Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

International audienceIn Brief 20S proteasomes are very heterogeneous protein complexes involved in many cellular processes. In the present study, we combined an MRM-based assay with the production and purification of entire SILAC labelled pro-teasome to monitor absolute quantities of the different 20S proteasome subtypes in various human cells and tissues. This method applied to adipocyte-derived stem cells (ADSCs) amplified under various conditions highlights an increased expression of immunoproteasome when this type of cell is primed with IFN␥ or amplified in a 20% O 2 environment. Graphical Abstract Highlights • Design of an MRM assay to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes. • Use of purified isotopically labelled 20S proteasome as internal standard for accurate quantification. • Variation in the expression of immunoproteasome in adipocyte-derived stem cells (ADSCs) grown under different O 2 levels might be causal for change in cells differentiation capacity. • The status of 20S proteasome during ADSCs expansion might constitute an additional relevant quality control parameter to contribute to predict, among other quality markers, their therapeutic capacity

Back to basics : Rethinking the diversity and pathogeny of the causal agent of Bacterial Leaf Spot of Lettuce

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Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics

To date, Mycobacterium tuberculosis (Mtb) remains the world’s greatest infectious killer. The rise of multidrug-resistant strains stresses the need to identify new therapeutic targets to fight the epidemic. We previously demonstrated that bacterial protein-O-mannosylation is crucial for Mtb infectiousness, renewing the interest of the bacterial-secreted mannoproteins as potential drug-targetable virulence factors. The difficulty of inventorying the mannoprotein repertoire expressed by Mtb led us to design a stringent multi-step workflow for the reliable identification of glycosylated peptides by large-scale mass spectrometry-based proteomics. Applied to the differential analyses of glycoproteins secreted by the wild-type Mtb strain—and by its derived mutant invalidated for the protein-O-mannosylating enzyme PMTub—this approach led to the identification of not only most already known mannoproteins, but also of yet-unknown mannosylated proteins. In addition, analysis of the glycoproteome expressed by the isogenic recombinant Mtb strain overexpressing the PMTub gene revealed an unexpected mannosylation of proteins, with predicted or demonstrated functions in Mtb growth and interaction with the host cell. Since in parallel, a transient increased expression of the PMTub gene has been observed in the wild-type bacilli when infecting macrophages, our results strongly suggest that the Mtb mannoproteome may undergo adaptive regulation during infection of the host cells. Overall, our results provide deeper insights into the complexity of the repertoire of mannosylated proteins expressed by Mtb, and open the way to novel opportunities to search for still-unexploited potential therapeutic targets

Substrates of the ASB2α E3 ubiquitin ligase in dendritic cells

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The protein kinase PknB negatively regulates biosynthesis and trafficking of mycolic acids in mycobacteria

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How to name those to blame: A practical taxonomical study of the Xanthomonas causing bacterial leaf spot of lettuce has major consequences for the boundaries delimitation of X. hortorum

International audienceAssessment of the taxonomy and diversity of the Xanthomonas strains causing bacterial leaf spot of lettuce (BLSL), commonly referred to as X. campestris pv. vitians, has been a long-lasting issue which held back the global efforts made to understand this pathogen. In order to provide a sound basis essential to its study, we conducted a polyphasic approach on a panel of strains obtained through sampling campaigns or acquired from collections. Results of a multilocus sequence analysis combined with phenotypic assays revealed that the pathotype strain of X. campestris pv. vitians does not match the description of the nomenspecies provided by Brown in 1918 [1]. However, LMG 938 = CFBP 8686 does fit this description. Therefore, we propose that LMG 938 = CFBP 8686 replaces LMG 937 = CFBP 2538 as pathotype strain of X. campestris pv. vitians. Then, overall genome relatedness indices (OGRIs) calculations and crossed-pathogenicity assays on lettuce allowed to validate the nomenclature X. hortorum pv. vitians comb. nov. originally proposed by Vauterin et al. in 1995 [2]. Interestingly, genome-based phylogenies exhibited an evolutionary continuum between closely related species X. hortorum and X. cynarae, confirmed by multiple OGRIs calculations between all the taxonomically relevant members of these two species. Phenotypic profiles characterized with Biolog GEN III microplates did not reveal any stable diagnostic traits legitimizing the distinction into two different species but a strong stable core phenotype. Thus, to be in accordance with nowadays standards in bacterial taxonomy and with respect to the global taxonomical framework of the genus Xanthomonas [3, 4], we propose the new combination of X. hortorum Vauterin et al. 1995 and X. cynarae (Trébaol 2000) Timilsina et al. 2018 into a new X. hortorum comb. nov. This new stable species would allow to integrate new uncharacterized members of X. hortorum isolated over the years on a wide range of host plants, making it a promising model for the study of genomic and ecologic determinants underlying adaptation to host plants. 1. Brown NA. Some bacterial diseases of lettuce. J Agric Res 1918;13:367. 2. Vauterin L, Hoste B, Kersters K, Swings J. Reclassification of Xanthomonas. Int J Syst Evol Microbiol 1995;45:472-489. 3. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 2018;68:461-466. 4. Rosselló-Móra R, Amann R. Past and future species definitions for Bacteria and Archaea. Syst Appl Microbiol 2015;38:209-216

Clarifying the taxonomy of the causal agent of bacterial leaf spot of lettuce through a polyphasic approach reveals that Xanthomonas cynarae Trébaol et al. 2000 emend. Timilsina et al. 2019 is a later heterotypic synonym of Xanthomonas hortorum Vauterin et al. 1995

International audienceAssessment of the taxonomy and diversity of Xanthomonas strains causing bacterial leaf spot of lettuce(BLSL), commonly referred to as Xanthomonas campestris pv. vitians, has been a long-lasting issue whichheld back the global efforts made to understand this pathogen. In order to provide a sound basis essentialto its study, we conducted a polyphasic approach on strains obtained through sampling campaigns oracquired from collections. Results of a multilocus sequence analysis crossed with phenotypic assaysrevealed that the pathotype strain does not match the description of the nomenspecies provided byBrown in 1918. However, strain LMG 938 = CFBP 8686 does fit this description. Therefore, we proposethat it replaces LMG 937 = CFBP 2538 as pathotype strain of X. campestris pv. vitians.Then, whole-genome based phylogenies and overall genome relatedness indices calculated on taxo-nomically relevant strains exhibited the intermediate position of X. campestris pv. vitians between closelyrelated species Xanthomonas hortorum and Xanthomonas cynarae. Phenotypic profiles characterized usingBiolog microplates did not reveal stable diagnostic traits legitimizing their distinction. Therefore, we pro-pose that X. cynarae Trébaol et al. 2000 emend. Timilsina et al. 2019 is a later heterotypic synonym ofX. hortorum, to reclassify X. campestris pv. vitians as X. hortorum pv. vitians comb. nov. and to transferX. cynarae pathovars in X. hortorum as X. hortorum pv. cynarae comb. nov. and X. hortorum pv. gardnericomb. nov. An emended description of X. hortorum is provided, making this extended species a promisingmodel for the study of Xanthomonas quick adaptation to different host

Who’s who : Resolving the taxonomy of the causal agent of bacterial leaf spot of lettuce through a polyphasic approach leads to combine Xanthomonas hortorum Vauterin et al. 1995 and Xanthomonas cynarae (Trébaol 2000) Timilsina et al. 2018

International audienceAssessment of the taxonomy and diversity of the Xanthomonas strains causing bacterial leaf spot of lettuce (BLSL), commonly referred as Xanthomonas campestris pathovar vitians, has been a long-lasting issue which held back the global efforts made to understand this disease and struggle against the damages it causes in the field. In order to restore sane bases essential to the study of this pathogen, we conducted an extensive polyphasic approach on a panel of strains obtained through sampling campaigns or collection strains acquisitions. Results of a multilocus sequence analysis crossed with pathogenicity assays revealed that BLSL-causing strains formed a monophyletic pathovar sparsely diversified in three clonal groups. However, genome-based phylogenies yielded only two major groups of strains. From the bigger picture perspective, phylogenies exhibited the intermediate position of pathovar vitians between close species X. hortorum and X. cynarae, resulting in a phylogenetic continuum regarding the known diversity of the genus. Finally, whole-genome comparisons of all type, pathotype or representative strains of these two species by multiple overall genome relatedness indices (OGRIs) calculation endorsed the hypothesis of one single specie. Carbon and nitrogen sources utilization tested using Biolog microplates did not revealed any particular phenotypic differences which would have legitimate the distinction into two species. Therefore, we propose the combination of X. hortorum, X. cynarae and X. campestris pv. vitians into a new X. hortorum comb. nov. with CFBP 4925 T = LMG 937 T remaining the type strain, and LMG 938 neoPT = NCPB 2248 neoPT becoming the pathotype strain of X. hortorum pv. vitians