Transcriptional Analysis of Murine Macrophages Infected with Different Toxoplasma Strains Identifies Novel Regulation of Host Signaling Pathways

et al. (2013) Transcriptional Analysis of Murine Macrophages Infected with Different Toxoplasma Strains Identifies Novel Regulation of Host Signaling Pathways. PLoS Pathog 9(12): e1003779. doi:10.1371/journal.ppat.1003779 Transcriptional Analysis of Murine Macrophages Infected with Different Toxoplasma Strains Identifies Novel Regulation of Host Signaling Pathways Mariane B. Melo 0 Quynh P. Nguyen 0 Cynthia Cordeiro 0 Musa A. Hassan 0 Ninghan Yang 0 Rene e McKell 0 Emily E. Rosowski 0 Lindsay Julien 0 Vincent Butty 0 Marie-Laure Darde 0 Daniel Ajzenberg 0 Katherine Fitzgerald 0 Lucy H. Young 0 Jeroen P. J. Saeij 0 David L. Sacks, National Institute of Health, United States of America 0 1 Massachusetts Institute of Technology, Department of Biology, Cambridge, Massachusetts, United States of America, 2 Internal Medicine Department, School of Medicine, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil, 3 Retina Service, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School , Boston , Massachusetts, United States of America, 4 Centre National de Re fe rence Toxoplasmose/Toxoplasma Biological Resource Center, Centre Hospitalier-Universitaire Dupuytren, Limoges, France, 5 Institut National de la Sante et de la Recherche Me dicale, Unite Mixte de Recherche 1094, Neuroe pide miologie Tropicale, Laboratoire de Parasitologie-Mycologie, Faculte de Me decine, Universite de Limoges, Limoges, France, 6 University of Massachusetts Medical School, Division of Infectious Diseases and Immunology , Worcester, Massachusetts , United States of America Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNb production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts. - Funding: JPJS was supported by National Institutes of Health R01-AI080621, a NERCE developmental grant AIO57159, the Pew Scholars Program in the Biomedical Sciences and a Robert A. Swanson Career Development award; MBM was supported by the Knights Templar Eye Foundation; MAH was supported by a Welcome Trust-MIT postdoctoral fellowship. NY was supported by an A*STAR NSS (PhD) graduate scholarship. ER was supported by Pre-Doctoral Grant in the Biological Sciences 5-T32- GM007287-33. ER was also supported by the Cleo and Paul Schimmel Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Toxoplasma gondii is a ubiquitous obligate intracellular protozoan parasite that can invade and replicate in almost all cells of a wide range of warm-blooded animals [1]. In humans it is the 2nd most important foodborne pathogen in terms of annual cost of illness and quality adjusted life year loss [2]. Normally a lifelong, largely asymptomatic, infection is established but in immunocompromised individuals and in congenital infections Toxoplasma infection can lead to severe disease and even death. Nevertheless, not all seropositive immunosuppressed patients have reactivating toxoplasmosis [3], and not all congenital infections lead to disease [4]. There is good evidence that both the host genetic background [57] and the genotype of the infecting strain [8,9] play a role in the severity of disease. Despite the existence of a sexual phase in its life cycle, which only occurs in felines, few strains dominate human infections in Europe and North America. Type II strains dominate in Europe, while in North America, types 12, II and III account for the majority of strains isolated from wild-life and patients [911]. Genotypes not belonging to these lineages are predominant in South America [1214]. A phylogenetic analysis of 956 strains, using single nucleotide polymorphisms (SNPs) identified in five loci, clustered these into 15 haplogroups, including type I, II and III [11,1517]. Using genome-wide SNPs, it was shown that even within these haplogroups there is often significant diversity and many strains did not fit into the 15 proposed haplogroups. Instead, many strains appear to have formed through recent recombination events [18]. Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii. The parasite is found throughout the world. When humans are infected, few have symptoms because a healthy immune system usually prevents the parasite from causing illness. Nevertheless, cases of severe disease in otherwise healthy individuals have been observed. These cases are usually a result of infection with less common atypical strains of Toxoplasma. Factors associated with virulence in the atypical strains are not well understood. Here, we infected host cells with 29 different strains of Toxoplasma, and performed highthroughput RNA sequencing of both host cells and parasites. We found significant differences in gene expression profiles between strains. Host cell transcriptional response also varied substantially depending on the infecting strain. Specifically, we found that a small group of atypical strains are able to induce production of type I interferons, which are immunomodulatory cytokines. Interferon production is a result of the elimination of internalized parasites through a novel killing mechanism. The dataset we generated is a valuable tool for identification of host cell targ (...truncated)