Illnesses caused by rickettsiales of the genera Ehrlichia and Anaplasma are a growing human health concern in recent years and are the second leading cause of tick-borne infections in the USA. They include human monocytic ehrlichiosis caused by E. chaffeensis. Despite the sophisticated systems of defense in vertebrate and tick hosts, the rickettsiales evade the host clearance. E. chaffeensis in macrophages and tick cells differ significantly in expression of several outer membrane proteins and effector proteins secreted via its secretary pathways. We reported that differential expression contributes to the varied host response and delayed clearance in a host. The central hypothesis of our funded project is that E. chaffeensis differentially regulates gene expression and that the host-specific gene expression is essential for its survival in vertebrate and tick cels.
Specific aims of the project are to 1) establish in vitro transcription system for mapping Ehrlichi promoters, 2) map transcriptional machinery of two differentially expressed genes of the p28-Omp locus, 3) evaluate the contributions of macrophage and tick cell environments for differential expression, and 4) develop methods to evaluate knockout mutations to assess the biological relevance of host cell specific differential expression. We successfully accomplished the goals of aims 1 and 2;although some progress was made in aim 3, we need to expand this area more in the renewed project.
Aim 4 is the most challenging because we are the first to develop methods in creating both targeted and random mutations in E. chaffeensis;we created mutations in several genomic locations and presented the first evidence in identifying genes essential for the pathogen's in vivo growth. Progress from the funded project forms the strong foundation for this renewal application.
Specific aims are 1) characterize E. chaffeensis RNA polymerase complex in support of understanding host-specific differential gene expression, 2) evaluate the significance of host-specific differential expression by characterizing mutations in three genes identified as essential for E. chaffeensis in vivo growth, and 3) perform mutational analysis and in vivo screening to identify additional genes essential for the E. chaffeensis pathogenesis in vertebrate and tick hosts.
The results from this study will provide important information for understanding E. chaffeensis pathogenesis, gene regulation and how the rickettsiale in vertebrate and tick hosts respond to the loss of expression from differentially expressed genes. This study will also allow us to determine how the tick transmitted pathogen persists and will aid in identifying targets for controlling E. chaffeensis infections.