The major goal of this project is to define host genes necessary for the replication of an arthropod (tick)-transmitted pathogen, Ehrlichia chaffeensis, in a model arthropod (Drosophila) system. The principal investigators completed a gene expression array analysis of Drosophila S2 cells infected with E. chaffeensis under permissive and non-permissive conditions. This analysis revealed 528 genes that were significantly upregulated (>1.5 fold higher than uninfected control) exclusively during permissive bacterial growth conditions. The working hypothesis is that one or more of these genes are necessary for optimal growth of E. chaffeensis in a host and disruption of those genes will affect bacterial growth. The first specific aim will be to infect adult flies carrying mutations in genes identified by our array. 110 fly lines that carry mutations in genes specifically upregulated during permissive conditions can be obtained as viable-fertile adults. Should the principal investigators identify any genes in this screen, they will use double stranded RNA to silence the gene to validate that silencing of that Drosophila gene will affect the replication of bacteria in Drosophila S2 hemocyte-like cells. The second specific aim will be use an RNAi library to identify host genes that are not expressed in mutant form in adult flies that are also essential for intracellular growth of E. chaffeensis. The availability of comprehensive RNAi screen at the The Drosophila RNAi Screening Center (DRSC) will allow us to investigate how down regulation of genes in S2 cells affects infection. The principal investigators anticipate that they will identify multiple genes that when disrupted will cause lower levels of infection by E. chaffeensis in vivo (in flies) and in vitro (in S2 cells). That outcome will allow the principal investigators to accept their experimental hypothesis and allow them to sort whether the required genes are necessary in a single or multiple biochemical/cellular pathways. The data obtained from this work will allow the principal investigators to focus on the identified genes to determine how manipulation of biochemical pathways with drugs in vertebrate or invertebrate (e.g. tick) systems affect bacterial growth. Therefore, this project presents a highly innovative way to identify host-dependent factors necessary for E. chaffeensis replication and will be useful for identifying translational applications to prevent human monocytic ehrlichiosis and perhaps other Rickettsial diseases.
This is a biomedically related project that addresses which host genes are necessary for the successful replication of an intracellular bacteria that is a human category C pathogen.
|Dib, Lea H; Ortega, M Teresa; Melgarejo, Tonatiuh et al. (2016) Establishment and characterization of DB-1: a leptin receptor-deficient murine macrophage cell line. Cytotechnology 68:921-33|
|Xie, Linglin; Fu, Qiang; Ortega, Teresa M et al. (2014) Overexpression of IL-10 in C2D macrophages promotes a macrophage phenotypic switch in adipose tissue environments. PLoS One 9:e86541|
|Dib, Lea H; Ortega, M Teresa; Fleming, Sherry D et al. (2014) Bone marrow leptin signaling mediates obesity-associated adipose tissue inflammation in male mice. Endocrinology 155:40-6|
|Chapes, Stephen K; Ortega, M Teresa (2013) Understanding macrophage differentiation during space flight: The importance of ground-based experiments before space flight. Recent Pat Space Technol 3:40-47|
|Chitko-McKown, Carol G; Chapes, Stephen K; Miller, Laura C et al. (2013) Development and characterization of two porcine monocyte-derived macrophage cell lines. Results Immunol 3:26-32|
|Drolia, Rishi; Von Ohlen, Tonia; Chapes, Stephen K (2013) Ehrlichia chaffeensis replication sites in adult Drosophila melanogaster. Int J Med Microbiol 303:40-9|
|Von Ohlen, Tonia; Luce-Fedrow, Alison; Ortega, M Teresa et al. (2012) Identification of critical host mitochondrion-associated genes during Ehrlichia chaffeensis infections. Infect Immun 80:3576-86|
|Ortega, M Teresa; Lu, Nanyan; Chapes, Stephen K (2012) Evaluation of in vitro macrophage differentiation during space flight. Adv Space Res 49:1441-1455|
|Ortega, M Teresa; Xie, Linglin; Mora, Silvia et al. (2011) Evaluation of macrophage plasticity in brown and white adipose tissue. Cell Immunol 271:124-33|