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1.
Sci Rep ; 10(1): 8492, 2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444655

RESUMO

Most currently used antibiotics originate from Streptomycetes and phosphate limitation is an important trigger of their biosynthesis. Understanding the molecular processes underpinning such regulation is of crucial importance to exploit the great metabolic diversity of these bacteria and get a better understanding of the role of these molecules in the physiology of the producing bacteria. To contribute to this field, a comparative proteomic analysis of two closely related model strains, Streptomyces lividans and Streptomyces coelicolor was carried out. These strains possess identical biosynthetic pathways directing the synthesis of three well-characterized antibiotics (CDA, RED and ACT) but only S. coelicolor expresses them at a high level. Previous studies established that the antibiotic producer, S. coelicolor, is characterized by an oxidative metabolism and a reduced triacylglycerol content compared to the none producer, S. lividans, characterized by a glycolytic metabolism. Our proteomic data support these findings and reveal that these drastically different metabolic features could, at least in part, due to the weaker abundance of proteins of the two component system PhoR/PhoP in S. coelicolor compared to S. lividans. In condition of phosphate limitation, PhoR/PhoP is known to control positively and negatively, respectively, phosphate and nitrogen assimilation and our study revealed that it might also control the expression of some genes of central carbon metabolism. The tuning down of the regulatory role of PhoR/PhoP in S. coelicolor is thus expected to be correlated with low and high phosphate and nitrogen availability, respectively and with changes in central carbon metabolic features. These changes are likely to be responsible for the observed differences between S. coelicolor and S. lividans concerning energetic metabolism, triacylglycerol biosynthesis and antibiotic production. Furthermore, a novel view of the contribution of the bio-active molecules produced in this context, to the regulation of the energetic metabolism of the producing bacteria, is proposed and discussed.

2.
Nat Commun ; 11(1): 2144, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358542

RESUMO

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology.


Assuntos
Saccharomycetales/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Biotecnologia/métodos , Genoma Fúngico/genética , Filogenia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Sci Rep ; 10(1): 2865, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071324

RESUMO

Antibiotic persistence is a transient phenotypic state during which a bacterium can withstand otherwise lethal antibiotic exposure or environmental stresses. In Escherichia coli, persistence is promoted by the HipBA toxin-antitoxin system. The HipA toxin functions as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neutralizes the toxin. E. coli HipA inactivates the glutamyl-tRNA synthetase GltX, which inhibits translation and triggers the highly conserved stringent response. Although hipBA operons are widespread in bacterial genomes, it is unknown if this mechanism is conserved in other species. Here we describe the functions of three hipBA modules in the alpha-proteobacterium Caulobacter crescentus. The HipA toxins have different effects on growth and macromolecular syntheses, and they phosphorylate distinct substrates. HipA1 and HipA2 contribute to antibiotic persistence during stationary phase by phosphorylating the aminoacyl-tRNA synthetases GltX and TrpS. The stringent response regulator SpoT is required for HipA-mediated antibiotic persistence, but persister cells can form in the absence of all hipBA operons or spoT, indicating that multiple pathways lead to persister cell formation in C. crescentus.

4.
Mol Plant Microbe Interact ; 33(2): 200-211, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31567040

RESUMO

The Gram-negative bacterium Ralstonia solanacearum, the causal agent of bacterial wilt, is a worldwide major crop pathogen whose virulence strongly relies on a type III secretion system (T3SS). This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. To date, very few data are available in plant-pathogenic bacteria concerning the role played by type III secretion (T3S) regulators at the posttranslational level. We have demonstrated that HpaP, a putative T3S substrate specificity switch protein of R. solanacearum, controls T3E secretion. To better understand the role of HpaP on T3S control, we analyzed the secretomes of the GMI1000 wild-type strain as well as the hpaP mutant using a mass spectrometry experiment (liquid chromatography tandem mass spectrometry). The secretomes of both strains appeared to be very similar and highlighted the modulation of the secretion of few type III substrates. Interestingly, only one type III-associated protein, HrpJ, was identified as specifically secreted by the hpaP mutant. HrpJ appeared to be an essential component of the T3SS, essential for T3S and pathogenicity. We further showed that HrpJ is specifically translocated in planta by the hpaP mutant and that HrpJ can physically interact with HpaP. Moreover, confocal microscopy experiments demonstrated a cytoplasmic localization for HrpJ once in planta. When injected into Arabidopsis thaliana leaves, HrpJ is able to trigger a necrosis on 16 natural accessions. A genome-wide association mapping revealed a major association peak with 12 highly significant single-nucleotide polymorphisms located on a plant acyl-transferase.


Assuntos
Arabidopsis , Proteínas de Bactérias , Ralstonia solanacearum , Virulência , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Estudo de Associação Genômica Ampla , Doenças das Plantas/microbiologia , Ralstonia solanacearum/genética , Ralstonia solanacearum/patogenicidade , Virulência/genética
5.
Cell Rep ; 29(12): 3974-3982.e4, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851927

RESUMO

The essentiality of fatty acid synthesis (FASII) products in the human pathogen Staphylococcus aureus is the underlying rationale for FASII-targeted antimicrobial drug design. Reports of anti-FASII efficacy in animals support this choice. However, restricted test conditions used previously led us to investigate this postulate in a broader, host-relevant context. We report that S. aureus rapidly adapts to FASII antibiotics without FASII mutations when exposed to host environments. FASII antibiotic administration upon signs of infection, rather than just after inoculation as commonly practiced, fails to eliminate S. aureus in a septicemia model. In vitro, serum lowers S. aureus membrane stress, leading to a greater retention of the substrates required for environmental fatty acid (eFA) utilization: eFAs and the acyl carrier protein. In this condition, eFA occupies both phospholipid positions, regardless of anti-FASII selection. Our results identify S. aureus membrane plasticity in host environments as a main limitation for using FASII antibiotics in monotherapeutic treatments.

6.
Front Microbiol ; 10: 1329, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31275266

RESUMO

Protein phosphorylation especially on serine/threonine/tyrosine residues are frequent in many bacteria. This post-translational modification has been associated with pathogenicity and virulence in various species. However, only few data have been produced so far on generally recognized as safe bacteria used in food fermentations. A family of kinases known as Hanks-type kinases is suspected to be responsible for, at least, a part of these phosphorylations in eukaryotes as in bacteria. The objective of our work was to establish the first phosphoproteome of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy fermentations in order to identified the proteins and pathways tagged by Ser/Thr/Tyr phosphorylations. In addition, we have evaluated the role in this process of the only Hanks-type kinase encoded in the S. thermophilus genome. We have constructed a mutant defective for the Hanks type kinase in S. thermophilus and established the proteomes and phosphoproteomes of the wild type and the mutant strains. To do that, we have enriched our samples in phosphopeptides with titane beads and used dimethyl tags to compare phosphopeptide abundances. Peptides and phosphopeptides were analyzed on a last generation LC-MS/MS system. We have identified and quantified 891 proteins representing half of the theoretical proteome. Among these proteins, 106 contained phosphorylated peptides. Various functional groups of proteins (amino acid, carbon and nucleotide metabolism, translation, cell cycle, stress response, …) were found phosphorylated. The phosphoproteome was only weakly reduced in the Hanks-type kinase mutant indicating that this enzyme is only one of the players in the phosphorylation process. The proteins that are modified by the Hanks-type kinase mainly belong to the divisome.

7.
Ecotoxicol Environ Saf ; 182: 109421, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31301592

RESUMO

The environmental contamination of soil by metal oxide nanomaterials is a growing global concern because of their potential toxicity. We investigated the effects of Mg doped ZnO (Mg-nZnO) nanoparticles on a model soil microorganism Bacillus subtilis. Mg-nZnO exhibited only a moderate toxic effect on B. subtilis vegetative cells but was able to prevent biofilm formation and destroy already formed biofilms. Similarly, Mg-nZnO (≤1 mg/mL) was moderately toxic towards Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and murine macrophages. Engineered Mg-nZnO produced H2O2 and O2•- radicals in solutions of various salt and organic molecule compositions. A quantitative proteomic analysis of B. subtilis membrane proteins showed that Mg-nZnO increased the expression of proteins involved in detoxification of ROS, translation and biofilm formation. Overall, our results suggest that Mg-nZnO released into the environment may hinder the spreading, colonization and biofilm formation by B. subtilis but also induce a mechanism of bacterial adaptation.


Assuntos
Bacillus subtilis/efeitos dos fármacos , Nanopartículas/toxicidade , Poluentes do Solo/toxicidade , Óxido de Zinco/toxicidade , Animais , Biofilmes , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Camundongos , Óxidos/metabolismo , Proteômica , Solo , Microbiologia do Solo , Staphylococcus aureus
8.
Sci Rep ; 9(1): 5243, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30918277

RESUMO

In a previous study on camel milk from Kazakhstan, we reported the occurrence of two unknown proteins (UP1 and UP2) with different levels of phosphorylation. Here we show that UP1 and UP2 are isoforms of camel αs2-CN (αs2-CNsv1 and αs2-CNsv2, respectively) arising from alternative splicing events. First described as a 178 amino-acids long protein carrying eight phosphate groups, the major camel αs2-CN isoform (called here αs2-CN) has a molecular mass of 21,906 Da. αs2-CNsv1, a rather frequent (35%) isoform displaying a higher molecular mass (+1,033 Da), is present at four phosphorylation levels (8P to 11P). Using cDNA-sequencing, αs2-CNsv1 was shown to be a variant arising from the splicing-in of an in-frame 27-nucleotide sequence encoding the nonapeptide ENSKKTVDM, for which the presence at the genome level was confirmed. αs2-CNsv2, which appeared to be present at 8P to 12P, was shown to include an additional decapeptide (VKAYQIIPNL) revealed by LC-MS/MS, encoded by a 3'-extension of exon 16. Since milk proteins represent a reservoir of biologically active peptides, the molecular diversity generated by differential splicing might increase its content. To evaluate this possibility, we searched for bioactive peptides encrypted in the different camel αs2-CN isoforms, using an in silico approach. Several peptides, putatively released from the C-terminal part of camel αs2-CN isoforms after in silico digestion by proteases from the digestive tract, were predicted to display anti-bacterial and antihypertensive activities.

9.
BMC Genet ; 20(1): 14, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696406

RESUMO

BACKGROUND: Whey acidic protein (WAP) is a major protein identified in the milk of several mammalian species with cysteine-rich domains known as four-disulfide cores (4-DSC). The organization of the eutherian WAP genes is highly conserved through evolution. It has been proposed that WAP could play an important role in regulating the proliferation of mammary epithelial cells. A bacteriostatic activity was also reported. Conversely to the other mammalian species expressing WAP in their milk, camel WAP contains 4 additional amino acid residues at the beginning of the second 4-DSC domain, introducing a phosphorylation site. The aim of this study was to elucidate the origin of this specificity, which possibly impacts its physiological functions. RESULTS: Using LC-ESI-MS, we identified in Camelus bactrianus from Kazakhstan a phosphorylated whey protein, exhibiting a molecular mass (12,596 Da), 32 Da higher than the original WAP (12,564 Da) and co-eluting with WAP. cDNA sequencing revealed a transition G/A, which modifies an amino acid residue of the mature protein (V12 M), accounting for the mass difference observed between WAP genetic variants. We also report the existence of two splicing variants of camel WAP precursors to mRNA, arising from an alternative usage of the canonical splice site recognized as such in the other mammalian species. However, the major camel WAP isoform results from the usage of an unlikely intron cryptic splice site, extending camel exon 3 upstream by 12-nucleotides encoding the 4 additional amino acid residues (VSSP) in which a potentially phosphorylable Serine residue occurs. Combining protein and cDNA sequences with genome data available (NCBI database), we report another feature of the camel WAP gene which displays a very rare GC-AG type intron. This result was confirmed by sequencing a genomic DNA fragment encompassing exon 3 to exon 4, suggesting for the GC donor site a compensatory effect in terms of consensus at the acceptor exon position. CONCLUSIONS: Combining proteomic and molecular biology approaches we report: the characterization of a new genetic variant of camel WAP, the usage of an unlikely intron cryptic splice site, and the occurrence of an extremely rare GC-AG type of intron.


Assuntos
Camelus/genética , Íntrons/genética , Leite/metabolismo , Sítios de Splice de RNA , Proteínas do Soro do Leite/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Variação Genética , Genômica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/metabolismo
10.
Sci Rep ; 8(1): 12276, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30115985

RESUMO

The increasing number of multidrug resistant bacteria raises a serious public-health concern, which is exacerbated by the lack of new antibiotics. Metal oxide nanoparticles are already applied as an antibacterial additive in various products used in everyday life but their modes of action have remained unclear. Moreover, their potential negative effects to human health are still under evaluation. We explored effects of mixed metal oxide Zn0.15Mg0.85O on Bacillus subtilis, as a model bacterial organism, and on murine macrophages. Zn0.15Mg0.85O killed planktonic bacterial cells and prevented biofilm formation by causing membrane damages, oxidative stress and metal ions release. When exposed to a sub-inhibitory amount of Zn0.15Mg0.85O, B. subtilis up-regulates proteins involved in metal ions export, oxidative stress response and maintain of redox homeostasis. Moreover, expression profiles of proteins associated with information processing, metabolism, cell envelope and cell division were prominently changed. Multimode of action of Zn0.15Mg0.85O suggests that no single strategy may provide bacterial resistance. Macrophages tolerated Zn0.15Mg0.85O to some extend by both the primary phagocytosis of nanoparticles and the secondary phagocytosis of damaged cells. Bacterial co-treatment with ciprofloxacin and non-toxic amount of Zn0.15Mg0.85O increased antibiotic activity towards B. subtilis and E. coli.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Óxido de Magnésio/química , Nanopartículas/química , Óxidos/química , Óxidos/farmacologia , Óxido de Zinco/química , Animais , Antibacterianos/toxicidade , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Biofilmes/efeitos dos fármacos , Ciprofloxacino/farmacologia , Sinergismo Farmacológico , Camundongos , Óxidos/toxicidade , Tamanho da Partícula , Plâncton/citologia , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo
11.
PLoS One ; 13(5): e0197026, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29746547

RESUMO

Nutritional suitability of milk is not only related to gross composition, but is also strongly affected by the microheterogeniety of the protein fraction. Hence, to go further into the evaluation of the potential suitability of non-bovine milks in human/infant nutrition it is necessary to have a detailed characterization of their protein components. Combining proven proteomic approaches (SDS-PAGE, LC-MS/MS and LC-ESI-MS) and cDNA sequencing, we provide here in depth characterization of the milk protein fraction of dromedary and Bactrian camels, and their hybrids, from different regions of Kazakhstan. A total 391 functional groups of proteins were identified from 8 camel milk samples. A detailed characterization of 50 protein molecules, relating to genetic variants and isoforms arising from post-translational modifications and alternative splicing events, belonging to nine protein families (κ-, αs1-, αs2-, ß-; and γ-CN, WAP, α-LAC, PGRP, CSA/LPO) was achieved by LC-ESI-MS. The presence of two unknown proteins UP1 (22,939 Da) and UP2 (23,046 Da) was also reported as well as the existence of a ß-CN short isoform (946 Da lighter than the full-length ß-CN), arising very likely in both genetic variants (A and B) from proteolysis by plasmin. In addition, we report, for the first time to our knowledge, the occurrence of a αs2-CN phosphorylation isoform with 12P groups within two recognition motifs, suggesting thereby the existence of two kinase systems involved in the phosphorylation of caseins in the mammary gland. Finally, we demonstrate that genetic variants, which hitherto seemed to be species- specific (e.g. ß-CN A for Bactrian and ß-CN B for dromedary), are in fact present both in Camel dromedarius and C. bactrianus.


Assuntos
Camelus/metabolismo , Quimera/metabolismo , Proteínas do Leite/metabolismo , Proteômica , Animais , Camelus/genética , Quimera/genética , Feminino , Cazaquistão , Espectrometria de Massas
12.
Front Microbiol ; 9: 486, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29619013

RESUMO

Bacillus subtilis cells can adopt different life-styles in response to various environmental cues, including planktonic cells during vegetative growth, sessile cells during biofilm formation and sporulation. While switching life-styles, bacteria must coordinate the progression of their cell cycle with their physiological status. Our current understanding of the regulatory pathways controlling the decision-making processes and triggering developmental switches highlights a key role of protein phosphorylation. The regulatory mechanisms that integrate the bacterial chromosome replication status with sporulation involve checkpoint proteins that target the replication initiator DnaA or the kinase phosphorelay controlling the master regulator Spo0A. B. subtilis YabA is known to interact with DnaA to prevent over-initiation of replication during vegetative growth. Here, we report that YabA is phosphorylated by YabT, a Ser/Thr kinase expressed during sporulation and biofilm formation. The phosphorylation of YabA has no effect on replication initiation control but hyper-phosphorylation of YabA leads to an increase in sporulation efficiency and a strong inhibition of biofilm formation. We also provide evidence that YabA phosphorylation affects the level of Spo0A-P in cells. These results indicate that YabA is a multifunctional protein with a dual role in regulating replication initiation and life-style switching, thereby providing a potential mechanism for cross-talk and coordination of cellular processes during adaptation to environmental change.

13.
Front Microbiol ; 8: 2168, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163446

RESUMO

A fascinating characteristic of most members of the genus Flavobacterium is their ability to move over surfaces by gliding motility. Flavobacterium psychrophilum, an important pathogen of farmed salmonids worldwide, contains in its genome the 19 gld and spr genes shown to be required for gliding or spreading in Flavobacterium johnsoniae; however, their relative role in its lifestyle remains unknown. In order to address this issue, two spreading deficient mutants were produced as part of a Tn4351 mutant library in F. psychrophilum strain THCO2-90. The transposons were inserted in gldD and gldG genes. While the wild-type strain is proficient in adhesion, biofilm formation and displays strong proteolytic activity, both mutants lost these characteristics. Extracellular proteome comparisons revealed important modifications for both mutants, with a significant reduction of the amounts of proteins likely transported through the outer membrane by the Type IX secretion system, indicating that GldD and GldG proteins are required for an effective activity of this system. In addition, a significant decrease in virulence was observed using rainbow trout bath and injection infection models. Our results reveal additional roles of gldD and gldG genes that are likely of importance for the F. psychrophilum lifestyle, including virulence.

14.
J Proteome Res ; 16(7): 2597-2613, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28560880

RESUMO

Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Glicólise/genética , Fosforilação Oxidativa , Proteômica/métodos , Streptomyces coelicolor/metabolismo , Streptomyces lividans/metabolismo , Aerobiose/genética , Aminoácidos/metabolismo , Anaerobiose/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Glucose/metabolismo , Anotação de Sequência Molecular , Oxigênio/farmacologia , Regulon/efeitos dos fármacos , Especificidade da Espécie , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/genética , Streptomyces lividans/efeitos dos fármacos , Streptomyces lividans/genética
15.
J Extracell Vesicles ; 6(1): 1305677, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28473884

RESUMO

Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.

16.
J Bacteriol ; 199(9)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28242718

RESUMO

Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection.IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different transporters is used: the maltose-PTS transporter MalT, or the MdxEFG-MsmX ABC transporter. MdxEFG-MsmX takes up longer maltodextrins as well as complex molecules, such as panose and isopanose.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Enterococcus faecalis/enzimologia , Enterococcus faecalis/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Polissacarídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Rim/microbiologia , Fígado/microbiologia , Maltose/farmacologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Mutação , Oligossacarídeos/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Trissacarídeos/farmacologia
17.
Mol Biol Cell ; 27(19): 2946-64, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27535430

RESUMO

During lactation, mammary epithelial cells secrete huge amounts of milk from their apical side. The current view is that caseins are secreted by exocytosis, whereas milk fat globules are released by budding, enwrapped by the plasma membrane. Owing to the number and large size of milk fat globules, the membrane surface needed for their release might exceed that of the apical plasma membrane. A large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globule membranes was used to decipher the cellular origins of the milk fat globule membrane. Surprisingly, differential analysis of protein profiles of these two organelles strongly suggest that, in addition to the plasma membrane, the endoplasmic reticulum and the secretory vesicles contribute to the milk fat globule membrane. Analysis of membrane-associated and raft microdomain proteins reinforces this possibility and also points to a role for lipid rafts in milk product secretion. Our results provide evidence for a significant contribution of the endoplasmic reticulum to the milk fat globule membrane and a role for SNAREs in membrane dynamics during milk secretion. These novel aspects point to a more complex model for milk secretion than currently envisioned.


Assuntos
Glicolipídeos/biossíntese , Glicolipídeos/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/metabolismo , Animais , Mama/metabolismo , Caseínas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Lactação/metabolismo , Lipídeos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Microdomínios da Membrana/metabolismo , Membranas/metabolismo , Camundongos , Leite/metabolismo , Proteômica/métodos , Proteínas SNARE/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/fisiologia
18.
Mol Microbiol ; 100(5): 788-807, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26858137

RESUMO

The bacterial phosphotransferase system (PTS) transports and phosphorylates sugars, but also carries out numerous regulatory functions. The ß-proteobacterium Neisseria meningitidis possesses an incomplete PTS unable to transport carbon sources because it lacks a membrane component. Nevertheless, the residual phosphorylation cascade is functional and the meningococcal PTS was therefore expected to carry out regulatory roles. Interestingly, a ΔptsH mutant (lacks the PTS protein HPr) exhibited reduced virulence in mice and after intraperitoneal challenge it was rapidly cleared from the bloodstream of BALB/c mice. The rapid clearance correlates with lower capsular polysaccharide production by the ΔptsH mutant, which is probably also responsible for its increased adhesion to Hec-1-B epithelial cells. In addition, compared to the wild-type strain more apoptotic cells were detected when Hec-1-B cells were infected with the ΔptsH strain. Coimmunoprecipitation revealed an interaction of HPr and P-Ser-HPr with the LysR type transcription regulator CrgA, which among others controls its own expression. Moreover, ptsH deletion caused increased expression of a ΦcrgA-lacZ fusion. Finally, the presence of HPr or phospho-HPr's during electrophoretic mobility shift assays enhanced the affinity of CrgA for its target sites preceding crgA and pilE, but HPr did not promote CrgA binding to the sia and pilC1 promoter regions.


Assuntos
Aderência Bacteriana , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , Neisseria meningitidis/patogenicidade , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose , Proteínas de Bactérias/genética , Células Epiteliais , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosfotransferases/química , Fosfotransferases/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Virulência
19.
Mol Cell Proteomics ; 15(2): 598-613, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26637540

RESUMO

Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume plant Medicago truncatula.


Assuntos
Proteínas de Bactérias/genética , Doenças das Plantas/microbiologia , Proteômica , Ralstonia solanacearum/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Chaperonas Moleculares/genética , Mutação , Doenças das Plantas/genética , Plantas/microbiologia , Ralstonia solanacearum/metabolismo , Ralstonia solanacearum/patogenicidade
20.
Eur J Hum Genet ; 24(6): 838-43, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26395558

RESUMO

Semaphorins are a large family of secreted and membrane-associated proteins necessary for wiring of the brain. Semaphorin 5A (SEMA5A) acts as a bifunctional guidance cue, exerting both attractive and inhibitory effects on developing axons. Previous studies have suggested that SEMA5A could be a susceptibility gene for autism spectrum disorders (ASDs). We first identified a de novo translocation t(5;22)(p15.3;q11.21) in a patient with ASD and intellectual disability (ID). At the translocation breakpoint on chromosome 5, we observed a 861-kb deletion encompassing the end of the SEMA5A gene. We delineated the breakpoint by NGS and observed that no gene was disrupted on chromosome 22. We then used Sanger sequencing to search for deleterious variants affecting SEMA5A in 142 patients with ASD. We also identified two independent heterozygous variants located in a conserved functional domain of the protein. Both variants were maternally inherited and predicted as deleterious. Our genetic screens identified the first case of a de novo SEMA5A microdeletion in a patient with ASD and ID. Although our study alone cannot formally associate SEMA5A with susceptibility to ASD, it provides additional evidence that Semaphorin dysfunction could lead to ASD and ID. Further studies on Semaphorins are warranted to better understand the role of this family of genes in susceptibility to neurodevelopmental disorders.


Assuntos
Transtorno do Espectro Autista/genética , Deleção Cromossômica , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Transtorno do Espectro Autista/complicações , Transtorno do Espectro Autista/diagnóstico , Criança , Pontos de Quebra do Cromossomo , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 5/genética , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico , Masculino , Herança Paterna , Translocação Genética
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