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

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210

^* To whom correspondence should be addressed. Email: rikihisa.1{at}osu.edu.

	Abstract

The type IV secretion (T4S) system is critical for the virulence of several pathogens. In the rickettsial pathogen, Ehrlichia chaffeensis, virB/D genes are split into two operons: virB3–virB6 (preceded by sodB) and virB8–virD4. 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 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 virB/D 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 named EcxR. Recombinant EcxR bound to the promoter regions upstream of five individual virB/D loci. EcxR also activated transcription of all five virB/D loci in lacZ reporter constructs. The expression of ecxR was positively autoregulated by EcxR. These results suggest that the five virB/D loci are coordinately regulated by EcxR to allow developmental stage–specific expression of the T4S system in E. chaffeensis.