Dataset Information

DIA-MS serum proteomics in dogs with leishmaniosis

ABSTRACT: Serum proteomes of healthy and Leishmania-infected dogs were analyzed by DIA-MS approach using non-depleted serum and 60 min gradient time in order to study host response to infection.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Canis Familiaris (dog) (canis Lupus Familiaris)

TISSUE(S): Blood Serum

DISEASE(S): Leishmaniasis

SUBMITTER: Anita Horvatic

LAB HEAD: Peter David Eckersall

PROVIDER: PXD039765 | Pride | 2023-05-10

REPOSITORIES: Pride

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Publications

Data Independent Acquisition Reveals In-Depth Serum Proteome Changes in Canine Leishmaniosis.

Martinković Franjo F Popović Marin M Smolec Ozren O Mrljak Vladimir V Eckersall Peter David PD Horvatić Anita A

Metabolites 20230228 3

Comprehensive profiling of serum proteome provides valuable clues of health status and pathophysiological processes, making it the main strategy in biomarker discovery. However, the high dynamic range significantly decreases the number of detectable proteins, obstructing the insights into the underlying biological processes. To circumvent various serum enrichment methods, obtain high-quality proteome wide information using the next-generation proteomic, and study host response in canine leishman ...[more]

PMID: 36984805

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Project description:In order to successfully survive in and to colonize the gastrointestinal tract, bacteria need to develop strategies to overcome bile acid stress. The most prominent bile acids are the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA). In this study, we investigated the stress response of E. faecalis and E. faecium to sublethal concentrations of these three bile acids on the proteome level using DIA-MS. As both species showed similar IC50 for DCA and CDCA in growth experiments and both were highly resistant towards CA, we assumed similar changes to their protein expression profiles. Moreover, we investigated proteomic differences of E. faecalis grown under aerobic or microaerophilic conditions. Our findings showed similarities, but also species-specific variations in the response to the different bile acids, which reveal potential differences in the adaptation process. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis, but to a lesser extent in E. faecium. Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a “general bile acid response”. Among these, ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Specific for all E. faecalis samples was the up-expression of several subunits of a V-type ATPase and the down-expression of proteins involved in pyruvate-, citrate- and folate metabolism. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions in comparison to aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously.

Project description:Background Homeobox gene associated regulatory networks are among the key determinants of early hematopoietic development. Previously, the ‘hematopoietic PBX interacting protein’ (HPIP) has been identified as a novel interacting partner of the TALE homeodomain protein PBX1, forming a microtubule signalling complex. Expression of HPIP has been associated with increased tumorigenicity of the MCF7 breast cancer cell line. We now demonstrate that HPIP is a novel regulatory protein in human hematopoiesis: constitutive expression of HPIP in human umbilical cord blood derived CD34+ cells increased the absolute number of clonogenic progenitors in liquid expansion culture as well as in methylcellulose assays with a significantly enhanced formation of erythroid colonies compared to the control (p?0.01, n=6). Limiting dilution LTC-IC assays confirmed the hematopoietic activity of the protein on primitive human progenitor cells with an over 5fold increase in the absolute number of LTC-ICs compared to non-transduced cells (n=8; p<0.05). In vivo HPIP expression induced a significant shift towards myeloid engraftment (n=8;p<0.05) and doubled the proportion of hCD34+CD38+ human cells in transplanted mice (p?0.05, n=8). Structure – function analyses identified the C - terminal nuclear receptor/PBX interacting domain (NRPID; LXXLL domain) as a critical domain for the hematopoietic activity of HPIP. Gene expression data by microarray and Q-RT-PCR analysis demonstrated that HPIP induced particularly differential expression of genes involved in the MAPK pathway and cytokine-cytokine interaction. Taken together, these data demonstrate that proteins involved in the organization of microtubular signalling complexes such as HPIP can act as regulators of early human hematopoiesis. Stable PG13 packaging cell lines were used to perform transductions of human umbilical cord blood derived CD34+ cells. Phoenix amphotropic cells were used for transient transduction of PG13 with wt and ?NRPID mutant viruses. Both cell lines were cultured in DMEM with 10% fetal bovine serum and plated on corning dishes for transfections and transient transduction ( 2.5 x 106 cells per 10 cm plate) a day prior to the experiment. Transient transductions were performed as described before in literature. Briefly, cells at 2x10e5/mL were prestimulated for 48 hours in Iscove´s IMDM( GIBCO-Invitrogen, Karlsruhe, Germany) containing a serum substitute (BITTM, Stem Cell Technologies), 10-4M ? mercaptoethanol (Sigma-Aldrich, Taufkirchen, Germany), supplemented with the following recombinant human cytokines: 100 ng/mL Flt-3 Ligand, 100 ng/mL SF, 20 ng/mL IL-3, 20 ng/mL G-CSF, and 20 ng/mL IL-6 (Immunotools, Friesoythe, Germany). After 48 Hours, cells were resuspended in filtered virus-containing medium (VCM) supplemented with the same five cytokine cocktail and polybrene (5 µg/mL) on tissue culture dishes (Corning). The dishes were pre-loaded with VCM twice, each time for 45 mins. The procedure was repeated for a total of three infections. Fresh RNA was prepared using RNA easy micro kit (Sigma) with ? 2x105 retrovirally transduced human cord blood CD34+HPIP-WT-YFP+, CD34+?NRPID-HPIP-YFP+ or CD34+YFP+ cells. Total RNA was reverse transcribed (Superscript II, Invitrogen) by using oligo (dT) to prime cDNA synthesis. In vitro transcription was performed using a the GeneChip® Two-Cycle cDNA Synthesis Kit, uniquely configured and tested for GeneChip® target labeling. Two cycles of double-stranded cDNA synthesis containing the T7 promoter sequence were performed from 10 to 100 ng of total RNA using this Kit.

Dataset Information

DIA-MS serum proteomics in dogs with leishmaniosis

Publications

Data Independent Acquisition Reveals In-Depth Serum Proteome Changes in Canine Leishmaniosis.

Similar Datasets

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