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Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis, a Gram-negative organism belonging to the order Rickettsiales, is responsible for an emerging infectious disease in humans, the human monocytic ehrlichiosis. E. chaffeensis also infects several other vertebrate hosts including dogs, goats, coyotes and white tailed deers. This organism is transmitted by an infected tick, Amblyomma americanum. The exact pathogenic mechanisms involved for the persistence of the pathogen in vertebrate hosts are still unclear. E. chaffeensis protein expression varies significantly in vertebrate and tick hosts. Differentially expressed proteins include the immunodominant outer membrane proteins encoded by the p28-Omp multigene locus. The p28-Omp 14 is expressed primarily in tick cells and the p28-Omp 19 is the major expressed protein in macrophages both under in vitro and in vivo conditions. The objective of this study is to prepare recombinant proteins and use them to assess the secondary structures and protein functions. The protein sequences were analyzed with the aid of bioinformatics programs to make structural predictions. The analysis suggested the presence of eight β barrel structures for both the p28-Omp proteins. The coding sequence of the p28-Omp genes were cloned and over expressions of proteins in in E. coli was accomplished by using the plasmid expression construct, pET28. The proteins were purified to near homogeneity and used to refold using detergents to mimic native protein structure in the bacterial outer membrane. Refolding of proteins was analyzed by two methods; SDS-PAGE and Circular Dichroism. The Circular dichroism spectroscopy analysis suggested the formation of β-sheet structures of proteins in micelles formed with the detergents. β-sheet structures may have been formed with the hydrophobic domains of the protein imbedded in the micelles. The hydrophilic segments (predicted by bio informatics analysis) may be exposed to the aqueous phase. The recombinant proteins were also iii used to prepare proteoliposomes and tested for the porin activity. The analysis demonstrated the porin activity for both p28-Omp 14 and 19 recombinant proteins by using mono-, di- and tetra- saccharides as well as for amino acid L-glutamine. This study forms the basis for initiating studies to compare the structural difference between the two differentially expressed proteins of E. chaffeensis.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis, an emerging tick-borne pathogen, causes human monocytic ehrlichiosis (HME). The relationship between E. chaffeensis and its target cells in ticks and vertebrates is critical as the organism must persist in them. We hypothesize that E. chaffeensis alters gene expression in support of adapting to dual hosts. In support of testing this hypothesis, we developed an ORF-based microarray and performed global transcriptional analysis on the pathogen grown in macrophage and tick cells. The analysis revealed the expression of about 30% of all the predicted E. chaffeensis genes, in macrophages or tick cell. Two-thirds of the transcribed genes are common for both host cell backgrounds. About 20% of the commonly expressed genes also varied in expression levels which ranged from two to five fold. Microarray data was verified by RT-PCR for a subset of randomly selected genes. Together, this is the first report describing the global host cell-specific gene expression patterns in E. chaffeensis. Differential gene expression may be an important adaptive mechanism used by E. chaffeensis for its continued survival in dual hosts. To test this hypothesis, we established many basic protocols and tools needed for performing mutational analysis in E. chaffeensis. Four antibiotic selection markers; gentamicin, chloramphenicol, spectinomycin and rifampin, and two promoters constitutively expressed in E. chaffeensis, genes rpsL and tr, were identified. Two regions of the genome were also identified for performing initial mutational analysis. Several plasmid constructs were also made. The optimal conditions for introducing these plasmids into host cell-free viable E. chaffeensis organisms were also established. The molecular evaluation of several E. chaffeensis transformants using these plasmids suggested that the plasmids gained entry, but failed to get integrated into the genome or remain in the bacteria for longer periods of time. In summary, we demonstrated global host cell-specific differential gene expression in E. chaffeensis by employing microarray analysis. Numerous host-specific expressed genes will be important for studies leading to effective methods of control. We also established several basic protocols and tools needed for performing mutational analysis useful in evaluating the impact of the loss of expression of uniquely expressed genes.

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