Ehrlichia chaffeensis, an obligatory intracellular bacterium of human monocytes-macrophages, causes human monocytic ehrlichiosis (HME). The bacterium has a biphasic developmental cycle in mammalian cells that alternates between a small ‘dense-cored cell’ (DC) and a large ‘reticulate cell’ (RC), as defined by morphological features, differential surface protein expression, and infectivity. However, how E. chaffeensis intracellular development is regulated is poorly understood.
The type IV secretion (T4S) system is critical for the virulence of several pathogens. E. chaffeensis T4S apparatus, 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. We found that the transcription of all five loci was down-regulated prior to the release of E. chaffeensis from host THP-1 cells and up-regulated at the initiation of the exponential growth. An E. chaffeensis 12.3-kDa hypothetical protein, EcxR, specifically bound to the promoter regions upstream of virB/D loci. EcxR also activated transcription of all five virB/D loci in lacZ reporter constructs. The expression of ecxR was positively auto-regulated 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.
The two-component regulatory system (TCS) composed of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through the activation or repression of specific genes. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host-cell-free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells one day post infection with closantel cleared the infection in dose-dependent manner. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro. A number of E. chaffeensis genes, including the six TCS genes, were down-regulated within 5-60 min post closantel treatment. These results suggest that these TCSs play an essential role in the infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes.
Caulobacter crescentus CtrA (cell cycle transcription regulator A) is a transcriptional regulator that allows the coordination of cell cycle progression and morphogenesis by controlling the expression levels of ~100 genes. In E. chaffeensis, CtrA mRNA and protein levels were down-regulated within 6 h post infection in synchronously infected cell culture, and significantly up-regulated at the late stage of infection prior to the bacterial release. C. crescentus CtrA-binding motif (CtrA box) was predicted in the upstream regions of more than 30 E. chaffeensis genes. Temporal mRNA expressions of these genes were divided into four patterns: 1) up-regulated at the late exponential phase, similar to CtrA, 2) up-regulated at the lag phase and down-regulated during the exponential phase, 3) up-regulated at the lag phase but not down-regulated during the exponential phase, 4) constitutively expressed. E. chaffeensis CtrA specifically bound to the promoter regions of E. chaffeensis ctrA and surE, and rCtrA transactivated surE promoter-lacZ reporter constructs. These results suggest that CtrA is a global regulator controlling the intracellular development of E. chaffeensis.
In E. chaffeensis, lipoproteins are required for bacterial infection to the host cells. OmpA (outer membrane protein A, also known as a peptidoglycan-associated lipoprotein) was E. chaffeensis surface exposed. E. chaffeensis binding and infection to the host cells were inhibited by rabbit anti-OmpA IgG. OmpA mRNA and protein expressions were down-regulated during the lag phase and the early exponential phase, but highly up-regulated at the late exponential phase. CtrA-binding site was predicted in the promoter region of ompA gene. CtrA bound to this region and activated OmpA expression. The results suggest that CtrA regulates ompA expression, consequently, E. chaffeensis infection of host cells.