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Global mapping of Salmonella enterica-host protein-protein interactions during infection.

ABSTRACT: Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.

SUBMITTER: Walch P

PROVIDER: S-EPMC8561747 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Global mapping of Salmonella enterica-host protein-protein interactions during infection.

Walch Philipp P Selkrig Joel J Knodler Leigh A LA Rettel Mandy M Stein Frank F Fernandez Keith K Viéitez Cristina C Potel Clément M CM Scholzen Karoline K Geyer Matthias M Rottner Klemens K Steele-Mortimer Olivia O Savitski Mikhail M MM Holden David W DW Typas Athanasios A

Cell host & microbe 20210707 8

Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously descri ...[more]

PMID: 34237247

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Project description:Intracellular bacterial pathogens inject a cocktail of effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. Salmonella enterica serovar Typhimurium (STm) uses two Type 3 secretion system (T3SS) to deliver a suite of effector proteins into host cells, however, knowledge of their host-targets remains sparse. To systematically map STm effector-host protein-protein interactions (PPIs) during active infection, we generated a library of 32 STm strains expressing affinity-tagged effector proteins from their endogenous chromosomal loci. Tagged-effector strains were then used to infect macrophages or epithelial cells, and PPIs were detected by Affinity-Purification Quantitative Mass-Spectrometry (AP-QMS) under both native and cross-linking conditions. We recovered 25 previously described effector-host PPIs, along with 421 novel interactions across the two cell lines tested, as well as several effector-effector PPIs. While effectors converged on specific host cellular processes, which varied across cell types, most had multiple host targets. Using reciprocal co-immunoprecipitations, we could validated 13 out of 22 new PPIs, demonstrating an accuracy of at least 59% for the AP-QMS approach. To illustrate the utility of this resource for discovering novel infection biology, we first showed that SseJ and SseL cooperate to control cholesterol transport at the Salmonella Containing Vacuole (SCV) via the Niemann-Pick C1 protein (NPC1). Second, we uncovered that PipB directly binds and recruits the organelle contact site protein PDZD8 to the SCV. Third, we elucidated a novel mechanism whereby the effector kinase SteC promotes host actin bundling, by directly interacting and phosphorylating formin-like proteins. Overall, we provide a systems-wide host-bacterial pathogen physical interactome resource, to our knowledge the first in an infection context, with broad implications for effector function and cooperation.

Project description:BackgroundIntestinal fibrosis is a common and serious complication of Crohn's disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear.MethodsChronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases.ResultsMice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts.ConclusionsIn summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.

Dataset Information

Global mapping of Salmonella enterica-host protein-protein interactions during infection.

Publications

Global mapping of Salmonella enterica-host protein-protein interactions during infection.

Similar Datasets

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