Molecular evaluation of Ehrlichia chaffeensis

by Sirigireddy, Kamesh Reddy

Abstract (Summary)
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.

Bibliographical Information:


School:Kansas State University

School Location:USA - Kansas

Source Type:Master's Thesis

Keywords:ehrlichia chaffeensis microarray genetic manipulation gene expression biology microbiology 0410 molecular 0307 veterinary science 0778


Date of Publication:01/01/2008

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