Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein

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Abstract
The type IV secretion (T4S) system is critical for the virulence of several pathogens. In the rickettsial pathogen Ehrlichia chaffeensis , the virBD genes are split into two operons, the virB3 - virB6 (preceded by sodB ) and virB8 - virD4 operons. Between these two operons, there are duplications of virB4 , virB8 , and virB9 . In this study we found that transcription of all five loci was downregulated prior to the release of E. chaffeensis from host THP-1 cells and was upregulated at the initiation of exponential growth. Electrophoretic mobility shift assays revealed an E. chaffeensis -encoded protein that specifically bound to the promoter regions upstream of the virBD loci. The protein was purified from the bacterial lysate by affinity chromatography using a biotinylated promoter region upstream of sodB . Mass spectrometry identified the protein as an E. chaffeensis 12.3-kDa hypothetical protein, which was designated EcxR. Recombinant EcxR bound to the promoter regions upstream of five individual virBD loci. EcxR also activated transcription of all five virBD loci in lacZ reporter constructs. The expression of ecxR was positively autoregulated by EcxR. These results suggest that the five virBD loci are coordinately regulated by EcxR to allow developmental stage-specific expression of the T4S system in E. chaffeensis.

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