Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media

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.

having a dense nucleoid, and cells with a larger, pleomorphic electron lucent form known as reticulate cells (RCs), which have a dispersed nucleoid (11,12). The DC is the infectious form, while the RC is noninfectious and replicates within a phagosome. Between 4 and 12 h, a phagocytized DC form transforms to the RC form and replicates within a phagosome until reverting to the DC form and undergoing subsequent release from the infected host cells by completing cell lysis or exocytosis (11,12).
The ability to grow obligate intracellular bacteria under axenic conditions can be a major advancement (13)(14)(15), as it will enable new paths of investigation, such as aiding the manipulation of the pathogenic organisms in the absence of host cells, clonal purification of bacterial mutants, and allowing detailed biochemical and genetic studies. 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). Similarly, we recently assessed the application of axenic media for E. chaffeensis, where we demonstrated both protein and DNA biosynthesis for the RC form of the pathogen (20). As protein synthesis and DNA replication are limited in the axenic media, we proposed several strategies to promote optimal cell-free bacterial replication (20). Furthermore, such methods to grow Anaplasma species pathogens in host cell-free media have yet to be developed.
Considering the potential advancements likely achieved by growth on axenic media of the pathogenic rickettsiae, we extended investigations in this follow-up study to improve the axenic growth conditions for E. chaffeensis and also initiated similar experiments for A. phagocytophilum. In this study, we present novel data demonstrating the purification of host cell-free phagosomes containing A. phagocytophilum or E. chaffeensis and then used the phagosomes to assess the bacterial protein and DNA synthesis under axenic medium conditions.

RESULTS
Rickettsia-containing phagosome purification and verification. 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 similarly E. chaffeensis-infected DH82 cells (Fig. 1). Confocal microscopy evaluation following staining with DAPI (49,6-diamidino-2-phenylindole) for nuclear material and phagosome membrane-specific Rab 5 monoclonal antibody, respectively, were used to confirm the purity of phagosomes containing the rickettsial organisms.
Assessment of protein synthesis in cell-free phagosomes in axenic media. Previously described axenic medium (20,21) was used with two different carbon/ energy sources-glucose-6-phosphate (G6P) or ATP-or by adding both metabolites to assess protein synthesis in cell-free phagosomes containing either A. phagocytophilum or E. chaffeensis. Rickettsiae containing cell-free phagosomes were incubated with the axenic media for 24 h at 37°C in a tri-gas incubator set to maintain 2.5% O 2 . The concentration of Cys and Met in the axenic media was reduced to 1 mM, and then the two radioactive amino acids were supplemented with 70 mCi of [ 35 S]Cys-Met. Protein synthesis was assessed by monitoring the [ 35 S]Cys-Met incorporation. Phagosomes isolated from the A. phagocytophilum-infected host cells and similarly from E. chaffeensiscontaining purified phagosomes utilized either G6P or ATP, as judged by the incorporation of [ 35 S]Cys-Met (Fig. 2). The protein synthesis was not significantly different for G6P or ATP alone, although the addition of both resulted in notable increase. The incorporation of radioactive amino acids was completely absent when chloramphenicol was added to the axenic medium to arrest protein synthesis.
Impact of pH on protein biosynthesis in cell-free phagosomes in the axenic media. We assessed the pH variations in promoting optimal protein biosynthesis in purified phagosomes containing either A. phagocytophilum or E. chaffeensis (Fig. 3).
Although the [ 35 S]Cys-Met incorporation was observed in the axenic media at pH variations from 5 to 8, the highest incorporation was observed for the media at pH 7 for both E. chaffeensis-and A. phagocytophilum-containing phagosomes.
Restricted protein biosynthesis observed when total protein synthesis was assessed by polyacrylamide gel electrophoresis and by Western blotting. We then assessed the level of protein biosynthesis by measuring protein levels relative to controls for both of the rickettsial organisms in host cell-free phagosomes maintained in the axenic media. Total protein profiles of the resolved proteins in a polyacrylamide gel following silver staining showed an increase in total proteins compared to the control reaction mixtures in which chloramphenicol was added ( Fig. 4A and B). However, the level of synthesized proteins appeared only moderately higher than that of the controls, as assessed for 24 h of incubation in the axenic media. Maximum total protein synthesis was observed for both A. phagocytophilum and E. chaffeensis phagosomes when the pH of the medium was adjusted to 7. These data were further confirmed following Western blot analysis ( Fig. 4C and D). Two different antibodies were used for this experiment: polyclonal sera raised against E. chaffeensis DnaK protein and a monoclonal antibody for the organism's outer membrane protein, P28-OMP19. The DnaK antibody detected a protein band of the expected size in E. chaffeensis, and this antibody also recognized a protein of similar size in A. phagocytophilum phagosome lysate, while the P28-OMP19 monoclonal antibody recognized only E. chaffeensis protein.
Independent of the variations in the pH of the axenic media, the increase in total protein synthesis was only moderate compared to the greatest difference in synthesis noted for the reaction for the media at pH 7 compared to the controls containing chloramphenicol.
DNA synthesis assessed in axenic media is analogous to protein biosynthesis. To determine if the axenic media also promoted DNA synthesis, rickettsia-containing phagosomes were incubated in the media containing [ 3 H]thymidine (Fig. 5). This experiment was also performed at variant pHs of the media. DNA synthesis was observed in the axenic media for purified phagosomes containing E. chaffeensis or A. phagocytophilum. Consistent with the [ 35 S]Cys-Met incorporation, the increase in DNA synthesis in the cell-free phagosomes incubated in the axenic media was observed, while maximum incorporation was detected for the media at pH 7.
RNA synthesis assessed in axenic media was also consistent with DNA and protein biosynthesis. We performed a quantitative reverse transcription-PCR (qRT-PCR) analysis targeting 16S rRNA for the purified phagosomes containing A. phagocytophilum or E. chaffeensis. The analysis was performed on RNA recovered at different hours of incubation in the axenic media for 0, 2, 6, 12, and 24 h. RNA samples in triplicate reactions were assessed for each incubation time point by TaqMan probe-based, real-time qRT-PCR targeting the bacterial 16S rRNA (Table 1). There was no significant change in the RNA expression levels between 0 h and 2 h of incubation for E. chaffeensis, while RNA copy numbers beyond 2 h for E. chaffeensis and for A. phagocytophilum starting at 2 h of incubation declined rapidly, possibly due to the rapid loss of viability of the cell-free rickettsia-containing phagosomes in the media after the first 2 h of incubation, where protein biosynthesis and DNA and RNA synthesis may have occurred.

DISCUSSION
The lack of methods to grow most of the obligate pathogens, including rickettsial pathogens, in the absence of host cells remains a major limiting factor hampering research progress (19,20). The importance of the application of the axenic medium method to grow an obligate pathogenic bacterium is well recognized from the rapid advances made following its development for Coxiella burnetii (14,(16)(17)(18)22). In an effort to develop such methods for rickettsial pathogens, we recently described the first evidence of application of axenic growth medium for E. chaffeensis (20). In that study, we demonstrated protein and DNA synthesis only in the RC form, but not in the DC form, in axenic medium formulations for host cell-free E. chaffeensis. We recognized that additional investigations are necessary to improve the experimental conditions optimal for promoting the continued replication of E. chaffeensis organisms (20). These observations are similar to a prior study demonstrating cell-free protein biosynthesis for C. trachomatis (19). Furthermore, cell-free medium growth studies have not been investigated to date for any Anaplasma species pathogens. We reasoned that the axenic medium growth of the cell-free RC form of E. chaffeensis may be improved if assessed with purified, host cell culture-derived E. chaffeensis in phagosomes in place of purified RCs. The phagosome microenvironment might mimic in vivo conditions. In the current study, therefore, we investigated this goal for E. chaffeensis. The investigations were also extended to A. phagocytophilum as there was no prior research focused on development of axenic media for any known Anaplasma species pathogens. Anaplasma species pathogens resulting from tick transmission cause significant morbidity and mortality in various agricultural and companion animals, besides impacting human health (23). Similarly, infections with several tick-borne Ehrlichia species pathogens impact the health of companion and agricultural animals and people (1,24). The availability of axenic medium culture systems for Anaplasma and Ehrlichia species pathogens, therefore, will greatly aid in advancing our understanding of these important microorganisms.
We first standardized the method to purify rickettsia-containing phagosomes and then utilized the purified phagosomes to investigate protein and DNA synthesis with axenic media under microaerophilic conditions. Similar to our prior observations for the host cell-free RC form of E. chaffeensis, the current study demonstrated protein biosynthesis and DNA synthesis in the axenic media for both E. chaffeensis-and A. phagocytophilum-containing phagosomes. Axenic medium-specific protein biosynthesis and DNA synthesis were confirmed for purified phagosomes with the inclusion of chloramphenicol in the media to serve as the protein synthesis inhibitor. The rickettsial protein and DNA biosynthesis was reported for the first time for phagosomes in vitro in the current study. While it is significant to demonstrate the data for host cell-free phagosomes containing rickettsial organisms, there was considerably less protein and DNA  biosynthesis. These data are similar to our prior observations for the purified RC form of E. chaffeensis (20). The assessment of total proteins synthesized in axenic media for phagosomes, as judged from the total protein analysis of silver-stained gels of resolved proteins and by Western blotting, suggested only a moderate enhancement of protein synthesis for both A. phagocytophilum and E. chaffeensis. These data are similar to our earlier reported axenic medium data for the host cell-free E. chaffeensis RCs (20).
Notably, contrary to our predictions, axenic media promoted only limited protein and DNA synthesis for Ehrlichia-or Anaplasma-containing phagosomes. Albeit the current study is a major step forward for improving growth on axenic media because this is the first study examining the utility of purified phagosomes containing two important rickettsial pathogens belonging to members of the Anaplasmataceae family, additional modifications are required to stimulate greater DNA and protein biosynthesis. We recognized that the in vitro-purified E. chaffeensis-and A. phagocytophilum-containing phagosomes rapidly lost their viability in the axenic medium culture conditions, as judged from the rapid loss of RNA copy number after about 2 h of incubation under the axenic medium conditions. The reasons for the rapid decline in viability are unclear. One possibility could be the extended time involved in the phagosome purification procedure. Alternatively, the axenic medium conditions may require further optimization. Importantly, it is evident that improvements should be made to sustain the viability during the axenic medium incubation time. We are currently investigating ways to reduce the purification time. Additional advances are also necessary to maintain the integrity of the recovered rickettsia-containing phagosomes, which may include the choice of harvest time following in vitro culture setup, as well as the host cell selection for propagation of A. phagocytophilum and E. chaffeensis, all of which may contribute to the stability of phagosomes and the promotion of growth on axenic media.
While the purification protocol is more laborious and time-consuming than purifying the RC form of bacteria, improvements may be possible to promote the bacterial growth in cell-free rickettsia-containing phagosomes. Transmission electron microscopy studies by us and other investigators demonstrated that mitochondria are closely associated with E. chaffeensis-containing phagosome vacuoles of infected host cells (11,25,26). E. chaffeensis and other Anaplasmataceae pathogens may benefit from mitochondria in multiple ways, including obtaining energy and metabolites. Axenic medium growth of A. phagocytophilum and E. chaffeensis in phagosomes recovered from in vitro cultures, therefore, may also be improved by the addition of purified mitochondria to the axenic media. Likewise, axenic medium growth for the RC forms of both Anaplasma and Ehrlichia species may be perfected in ways to promote bacterial synthesis in vitro. We believe that the complementary approaches using host cell-free rickettsia-containing phagosomes and RC forms may need to be simultaneously investigated to advance the goal of developing optimal protocols for axenic growth of Anaplasmataceae pathogens.
The current study describing the established methods of phagosome purification and their use in promoting protein and DNA biosynthesis in vitro under axenic medium culture conditions forms a strong foundation for making further improvements to promote sustained and enhanced replication of pathogenic rickettsiae in the absence of host cell support. Future investigations may also be extended to important tick-borne obligate rickettsial pathogens of the genera Anaplasma, Ehrlichia, and Neorickettsia to promote axenic medium growth to induce rapid bacterial replication and progression from the RC form to DC form. The host cell support-free axenic growth of obligate Anaplasmataceae pathogens will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

MATERIALS AND METHODS
Cell lines and cultivation of A. phagocytophilum and E. chaffeensis. Cells of the human promyelocytic cell line HL-60 (ATCC CCL-240; ATCC, Manassas, VA), uninfected or infected with A. phagocytophilum strain NCH-1, were cultured in complete RPMI 1640 medium (Gibco/Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen/Thermo Fisher Scientific, Waltham, MA) and 2 mM L-glutamine (Mediatech, Manassas, VA) by following the protocols described in reference 7. Cultivation of E. chaffeensis in DH82 cells was performed in complete RPMI 1640 medium (Gibco/Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen/Thermo Fisher Scientific) and 2 mM L-glutamine (Mediatech) as per protocols described previously (20). To prepare cell-free inocula, about 80 to 100% A. phagocytophilum-infected HL-60 cells or E. chaffeensis-infected DH82 cells from T25 flasks were harvested by centrifugation at 400 Â g for 10 min at 4°C. The pellets were resuspended in 5 ml of serum-free medium, and the cells were disrupted with glass beads by vortexing twice for 30 s. The cell debris and unbroken cells were removed by centrifugation at 200 Â g for 10 min at 4°C. The supernatant was passed through a 2.7-mm-pore syringe filter (Whatman, Pittsburgh, PA). HL-60 and DH82 cells were infected with host cell-free A. phagocytophilum and E. chaffeensis, respectively (at a multiplicity of infection of 100:1 bacteria to host cells) for 2 h to allow internalization. Noningested rickettsial organisms were removed by washing with phosphate-buffered saline (PBS), and the cells were incubated for 36 to 48 h in a T150 flask. The infected host cell cultures were harvested by centrifugation at 600 Â g for 5 min at 4°C and used for purification of the host cell-free phagosomes, as outlined below.
Purification of phagosomes. Purification of phagosomes from the A. phagocytophilum-infected HL-60 cells and E. chaffeensis-infected DH82 cells was performed by subjecting them to discontinuous sucrose density gradient centrifugation (27) in combination with magnet-assisted cell sorting (MACS) as described previously (28,29), with some minor modifications. In brief, infected host cells were pelleted at 4°C for 5 min at 350 Â g. The cells were washed twice with PBS and once with homogenization buffer (250 mM sucrose, 0.5 mM EGTA, and 20 mM HEPES-KOH at pH 7.2). The cells were then resuspended in homogenization buffer with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The cells were then homogenized at 4°C using a 10-ml syringe with a 23.5-gauge needle; typically, 10 to 15 strokes were used to disrupt the cells. Homogenization was carried out until approximately 90% of cells were disrupted without major breakage of nuclei, as monitored by light microscopy. The unlysed host cells and nuclei were then pelleted in a 15-ml tube centrifuged at 300 Â g at 4°C for 5 min. The resulting supernatant containing phagosomes, which was designated the postnuclear supernatant (PNS), was brought to a final concentration of 39% sucrose. The discontinuous sucrose gradient was made by layering 1 ml of PNS in 39% sucrose onto 2 ml of 55% sucrose layered onto 1 ml of 65% sucrose. We then layered 2 ml of 25% sucrose followed by 2 ml of 10% sucrose solution. The gradient was centrifuged at 100,000 Â g for 1 h at 4°C with an S50-ST swinging bucket rotor in a Sorvall MTX150 ultracentrifuge (Waltham, MA). Phagosomes were then recovered from the interface between 55% and 65% sucrose using a 16-gauge needle carefully without disrupting other gradient fractions. Subsequently, the MACS separation step was performed by incubation of the crude phagosomes with rabbit Rab5 antibody (1:1,000; Cell Signaling Technology, Danvers, MA) for 1.5 h, followed by incubation with a secondary goat anti-rabbit antibody with magnetic beads (1:100; Miltenyi Biotec, San Diego, CA) for another 1.5 h, and then loaded onto a MACS LS separation column (Miltenyi Biotec, San Diego, CA). The column was washed three times with 2 ml HSMG (20 mM HEPES, 250 mM sucrose, 1.5 mM MgCl 2 , 0.5 mM EGTA at pH 7.4), and on removal of the magnet, the phagosomes were eluted in 3 ml HSMG. The elution product was placed into 10 ml of PBS (4°C) and centrifuged at 40,000 Â g for 30 min at 4°C with an S50-ST swinging bucket rotor in a Sorvall MTX150 ultracentrifuge. The final purified pellets were resuspended in PBS for immediate use in the cell-free axenic medium assessment experiments.
Confocal microscopy analysis. A fraction of purified phagosomes was plated onto 8-well culture chamber slide to adhere for 1 h, and then stained with rabbit Rab5 (1:1000) (Cell Signaling Technology, Danvers, MA) antibody for 1.5 h and with Alexa Fluor 488-conjugated goat anti-rabbit antibody for 1 h. The slides were washed with PBS and mounted with mounting medium containing DAPI. For staining of infected HL-60 cells, the infected HL-60 cells were plated onto 8-well culture chamber slide and allowed to adhere for 1 h in the 37°C incubator. The cells were fixed with 4% formaldehyde for 10 min at room temperature and permeabilized with 0.1% TX-100 in PBS for 10 min. Subsequently, the cells were stained with rabbit Rab 5 antibody (1:1,000; Cell Signaling Technology) overnight at 4°C. The antigen slides were washed with PBS to remove unbound primary antibody and incubated with second antibody (Alexa Fluor 488-conjugated goat anti-rabbit antibody) for 1 h. The slides were washed 3 times with PBS and mounted with the mounting medium containing DAPI. The slides were then examined with a Zeiss LSM 700 laser scanning confocal microscope (Carl Zeiss Optronics GmbH, Oberkochen, Germany).
Preparation of axenic media. The axenic media were prepared essentially as described previously as per the compositions and concentrations outlined by Omsland et al. (21). As per the experimental variability, the medium contained glucose 6-phosphate (G6P), ATP, or both. Similarly, the pH of the media was modified as per the experimental need.
Protein synthesis by [ 35 S]Cys-Met incorporation. Protein synthesis in cell-free purified phagosome fractions containing E. chaffeensis or A. phagocytophilum was measured by incorporation of [ 35 S]Cys-Met (PerkinElmer, Waltham, MA) as we described previously (20). Microcentrifuge tubes containing purified phagosome fractions in 500 ml of axenic medium supplemented with 70 mCi of [ 35 S]Cys-Met were incubated at 37°C for 24 h in a tri-gas humidified incubator set to maintain 2.5% O 2 . The phagosomes containing rickettsial organisms were pelleted at the end of incubation by centrifugation at 15,000 Â g for 15 min at 4°C, washed with K-36 buffer (0.05 M K 2 HPO 4 , 0.05 M KH 2 PO 4 , 0.1 M KCl, 0.15 M NaCl, at pH 7.0) twice, and disrupted by adding 30 ml of 2Â SDS-PAGE sample buffer and by boiling for 5 min (20). Ten microliters of lysate each was then transferred to a tube containing 5 ml of Biosafe liquid II and used for quantification of [ 35 S]Cys-Met incorporation using the protocol 4 ( 35 S) in a liquid scintillation counter (Tri-Carb 2100TR; PerkinElmer). For visualization of the radiolabel incorporation into bacterial proteins, equal volumes of sample lysates were resolved by SDS-PAGE, and the gel was dried and exposed to an X-ray film. Similarly, cell-free growth experiments were carried out in the absence of [ 35 S]Cys-Met, resolved on an SDS-PAGE gel, and stained using a silver staining kit (Thermo Fisher Scientific, Waltham,