Abstract
Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens-Anaplasma phagocytophilum and Ehrlichia chaffeensis-in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.
Highlights
Members of the Anaplasmataceae family, including Anaplasma phagocytophilum and Ehrlichia chaffeensis, are obligate, Gram-negative, intracellular rickettsiae responsible for causing in humans the acute febrile illnesses human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME) [1], respectively
We adopted the ultracentrifugation method using discontinuous sucrose density gradient coupled with magnet-assisted cell sorting (MACS) to purify phagosomes from A. phagocytophiluminfected HL-60 cells and E. chaffeensis-infected DH82 cells (Fig. 1)
Phagosomes isolated from the A. phagocytophilum-infected host cells and from E. chaffeensiscontaining purified phagosomes utilized either glucose 6-phosphate (G6P) or ATP, as judged by the incorporation of [35S]Cys-Met incorporation using the protocol 4 (35S)]Cys-Met (Fig. 2)
Summary
Members of the Anaplasmataceae family, including Anaplasma phagocytophilum and Ehrlichia chaffeensis, are obligate, Gram-negative, intracellular rickettsiae responsible for causing in humans the acute febrile illnesses human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME) [1], respectively These pathogens cause infections in several vertebrate hosts [1]. The development of axenic medium for growth and its application are well documented in another important obligate bacterium, Coxiella burnetii, and the method aided greatly in studies focused on biochemical and genetic studies of the pathogen [16,17,18] While such efforts have been attempted for another important bacterial pathogen, Chlamydia trachomatis, only limited protein synthesis was reported [19]. We present novel data demonstrating the purification of host cell-free phagosomes containing A. phagocytophilum or E. chaffeensis and used the phagosomes to assess the bacterial protein and DNA synthesis under axenic medium conditions
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