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Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium.

ABSTRACT: Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food--and water--borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.

SUBMITTER: Bowden SD

PROVIDER: S-EPMC4010460 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium.

Bowden Steven D SD Hopper-Chidlaw Amanda C AC Rice Christopher J CJ Ramachandran Vinoy K VK Kelly David J DJ Thompson Arthur A

PloS one 20140505 5

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food--and water--borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic ge ...[more]

PMID: 24797930

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Project description:To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors.

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.

Project description:The gut microbiota confers resistance to pathogens of the intestinal ecosystem, yet the dynamics of pathogen-microbiome interactions and the metabolites involved in this process remain largely unknown. Here, we use gnotobiotic mice infected with the virulent pathogen Salmonella enterica serovar Typhimurium or the opportunistic pathogen Candida albicans in combination with metagenomics and discovery metabolomics to identify changes in the community and metabolome during infection. To isolate the role of the microbiota in response to pathogens, we compared mice monocolonized with the pathogen, uninfected mice "humanized" with a synthetic human microbiome, or infected humanized mice. In Salmonella-infected mice, by 3 days into infection, microbiome community structure and function changed substantially, with a rise in Enterobacteriaceae strains and a reduction in biosynthetic gene cluster potential. In contrast, Candida-infected mice had few microbiome changes. The LC-MS metabolomic fingerprint of the cecum differed between mice monocolonized with either pathogen and humanized infected mice. Specifically, we identified an increase in glutathione disulfide, glutathione cysteine disulfide, inosine 5'-monophosphate, and hydroxybutyrylcarnitine in mice infected with Salmonella in contrast to uninfected mice and mice monocolonized with Salmonella These metabolites potentially play a role in pathogen-induced oxidative stress. These results provide insight into how the microbiota community members interact with each other and with pathogens on a metabolic level.IMPORTANCE The gut microbiota is increasingly recognized for playing a critical role in human health and disease, especially in conferring resistance to both virulent pathogens such as Salmonella, which infects 1.2 million people in the United States every year (E. Scallan, R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, et al., Emerg Infect Dis 17:7-15, 2011, https://doi.org/10.3201/eid1701.P11101), and opportunistic pathogens like Candida, which causes an estimated 46,000 cases of invasive candidiasis each year in the United States (Centers for Disease Control and Prevention, Antibiotic Resistance Threats in the United States, 2013, 2013). Using a gnotobiotic mouse model, we investigate potential changes in gut microbial community structure and function during infection using metagenomics and metabolomics. We observe that changes in the community and in biosynthetic gene cluster potential occur within 3 days for the virulent Salmonella enterica serovar Typhimurium, but there are minimal changes with a poorly colonizing Candida albicans In addition, the metabolome shifts depending on infection status, including changes in glutathione metabolites in response to Salmonella infection, potentially in response to host oxidative stress.

Dataset Information

Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium.

Publications

Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium.

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