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Changes in Potential Pathogenicity-Associated Proteins of Helicobacter cinaedi Upon Infection of Macrophage Cells

ABSTRACT: Helicobacter cinaedi is a spiral-shaped Gram-negative, enterohepatic bacterium classified as a conditional pathogen (pathogenicity group 2). It is known to cause bacteremia and a variety of other diseases in humans. Notably, H. cinaedi has been shown to induce changes in cell morphology, including the transition to “foam cells”, when interacting with macrophages. To uncover protein factors involved in the pathogenesis of Helicobacter cinaedi, we performed a detailed mass spectrometric analysis of the proteome of strain BAA-847. To the best of our knowledge, this is the first such comprehensive study examining the bacterium's protein profile both under standard culture conditions and after infection of M1-type macrophage cells. We identified 1,575 proteins in the H. cinaedi proteome, and of these, 109 proteins were differentially upregulated after infection of macrophage cells. For these proteins, we performed a detailed functional analysis and proposed a model to explain their potential roles during Helicobacter cinaedi infection. In addition to the known pathogenicity factors HcaA, Cdt, and AhpC, we discovered several poorly understood proteins that are involved in immune response evasion, adaptation for survival within host cells, and possess toxic or other potentially pathogenic functions. Among the differentially upregulated proteins, we found those that enable the bacterium to utilize intracellular cholesterol as a carbon source. Additionally, we identified proteins involved in a toxin injection mechanism that disrupts host cell metabolism, which may be linked to the foam cell formation induced by Helicobacter cinaedi.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Helicobacter Cinaedi Ccug 18818 = Atcc Baa-847

TISSUE(S): Cell Culture

SUBMITTER: Georgij Arapidi

LAB HEAD: Georgij Arapidi

PROVIDER: PXD064480 | Pride | 2025-06-01

REPOSITORIES: Pride

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			Action	DRS
	Gera_20221107_Sh325-Sh327_HelicobacterCinaediProteome_UniProtKB_Experiment_Condensed.sf3	Other
	Sh-325.dat	Other
	Sh-325.mgf	Mgf
	Sh-325.mzid	Mzid
	Sh-325.mzid_Sh-325.MGF	Mzid

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Project description:OBJECTIVE: The liver X receptor M-NM-1 (LXRM-NM-1) is a ligand-dependent nuclear receptor and the major regulator of reverse cholesterol transport in macrophages. This makes it an interesting target for mechanistic study and treatment of atherosclerosis. METHODS AND RESULTS: We optimized a promising stilbenoid structure (STX4) in order to reach nanomolar effective concentrations in LXRM-NM-1 reporter-gene assays. STX4 displayed the unique property to activate LXRM-NM-1 effectively but not its subtype LXRM-NM-2. The potential of STX4 to increase transcriptional activity as an LXRM-NM-1 ligand was tested with gene expression analyses in THP1-derived human macrophages and oxLDL-loaded human foam cells. Only in foam cells but not in macrophage cells STX4 treatment showed athero-protective effects with similar potency as the synthetic LXR ligand T0901317 (T09). Surprisingly, combinatorial treatment with STX4 and T09 resulted in an additive effect on reporter-gene activation and target gene expression. In physiological tests the cellular content of total and esterified cholesterol was significantly reduced by STX4 without the undesirable increase in triglyceride levels as observed for T09. CONCLUSIONS: STX4 is a new LXRM-NM-1-ligand to study transcriptional regulation of anti-atherogenic processes in cell or ex vivo models, and provides a promising lead structure for pharmaceutical development. 2 treatment groups (STX4 vs. DMSO in foam cells), 3 biological replicates per treatment (6 samples in total), each representing one well from cell culture plate 2 treatment groups (STX4 vs. DMSO in macrophages), 4 biological replicates per treatment (8 samples in total), each representing one well from cell culture plate 2 treatment groups (T0901317 vs. DMSO in foam cells), 3-4 biological replicates per treatment (7 samples in total), each representing one well from cell culture plate 2 treatment groups (T0901317 vs. DMSO in macrophages), 4 biological replicates per treatment (8 samples in total), each representing one well from cell culture plate

Project description:Background: Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide, most of which are caused by atherosclerosis. Discerning processes that participate in macrophage-to-foam cell formation are critical for understanding the basic mechanisms underlying atherosclerosis. To explore the molecular mechanisms of foam cell formation, the differentially expressed proteins were identified. Methods: In this paper, human monocytes, macrophage colony-stimulating factor induced macrophages, and oxidized low-density lipoprotein induced foam cells were cultured, and tandem mass tag (TMT) labeling combined with mass spectrometry (MS) were performed to find associations between foam cell transformation and proteome profiles. Results: Totally, 5146 quantifiable proteins were identified, among which 1515 and 182 differentially expressed proteins (DEPs) were found in macrophage/monocyte and foam cell/macrophage, respectively, using a cutoff of 1.5-fold change. Subcellular localization analysis revealed that downregulated DEPs of macrophages/monocytes were mostly located in the nucleus and upregulated DEPs of foam cells/macrophages mostly located in the plasma membrane and extracellular. Functional analysis of DEPs demonstrated that cholesterol metabolism related proteins were upregulated in foam cells, whereas the immune response-related proteins were downregulated in foam cells. The protein-interaction network showed that the DEPs with the highest interaction intensity between macrophages and foam cells were mainly concentrated in lysosomes and the endoplasmic reticulum. Conclusions: This study for the first time to perform quantitative proteomic investigation by TMT labeling and LC-MS/MS to identify differentially expressed proteins in human monocyte, macrophage, and foam cell. The results confirmed cholesterol metabolism was upregulated in foam cells, while immune response was suppressed, which suggested that foam cells were not the population that promote inflammation. In addition, KEGG enrichment analysis and protein-protein interaction indicated that the differentially expressed proteins locating in the endoplasmic reticulum and lysosomes may be key targets to regulate foam cell formation. These data provide a basis for identifying the potential proteins associated with the molecular mechanism involved in the transformation of macrophages to foam cells.

Project description:We previously established long-term 3D organoid culture systems for several murine tissues (intestine, stomach, pancreas and liver) as well as human intestine and pancreas. Here, we describe culture conditions to generate long-term 3D culture from human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. Human gastric cultures can expand indefinitely in 3D Matrigel. Cultures can be generated from normal tissue, from single sorted stem cells, or from tumor tissue. Organoids maintain many characteristics of the respective tissue in terms of histology, marker expression and euploidy. Organoids from normal tissue express markers of four lineages of the stomach and self-organize in gland and pit-domains. They can be directed to specifically express either lineages of the gastric gland, or the gastric pit by addition of Nicotinamide and withdrawal of Wnt. While gastric pit lineages react marginally to bacterial infection, gastric gland lineages mount a strong inflammatory response. The gastric culture system provides a unique tool to study gastric pathologies. We generated 2 sets of experiments. The first set contains organoids in 4 conditions: (1) organoids in expansion condition ENRWFGNiTi ("gland-type organoids") from 3 donors, (2) organoids as in 1 but differentiated for 4 days in differentiation condition ENR_FGNiTi ("pit'type organoids"), (3) organoids as in 1 but infected with Helicobacter pylori strain P12 MOI 50 for 2 h, (4) organoids as in 2 but infected as in 3. All 4 conditions were tested on the same organoid line in parallel. This experiment was conducted independently with cultures from 3 different donors. The second set of experiments compares freshly isolated glands with organoids. Samples from 2 patients were analyzed. Each patient received a total gastrectomy. From each patient, glands from corpus region or pyloric antrum were isolated. From each isolation, one aliquod was stored for microarray analysis and one aliquod used to generate organoids. Organoids and glands were subsequently lysed and analyzed in parallel.

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Changes in Potential Pathogenicity-Associated Proteins of Helicobacter cinaedi Upon Infection of Macrophage Cells

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