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NleH1/2 proteomic analysis scripts

ABSTRACT: Combined script for the generation of images within paper. Three experiments in total 1) Phosphopeptide enrichment of Three biological replicates of each condition (uninfected, WT infected , detla NleH1 NleH2 infected, pNleH1 infected and pNleH2 infection) 2) Total proteome analysis to complement phosphoproteomic study aimed at identifying the substrate of NleH1/2 3) In vitro assays of EPS8 with catalytically active and inactive NleH1 and NleH2.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

SUBMITTER: Nichollas Scott

LAB HEAD: Professor Elizabeth L. Hartland

PROVIDER: PXD031117 | Pride | 2022-08-07

REPOSITORIES: Pride

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			Action	DRS
	EPEC_WT_infection_Nsco_20170213_TiOx_Gina_samples3.raw	Raw
	Phosphoproteomic_proteomic_analysis.zip	Other
	checksum.txt	Txt

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Project description:Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. Recently we demonstrated that infection of human DC with intracellular bacterium Listeria monocytogenes (L.monocytogenes) leads to the induction of the immunoinhibitory enzyme indoleamine 2,3-dioxygenase (Popov et al., J Clin Invest, 2006), while in the previous studies L.monocytogenes infection was associated with a rather stimulatory DC phenotype. To clarify this discrepancy we performed comparative microarray analysis of immature mo-DC (immDC), mature stimulatory mo-DC (matDC) and mature inhibitory DC either stimulated with prostaglandin E2 (PGE2-DC) or infected with L.monocytogenes (infDC). Studying infection of human myeloid DC with Listeria monocytogenes, we found out, that infected DC are modified by the pathogen to express multiple inhibitory molecules, including indoleamine 2,3-dioxygenase (IDO), cyclooxygenase-2, interleukin 10 and CD25, which acts on DC as IL-2 scavenger. All these inhibitory molecules, expressed on regulatory DC (DCreg), are strictly TNF-dependent and are in concert suppressing T-cell responses. Moreover, only DCreg can efficiently control the number of intracellular listeria, mostly by IDO-mediated mechanisms and by other factors, remaining to be identified. Analyzing publicly acessible data of transcriptional changes in DC and macrophages, infected by various pathogens and parasites (GEO, GSE360), we noticed that infection of these cells with Mycobacterium tuberculosis causes transcriptional response, comparable with the one caused by listeria in human DC. In fact, granuloma in tuberculosis and listeriosis in vivo are enriched for myeloid DC and macrophages characterized by regulatory phenotype. In summary, regulatory myeloid DC and macrophages may play a dual role during life-threatening granulomatous infections, such as tuberculosis: on one hand, regulatory myeloid cells promote pathogen containment by efficiently killing intracellular bacteria, on the other hand these cells inhibit granuloma-associated T cells and thereby might be involved in the retention of TNF-controlled granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. Experiment Overall Design: Transcriptional profiles of immature mo-DC (immDC, n=3), mature stimulatory mo-DC (matDC, n=3) and mature inhibitory mo-DC either stimulated with prostaglandin E2 (PGE2-DC, n=3) or infected with L.monocytogenes (infDC, n=3) were compared on Affymetrix HG-U133A platform. All samples represent biological replicates and were processed separately throughout the experiment.

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NleH1/2 proteomic analysis scripts

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