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Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis.

ABSTRACT: Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen) and surE (stationary-phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis SurE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in co-ordinating development of the stress resistance for passage from the present to the next host cells through its regulon.

SUBMITTER: Cheng Z

PROVIDER: S-EPMC3241975 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis.

Cheng Zhihui Z Miura Koshiro K Popov Vsevolod L VL Kumagai Yumi Y Rikihisa Yasuko Y

Molecular microbiology 20111107 5

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptid ...[more]

PMID: 22014113

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Project description:UNLABELLED:How the obligatory intracellular bacterium Ehrlichia chaffeensis begins to replicate upon entry into human monocytes is poorly understood. Here, we examined the potential role of amino acids in initiating intracellular replication. PutA converts proline to glutamate, and GlnA converts glutamate to glutamine. E. chaffeensis PutA and GlnA complemented Escherichia coli putA and glnA mutants. Methionine sulfoximine, a glutamine synthetase inhibitor, inhibited E. chaffeensis GlnA activity and E. chaffeensis infection of human cells. Incubation of E. chaffeensis with human cells rapidly induced putA and glnA expression that peaked at 24 h postincubation. E. chaffeensis took up proline and glutamine but not glutamate. Pretreatment of E. chaffeensis with a proline transporter inhibitor (protamine), a glutamine transporter inhibitor (histidine), or proline analogs inhibited E. chaffeensis infection, whereas pretreatment with proline or glutamine enhanced infection and upregulated putA and glnA faster than no treatment or glutamate pretreatment. The temporal response of putA and glnA expression was similar to that of NtrY and NtrX, a two-component system, and electrophoretic mobility shift assays showed specific binding of recombinant E. chaffeensis NtrX (rNtrX) to the promoter regions of E. chaffeensis putA and glnA. Furthermore, rNtrX transactivated E. chaffeensis putA and glnA promoter-lacZ fusions in E. coli. Growth-promoting activities of proline and glutamine were also accompanied by rapid degradation of the DNA-binding protein CtrA. Our results suggest that proline and glutamine uptake regulates putA and glnA expression through NtrY/NtrX and facilitates degradation of CtrA to initiate a new cycle of E. chaffeensis growth. IMPORTANCE:Human monocytic ehrlichiosis (HME) is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. HME is caused by infection with E. chaffeensis, an obligatory intracellular bacterium in the order Rickettsiales, which includes several category B/C pathogens, such as those causing Rocky Mountain spotted fever and epidemic typhus. The limited understanding of the mechanisms that control bacterial growth within eukaryotic cells continues to impede the identification of new therapeutic targets against rickettsial diseases. Extracellular rickettsia cannot replicate, but rickettsial replication ensues upon entry into eukaryotic host cells. Our findings will provide insights into a novel mechanism of the two-component system that regulates E. chaffeensis growth initiation in human monocytes. The result is also important because little is known about the NtrY/NtrX two-component system in any bacteria, let alone obligatory intracellular bacteria. Our findings will advance the field's current conceptual paradigm on regulation of obligatory intracellular nutrition, metabolism, and growth.

Dataset Information

Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis.

Publications

Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis.

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