu étudiées malgré leur probable implication dans des fonctions physiologiques essentielles chez ce bacille. La mise à disposition de la séquence du génome de M. tuberculosis a permis d'identifier les gènes codant ces enzymes chez ce microorganisme. Mon travail de thèse a consisté, dans un premier temps, en la caractérisation biochimique, structurale et fonctionnelle d'une enzyme lipolytique potentielle, la Rv0183, chez M. tuberculosis. Nous avons démontré que la Rv0183 est une monoacylglycérol lipase localisée non seulement dans l'enveloppe mycobactérienne mais aussi dans le milieu de culture. Le rôle physiologique de la Rv0183 a été extrapolé grâce à l'étude de son homologue chez M. smegmatis, la MSMEG_0220. Cette protéine, en plus de son implication dans l'hydrolyse de lipides exogènes, semble jouer un rôle structural au niveau de l'enveloppe mycobactérienne. Dans un deuxième temps, nous nous sommes intéressés à la caractérisation de deux autres protéines secrétées par M. tuberculosis apparentées à la famille des cutinases : la Rv1984c et la Rv3452. Bien qu'elles présentent une identité de séquence de 50%, l'étude des propriétés cinétiques de ces protéines a montré une divergence dans leur spécificité de substrat. Si la Rv1984c pourrait être définie comme une lipase, la Rv3452 est une phospholipase de type A2. Etant secrétées par M. tuberculosis, une évaluation de la cytotoxicité de ces protéines a montré que contrairement à la Rv1984c, la Rv3452 était capable d'induire la lyse de macrophages suggérant une implication de cette protéine dans la virulence de M. tuberculosis. Un dernier aspect de mon travail a été d'étudier la lipolyse in situ chez M. smegmatis. En utilisant la microscopie de florescence en temps réel, nous avons suivi, l'hydrolyse des réserves intracytoplasmiques chez ce bacille placé en milieu appauvri. Ce phénomène a été bloqué par la la tétrahydrolipstatine confirmant l'implication d'enzymes lipolytiquesLipolytic enzymes in Mycobacterium tuberculosis, the etiologic agents of tuberculosis, remain poorly studied despite their probable involvement in bacteria cell life. Genomic DNA from M. tuberculosis analysis has permitted the identification of genes encoding lipolytic enzymes. My thesis work was consisted, on the one hand, of biochemical, structural and physiological characterization of Rv0183, a putative lipolytic enzyme from M. tuberculosis. We have demonstrated that Rv0183 is a monoacylglycerol lipase which is located not only in the cell envelope but also in the culture medium filtrate. The physiological function of Rv0183 was assessed by studying its ortholog in M. smegmatis, the MSMEG_0220. It has been shown that this protein was involved not only in the exogenous lipid degradation but also in the cell envelop architecture. On the other hand, we have investigated on the biochemical characterization of two secreted proteins of M. tuberculosis belonging to the cutinase family, Rv1984c and Rv3452. Despite 50% identity in their amino acid sequence, these enzymes show distinct substrate specificities. Rv1984c hydrolyses carboxyl esters and monoacylglycerols whereas Rv3452 behaves as a phospholipase A2 able to induce macrophage lysis suggesting an implication of this enzyme in M. tuberculosis virulence. Finally, we have studied the in situ lipolysis in M. smegmatis. Thanks to time-lapse fluorescence microscopy, we have monitored the intracytoplasmic lipids hydrolysis in this bacterium incubated under nutrient starvation. Lipid degradation was inhibited by tetrahydrolipstatin confirming the involvement of lipolytic enzymes in this processAIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Lipid Droplets Breakdown: Adipose Triglyceride Lipase Leads the Way

International audiencePreviously in Current Protein & Peptide Science, Cerk et al. (2018) reported an updated review of concepts and knowledge regarding structure, function and regulatory mechanism of the adi-pose triglyceride lipase (ATGL), one of the key-enzyme required for intracellular lipolysis. Until recently, lipid droplets (LDs) were just considered as neutral lipid storage sites, thus providing energy through lipolysis and β-oxidation pathway when required during stressful conditions [1]. Nowadays, it is clearly established that LDs are well-organized and extremely dynamic cellular organelles, conserved in eubacteria, fungi, plants and animals plants and animals where they are essential for lipid homeostasis and energy maintenance [2]. Alteration of these two critical physiological processes can lead to important metabolic disorders [3]. The " conventional snapshot representation " of LDs remains a suitable model for defining their general composition which is based on a central organic core of neutral lipids (mainly triacylglycerol (TAG) and sterol esters) surrounded by a phospholipid monolayer [4], associated with a wide range of structural [5], enzymatic [6] and membrane-trafficking proteins [7]. However, we also know that proteins and lipid species of such structures can be extremely diversified depending on the different cell types or metabolic status. Interestingly, LDs anabolism and catabo-lism are well-balanced, and tightly-controlled biological mechanisms involving a large number of actors at both tran-scriptional, translational and post-translational levels [8]. TAG-containing LDs breakdown is achieved during extended starvation period or enhanced energy demand, and this phenomenon is mainly mediated by three distinct lipolytic enzymes (i.e., the adipose triglyceride lipase (ATGL), the hormone-sensitive lipase (HSL) and the mono-glyceride lipase (MGL)) that act sequentially to finally generate free fatty acids (FFA) and glycerol molecules [8]. Since ATGL catalyzes the first step of this essential lipolytic pathway , it is thus crucial to fully define the physiological func-tion(s) and structural properties of this protein, but also to get insights onto the regulatory mechanisms governing ATGL action towards LDs [9]

Characterization of RV1683, a bifunctional enzyme involved in storage and mobilization of mycobacterial lipid droplets

National audienc

Etude In vitro de Phospholipases mycobactériennes impliquées dans la virulence

Les phospholipases et en particulier les phospholipases C sont d'importants facteurs de virulence chez de nombreuses bactéries pathogènes (C. perfringens, B. Cereus et P. aeruginosa). Cependant, peu de choses sont connues sur l'implication de ces enzymes dans le processus de virulence des mycobactéries. Bien que l'étude des mutants des phospholipases C de M. tuberculosis dans un modèle d'infection chez la souris ait permis de proposer une implication de ces protéines dans la virulence de ce bacille, leurs propriétés biochimiques, leur mode d'action et leur rôle physiologique exact restent à élucider. Ce manque de données biochimiques sur les phospholipases mycobactériennes peuvent être attribuée à la difficulté à produire et à purifier des quantités importantes de ces enzymes. Dans le but de mieux caractériser le rôle physiologique des phospholipase mycobactériennes, l'objectif de ma thèse a été de mettre au point des conditions d'expression hétérologue permettant la production des phospholipases C mycobactériennes recombinantes (rPLC) dans différents systèmes d'expression (E. coli, Pichia pastoris et baculovirus/cellules d'insectes). Ces systèmes d'expression n'ayant pas donné des résultats satisfaisants, nous avons développé une méthode efficace d'expression de ces protéines en utilisant M. smegmatis.Ce système d'expression nous a permis de produire et de purifier les quate PLC (PLC-A, PLC-B, PLC-C et PLC-D) de M. tuberculosis et la PLC de M. Abscessus sous forme soluble et active. Nous avons pour la première fois montré que ces protéines purifiées avaient un effet cytotoxique sur les macrophages de souris en culture mais ne présentaient aucune activité hémolytique. en utilisant des marquages radioactifs, nous avons confirmé que l'effet cytotoxique observé était lité à l'hydrolyse des phospholipides des membranaires des cellules hôtes. Pour la première fois, nous avons pu confirmer que ces PLC sont directement impliquées dans le processus d'infection et de virulence.Un autre aspect de mon travail de thèse a concerné l'étude de deux autres protéines sécrétées par M. tuberculosis appartenant à la famille des cutinases : la Rv1984c et la Rv3452. Après les avoir produites et purifiées chez E. Coli, nouq avons montré que malgré ces deux protéines présentent 50% d'identité de séquence en acides aminés, elles ont des spécificités de substrat différentes et probablement un rôle physiologique différent. La Rv1984c est une lipase capacle d'hydrolyser des lipides à chaines moyennes, alors que la Rv3452 est une phospholipase de type A2 et est capable d'induire la lyse de macrophage de souris en culture.Phospholipases, particularly phospholipases C, are important virulence factors in several pathogenic bacteria (C. perfringens, B. cereus, L. monocytogenese and P. aeruginosa). However, little is know on the involvement of thses enzymes in mycobacteria pathogenesis. Although study on M. tuberculosis phospholipases C mutants in a mouse aerosol model of infection gave rise to the contribution of these proteins in virulence process, but their exact biochemical properties, mechanism of action and physiological role remain to be elucidated. This lack of data on mycobacterial phospholipases is mainly due to the difficulty to produce and purify these enzymes in large scale.With the aim to better characterise the physiological role of mycobacterial phospholipases, the main challenge of my thesis was to develop an efficient method for expression and purification of recombinant mycobacterial phospholipases C. Since no satisfactory results have been obtained with standard expression systems (E. coli, Pichia pastoris and baculovirus / insect cells), we develop a robust expression technique for these proteins using M. smegmatis as expression system.This allowed us to produce and purify all four PLC (PLC-A, PLC-B, PLC-C and PLC-D) of M. tuberculosis and the PLC of M. abscessus in soluble and active form. For the first time, we have show, that purified proteins have cytotoxic effect on mouse macrophages but have not haemolytic activity. Using radiolabelled lipids, we have confirmed that this first direct evidence that PLC are involved in infection and virulence processes. Another aspect of my thesis work concerned the study of two other secreted proteins of M. tuberculosis belonging to the cutinase family : the Rv 1984c ant the Rv3452. Recombinant proteins obtains in E. coli were found to have distinct substrate specificities and most likely distict physiological role, despite showing 50% amino acids sequence identity. Rv1984c is a lipase and is able to hydrolyse lipids with medium chains lengthn whereas Rv3452 is type A2, phospholipase and i able to induce macrophage lysis.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Identification of Residues Involved in Substrate Specificity and Cytotoxicity of Two Closely Related Cutinases from <em>Mycobacterium tuberculosis</em>

International audienceThe enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. Bysite-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production

Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage.

Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth