Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium

Heffron F (2009) Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium. PLoS Pathog 5(2): e1000306. doi:10.1371/journal.ppat.1000306 Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium Hyunjin Yoon 0 Jason E. McDermott 0 Steffen Porwollik 0 Michael McClelland 0 Fred Heffron 0 Jorge E. Galan, Yale University School of Medicine, United States of America 0 1 Department of Molecular Microbiology and Immunology, Oregon Health & Science University , Portland, Oregon , United States of America, 2 Pacific Northwest National Laboratories , Richland, Washington , United States of America, 3 The Sydney Kimmel Cancer Center , San Diego, California , United States of America 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. - Funding: This work was funded by NIH RO1 AI022933 and DE-ACO5-76RL01830 and by the National Institute of Allergy and Infectious Diseases (NIH/DHHS through interagency agreement Y1-AI-4894-01). Competing Interests: The authors have declared that no competing interests exist. Gastrointestinal infections are the second most common cause of childhood mortality in the developing world and Typhoid alone (caused by serovar Typhi) is estimated to result in 500,000 deaths per year [1]. In addition to fluid and electrolyte loss, non-typhoidal Salmonella often results in septicemia in children and in HIV infected adults in developing countries with a fatality rate of 25% or greater [2]. Salmonella enteriditis serotype Typhimurium (referred to simply as Salmonella or Typhimurium below) is a paradigm for understanding intracellular pathogenesis because of its established genetics and simple and inexpensive animal model - the mouse. All strains of Salmonella enteriditis share at least 95% sequence identity; the differences are associated with growth in a specific host or survival in an environmental niche. More than 4% of the entire genome is required for Typhimurium to infect the mouse [3]. These genes are widely distributed around the entire circular chromosome including many genes not involved in metabolic processes nor required for growth under laboratory conditions. Numerous studies have assigned a small fraction of these genes to specific steps in mouse infection but most are still a mystery. Many virulence genes are attributable to horizontally acquired DNA sequences that are not present in nonpathogenic but related bacteria. These regions include two 40 kb stretches of DNA termed Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2) [49]. SPI-1 and SPI-2 encode a secretion apparatus resembling a needle and related to the bacterial flagella that uses the proton motive force to secrete proteins directly into the cytoplasm of the eukaryotic cell [10]. Secretion can take place from extracellular bacteria that are juxtaposed to the surface of the cell or from intracellular bacteria located in vacuoles. The two type III secretion systems are expressed under different environmental conditions and play distinct roles in pathogenesis. SPI-2 is known to be required for systemic infection whereas SPI-1 plays an essential role during intestinal infection and in mouse persistence [1114]. During the course of systemic infection in mice, bacteria are found within neutrophils, monocytes, dendritic cells, and B and T cells but are not found extracellularly until the last one or two days immediately before death of the mouse [1517]. How Salmonella survives and replicates within the host and how it expresses virulence genes at the appropriate time during systemic infection is little understood and the subject of this work. Technological advances in the last 10 years such as microarrays, whole genome sequences, and global proteomics have provided a more complete picture of gene expression for a number of bacteria. The goal of the current work is to develop a predictive model for hostpathogen interactions that will provide insight into how Salmonella responds to specific conditions in the host. This approach was based on identification of regulators that were necessary for We have used the intracellular pathogen Salmonella to investigate how pathogenic bacterium correctly expresses virulence factors during infection. After ingestion by its host, Salmonella confronts multiple challenging microenvironments such as acidic pH in the stomach, nutrient starvation, and host innate immune responses. However, Salmonella overcomes host defenses through expression of an array of virulence factors. We defined 14 Salmonella regulators out of 83 tested that are required for systemic infection. We then analyzed expression of Salmonella genes in each mutant strain grown under various conditions. Surprisingly, these regulators coordinately activate expression of a subset of virulence genes required for intracellular survival and replication. Computer-aided analysis based on our data established a regulatory network in which all 14 regulators are connected. Furthermore, the an (...truncated)