Ehrlichia species pathogenesis and vaccine development

Abstract

Ehrlichia chaffeensis and Ehrlichia canis are tick-transmitted, small, obligate intracellular bacteria belonging to the family Anaplasmataceae within the order Rickettsiales. These organisms are known to cause infections in dogs and humans with a primary tropism to monocytes and macrophages. Depending on the host's health status and age, both pathogens can cause a range of clinical signs, from asymptomatic to severe diseases, potentially leading to death or permanent disabilities. E. chaffeensis infections are predominantly reported in North America in humans and dogs, while E. canis infections are documented worldwide, mostly in dogs, with isolated infections documented in humans in parts of South American countries, Mexico, and Europe. Currently, there are no vaccines for any Ehrlichia species, and the overall understanding of essential bacterial genes and host responses to these pathogens remains limited. Therefore, studies focused on vaccine development for both species and on understanding pathogenesis through bacterial protein characterization are of critical importance. Using the previously developed live-modified vaccine (MLV) with disruption in the gene ECH_0660 encoding for the phage head-to-tail connector protein (Phtcp) of E. chaffeensis, we investigated to determine whether this MLV prepared from the human infection-derived Arkansas isolate would provide protection against genetically distinct human isolates of E. chaffeensis, Heartland and Wakulla (chapter 2). Vaccine research was extended to E. canis, targeting first the deletion of the phtcp homolog without introducing an antibiotic-resistant cassette, and then using it to determine whether it serves as a modified live vaccine in conferring protection against wild-type infection challenge (chapter 3); both studies were carried out using the canine infection challenge model. Considering our research group's substantial progress in demonstrating the critical nature of functional phtcp for in vivo growth and persistence for E. chaffeensis, research was extended to characterize this gene and its neighboring six gene cluster spanning ECH_0659-ECH_0665 as four of the 7 gene segment having origins of a phage genome (chapter 4). Finally, investigations were extended to characterize E. chaffeensis type four secretion system (T4SS) effector protein (Etf1), by utilizing the genetically modified mutational construct expressing it as a fusion protein with a C-terminal enhanced green fluorescent protein (EGFP) tag. Our results demonstrated that 1) the phtcp mutant strain of E. chaffeensis as an MLV provide a broad protection against genetically distinct virulent strains of the pathogen; 2) similarly, prior infection of the phtcp homolog mutant in E. canis provides immune protection against subsequent wild-type challenge in the canine host; 3) genes from the 7-gene segment spanning ECH_0659 to ECH_0665 are differentially expressed in distinct strains of E. chaffeensis, and all 7 proteins were expressed and localized on the bacterial and/or phagosomal membranes, with potentially forming complexed by ECH_0661 and ECH_0663, and 4) Etf-1 translocation into host cell cytoplasm was confirmed with tracking its expression using EGFP; coimmunoprecipitation studies revealed its interaction with multiple host proteins involved in transport including its direct interactions with ER chaperone proteins. Similarly, Etf1 interacts with trans-Golgi networks but not with cis-Golgi networks. In conclusion, the studies highlight the importance of identifying bacterial essential genes and characterizing proteins encoded by them for defining pathogenesis and in developing methods of prevention.