BIOSYNTHETIC ACTIVITY OF THE ANAPLASMA PHAGOCYTOPHILUM AND EHRLICHIA CHAFFEENSIS PHAGOSOME IN A HOST CELL-FREE MEDIUM
20210024879 ยท 2021-01-28
Inventors
Cpc classification
C12N2500/42
CHEMISTRY; METALLURGY
C12N2500/60
CHEMISTRY; METALLURGY
International classification
Abstract
Axenic media and methods for growing E. Chaffeensis and/or A. phagocytophilum are provided. In general, the axenic media includes intracellular phosphate buffer (IPB), a carbon source, FBS, a mixture of amino acids, and at least one further component selected from the group consisting of glucose 6-phosphate (G6P), ATP, DTT, GTP, UTP, CTP, and any combination thereof.
Claims
1. A composition comprising: a) intracellular phosphate buffer (IPB); b) a carbon source; c) FBS; d) a mixture of amino acids; and e) at least one further component selected from the group consisting of glucose 6-phosphate (G6P), ATP, DTT, GTP, UTP, CTP, and any combination thereof.
2. The composition of claim 1 further comprising phagosomes and/or mitochondria.
3. (canceled)
4. The composition of claim 1, further comprising a bacterium from the family Anaplasmataceae.
5. The composition of claim 4, wherein the bacterium is selected from the group consisting of Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Ehrlichia ruminantium, Ehrlichia muris euclarancis, Anaplasma marginale, Anaplasma phagocytophilum, and any combination thereof.
6. The composition of claim 1, wherein said IPB comprises a mixture of at least one phosphate, at least one gluconate, and at least one chloride.
7. The composition of claim 6, wherein said at least one phosphate is selected from the group consisting of potassium phosphate, sodium phosphate, and any combination thereof, and/or wherein said at least one chloride is selected from the group consisting of potassium chloride, magnesium chloride, and any combination thereof.
8. The composition of claim 6, wherein said at least one phosphate is included at a final concentration of at least 1 mM and/or wherein said gluconate is included at a final concentration between 70 mM and 150 mM, and/or wherein said at least one chloride is included in the medium at a final concentration between 0.5 mM and 12 mM.
9. The composition of claim 6, wherein said phosphate comprises a mixture of potassium phosphate and sodium phosphate.
10-12. (canceled)
13. The composition of claim 1, wherein said carbon source is alpha ketoglutarate and/or sodium acetate.
14. The composition of claim 1, wherein said carbon source is included at a concentration of at least 0.1 mM.
15. The composition of claim 1, wherein said mixture of amino acids includes at least 5 different amino acids.
16. The composition of claim 1, wherein any two of GTP, UTP, and CTP are present.
17. The composition of claim 1, wherein when ATP is present, a quantity of DTT is also included.
18. A method of cell free protein synthesis of a bacterium from the family Anaplasmataceae comprising the step of contacting the bacteria with the composition of claim 1 for a period of time sufficient to permit protein synthesis.
19. The method of claim 18, wherein the time period is at least 12 hours and/or wherein said bacteria are surrounded by said composition and/or wherein said composition has a pH between 5 and 9 and/or wherein the cell free protein synthesis is performed at a temperature between 24 C. to 40 C.
20-23. (canceled)
24. The method of claim 18, wherein said bacterium is selected from the group consisting of Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Ehrlichia ruminantium, Ehrlichia muris euclarancis, Anaplasma marginale, Anaplasma phagocytophilum, and any combination thereof.
25. A method of cell free nucleic acid synthesis of a bacteria from the family Anaplasmataceae comprising the step of contacting the bacteria with the composition of claim 1 for a period of time sufficient to permit nucleic acid synthesis.
26. The method of claim 25, wherein the time period is at least 12 hours and/or wherein said bacteria are surrounded by said composition and/or wherein said composition has a pH between 5 and 9 and/or wherein the cell free protein synthesis is performed at a temperature between 24 C. to 40 C.
27-28. (canceled)
29. The method of claim 25, wherein the time period is between 1 day and 7 days.
30. (canceled)
31. The method of claim 25, wherein said bacterium is selected from the group consisting of Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Ehrlichia ruminantium, Ehrlichia muris euclarancis, Anaplasma marginale, Anaplasma phagocytophilum, and any combination thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Color Drawings
[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION
[0049] The following detailed description and examples set forth preferred materials and procedures used in accordance with the present disclosure. It is to be understood, however, that this description and these examples are provided by way of illustration only, and nothing therein shall be deemed to be a limitation upon the overall scope of the present disclosure. It should be appreciated that when typical reaction conditions (e.g., temperature, reaction times, etc.) have been given, the conditions both above and below the specified ranges can also be used, though generally less conveniently. The examples are conducted at room temperature (about 23 C. to about 28 C.) and at atmospheric pressure unless noted otherwise. All parts and percents referred to herein are on a weight basis and all temperatures are expressed in degrees centigrade unless otherwise specified. Further unless noted otherwise, all components of the disclosure are understood to be disclosed to cover comprising, consisting essentially of, and consisting of claim language as those terms are commonly used in patent claims.
EXAMPLE 1
[0050] This example evaluates the possibility of developing axenic culture methods for E. chaffeensis using a medium previously described for C. trachomitis, referred to as the CIP-1 medium. The results are described using the axenic medium for its value in supporting both protein and DNA biosynthesis in DC and RC forms of E. chaffeensis in the absence of host cells.
[0051] Materials and Methods
[0052] Cultivation of E. chaffeensis: E. chaffeensis was cultivated in canine macrophage cell line, DH82 as described previously. Similarly, E. chaffeensis in Vero cells (ATCC, Manasas, Va.) was cultured in the complete MEM medium (Gibco/ThermoFisher Scientific, Waltham, Mass.) supplemented with 7% fetal bovine serum (Invitrogen/ThermoFisher Scientific, Waltham, Mass.) and 2 mM L-glutamine (Mediatech, Manassas, Va.). Cultivation of E. chaffeensis in HL60 cells (ATCC, Manassas, Va.) in complete RPMI 1640 medium (Gibco/ThermoFisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen/ThermoFisher Scientific, Waltham, Mass.) and 2 mM L-glutamine (Mediatech, Manassas, Va.), by following the protocols described elsewhere for Anaplasma phagocytophilum strain NCH-1. To prepare cell-free Ehrlichia inocula, about 80-100% E. chaffeensis-infected DH82 cells in a T25 flask were harvested by centrifugation at 400g for 10 minutes 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 seconds. The cell debris and unbroken cells were removed by centrifugation at 200g for 10 minutes at 4 C. The supernatant was passed through a 2.7 m pore-size syringe filter (Whatman, Pittsburgh, Pa.). HL60 cells were incubated with host cell-free E. chaffeensis (multiplicity of infection of 100:1, bacteria to host cell) for 120 minutes to allow for internalization. Non-ingested E. chaffeensis were removed by washing with PBS, and the cells were incubated for an additional 3 days in T150 flask. Similar infection protocol is followed when infecting Vero cells or DH82 cells. When the infectivity reached to 80-90%, the infected host cell cultures were harvested by centrifugation at 500g for 5 minutes at 4 C. and used for purifying the host cell-free bacteria, as outlined below.
[0053] Purification of E. chaffeensis: E. chaffeensis organisms in the forms of dense-core cells (DCs) and reticulate cells (RCs) were purified by subjecting to renografin density gradient centrifugation as described previously with some minor modifications. In brief, pellets of infected host cells were suspended in sterile PBS. The cells were then homogenized at 4 C. using a 10 ml syringe with a 23.sup.-G needle; typically 10-15 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 disrupted cell suspension was centrifuged at 500g for 5 minutes at 4 C. The supernatant was collected and filtered through 2.7 m sterile syringe filter. The filtered supernatant was then centrifuged at 15,000g for 15 minutes at 4 C. The pellet was resuspended into sterile PBS and 2 mL of the suspension was layered over discontinuous renografin gradients (3 mL 25%, 4 mL of 35% renografin in PBS, vol/vol). These gradients were centrifuged at 100,000g for 1 h at 4 C. using a Swinging Bucket rotor (S50-ST) in a Sorvall MTX150 ultracentrifuge (Waltham, Mass.). Fractions at the interfaces of PBS-25% and 25-35% renografin were collected using a sterile syringe, diluted with three volumes of PBS, and then centrifuged at 15,000g for 15 minutes at 4 C. The pellets were washed with PBS to remove residual renografin by repeating the centrifugation step 15,000g for 15 minutes at 4 C., and then the final purified pellets were resuspended in PBS for use in the cell free activity experiments.
[0054] Preparation of axenic medium: The axenic medium was prepared according to the previous study on C. trachomatis cultured in axenic medium, and the compositions and concentrations of each component in the axenic medium were listed in Table 1. Depending on the experiment carried out, the medium contained or excluded glucose 6-phoshate (G6P) or adenosine triphosphate (ATP) or alpha ketoglutarate or sodium acetate to serve as carbon sources. Similarly, pH of the media was modified as per the experimental need.
TABLE-US-00001 TABLE 1 CIP-1 medium recipe mg/100 Final Component Formula ml concentration 2X IPB.sup.a Potassium phosphate KH.sub.2PO.sub.4 136 5 mM Sodium phosphate Na.sub.2HPO.sub.4 7H.sub.2O 536 10 mM Potassium gluconate C.sub.6H.sub.11KO.sub.7 5140 110 mM Potassium chloride KCl 120 8 mM Magnesium chloride MgCl.sub.2 6H.sub.2O 40 1 mM Nutrients Dithiothreitol 0.5 mM Glucose-6-phosphate 0.5 mM Adenosine triphosphate 1 mM Fetal bovine serum 1% 20 amino acids.sup.b 25 M Cytidine 5-triphosphate 50 M Guanosine 5- 50 M triphosphate Uridine 5-triphosphate 50 M .sup.aIntracellular Phosphate Buffer. Adjust final volume of complete medium to 1X. .sup.bFor radiolabeling, the concentration of cysteine and methionine are reduced to 1 M. Medium pH adjusted to 7.2-3 using 6N potassium hydroxide prior to sterile filtration.
[0055] Protein synthesis by .sup.35S-cysteine-methionine incorporation: Protein synthesis in cell-free purified fractions of E. chaffeensis was measured by incorporation of .sup.35S-Cys-Met (Perkin Elmer, Waltham, Mass.) as described by Omsland, A., et al., in Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium. Proc Natl Acad Sci USA. 109, 19781-19785 (2012), the teachings and content of which are hereby incorporated by reference. For normalization of bacterial total protein content, the suspension of E. chaffeensis cell-free fractions were lysed in 1% (wt/vol) SDS for 5 minutes at 100 C. and the total protein concentration was determined using Protein Assay kit (Bio-Rad, Hercules, Calif.). Subsequently, the suspensions of E. chaffeensis cell-free fractions were equally split into micro-centrifuge tubes at the amount of 30 g total protein. Partially opened micro-centrifuge tubes containing 500 L of medium supplemented with 70 Ci of .sup.35S-Cys-Met were incubated at 37 C. for 24 hours in a tri-gas incubator set to maintain 2.5% O.sub.2. E. chaffeensis cell-free organisms were pelleted at the end of incubation by centrifugation at 15,000g for 15 minutes at 4 C., washed with K-36 buffer (0.05M K.sub.2HPO.sub.4, 0.05M KH.sub.2PO.sub.4, 0.1M KCl, 0.15M NaCl, pH7.0) twice, and disrupted by adding 30 L of 2SDS-PAGE sample buffer and by boiling for 5 minutes. Ten L of lysate each was then transferred to a tube containing 5 mL of biosafe liquid II and used for quantification of .sup.35S-Cys-Met incorporation using the protocol 4 (.sup.35S) in a liquid scintillation counter (TRI-CARB 2100TR, PerkinElmer, Waltham, Mass.). For visualizing the radiolabel incorporation into bacterial proteins, equal volumes of sample lysates were also separated in an 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 .sup.35S-Cys-Met, resolved on an SDS-PAGE gel, and stained using silver nitrate staining kit (Pierce/ThermoFisher Scientific) as per the manufacturer's recommendations.
[0056] DNA synthesis by .sup.3H-thymidine incorporation: Purified E. chaffeensis cell-free fractions were also assessed for incorporation of .sup.3H-thymidine (Perkin Elmer, Waltham, Mass.) into the bacterial DNA simultaneously with the incorporation .sup.355-Cys-Met into proteins. Briefly, E. chaffeensis cell-free organisms were incubated for 48 hours at 37 C. with 2.5% O.sub.2 in micro-centrifuge tubes containing 500 L of medium supplemented with 20 Ci of .sup.3H-thymidine and 70 Ci of .sup.35S-Cys-Met. E. chaffeensis were pelleted at 15,000g for 15 minutes at 4 C., washed with K-36 twice, lysed in 30 L of 2SDS-PAGE sample buffer and then boiled for 5 min. 10 L of lysate each was added into 5 mL of Biosafe liquid II (Grainger, Hartford, Conn.) and used for quantification of .sup.3H-thymidine incorporation using the protocol 10 (.sup.3H) and .sup.35S-Cys-Met incorporation (the protocol 4, .sup.35S) by liquid scintillation counting (TRI-CARB 2100TR, PerkinElmer, Waltham, Mass.), respectively.
[0057] Sodium dodecyl sulfate polyacrylamide gel electrophoresis: Five L of NuPAGE SDS sample buffer and 2 L of NuPAGE reducing agent (Invitrogen/ThermoFisher Scientific) were added to each of 10 L of sample solution following cell-free incubation experiments in the axenic medium, boiled for 5 min, and then loaded onto a Mini-PROTEAN Precast Bis-Tris 4% to 14% gels (Bio-rad, Hercules, Calif.) and subjected to electrophoresis (100 mA/gel for 60 minutes). The gels were then stained with Silver staining kit (Pierce/ThermoFisher Scientific) according to the manufacturer's recommendations.
[0058] Western blot analysis to assess protein synthesis: For detection of the DnaK and ClpB proteins of E. chaffeensis, the above described electrophoresed proteins were transferred onto a nitrocellulose membrane (Thermo Fisher Scientific, Waltham, Mass.) by subjecting to electro-blotting using an electrophoretic transfer unit (Bio-Rad). Protein transfer buffer was prepared as per the manufacturer's instructions and used in the protein transfer protocols. Subsequently, E. chaffeensis ClpB or DnaK expressions were assessed using the polyclonal rabbit antisera raised against a recombinant E. chaffeensis proteins for ClpB or DnaK, respectively. Secondary anti-rabbit antibody conjugated with horseradish peroxidase (Sigma-Aldrich, St. Louis, Mo., USA) and Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Mass., USA) were used for the signal detection, respectively.
[0059] Preparation of E. chaffeensis cultures for use in transmission electron microscopy: Purified E. chaffeensis DCs and RCs by renografin density gradient centrifugation were resuspended in PBS and used in transmission electron microscopy analysis by following the methods described previously. Briefly, all centrifugation steps used in preparing the TEM samples were performed at 4 C. for 5 minutes at 200g, unless otherwise specified. The cultures in PBS were fixed with 1 ml of Karnovsky's fixative containing 2% paraformaldehyde, 2.5% gluteraldehyde in 0.1 M cacodylate buffer (pH7.4) overnight. The cell-free E. chaffeensis organisms were then washed three times with 1 ml of 0.1 M cacodylate buffer and were incubated in 1 ml of 1% osmium tetraoxide in 0.1 M cacodylate buffer for one hour, washed three times using deionized water and then resuspended in 2% trypsin soy agar solution. Each sample was diced with a teflon coated razor blade and placed in a wheaton glass vials with 50% ethanol at room temperature for 15 min, then stained with 70% ethanolluranyl acetate in the dark for one hour at room temperature. The bacterial suspension in soy agar was then subjected to dehydration process with an ethanol gradient of increased concentrations from 50% to 100%. All samples embedded in the soy agar-gluteraldehyde resin were subsequently transferred to silicon molds to allow for polymerization to be completed. All blocks were examined under a dissecting scope to identify a sample that was flush to the end of the block using an Ultracut E-Reichert-Jung ultramicrotome. Sections of 0.5 m size were cut in the range of 75-90 nm, and transferred to Athene Thin Bar copper grids (Ted Pella, Redding, Calif.). The grids were stained with uranyl acetate in 70% ethanol followed by Reynold's lead citrate. The stained grids were examined under a Hitachi H-300 electron microscope (Hitachi High-Tech, San Jose, Calif.).
[0060] Results
[0061] Protein synthesis in cell-free replicating form of E. chaffeensis assessed in axenic media. The axenic medium used for C. trachomatis is a complex mixture containing intracellular phosphate buffer (IPB) supplemented with 1% FBS, 25 M of equimolar mix of all 20 amino acids (AA), 0.5 mM glucose 6-phosphate (G6P) or 1.0 mM ATP with or without 0.5 mM DTT, and 50 M each of GTP, UTP, and CTP as described in, except when using alpha ketoglutarate (0.5 mM) or sodium acetate (0.5 mM) as the carbon source. When assessing .sup.35S Cys-Met incorporation, concentration of these two cold amino acids were reduced to 1 M and then the two radioactive amino acids were supplemented with 70 Ci of .sup.35S-Cys-Met. Protein synthesis in the axenic media is verified with inclusion of chloramphenicol (CHL) or rifampin (RIF), which serve as negative controls as protein synthesis would not be expected in the presence of these antibiotics. In the current study, we prepared the medium with or without an energy source and used it to determine if it supports E. chaffeensis protein synthesis in the absence of a host cell. E. chaffeensis dense-core cells (DCs) and reticulate cells (RCs) were purified from the infected Vero cells or DH82 cells by renografin gradient centrifugation which fractionated as PBS/25% renografin fraction (top layer) and 25-35% renografin fraction (bottom layer), respectively. Incubation of the purified Ehrlichia organisms promoted the protein synthesis in the bottom layer of the renografin fraction where the RCs appeared to have concentrated, while the protein synthesis was nearly absent or weaker in the top fraction, which is likely to contain DCs (
[0062] In particular,
[0063] The presence of DCs and RCs in the top and bottom layers of the renografin-purified fractions was confirmed by multiple experiments; Western blot analysis, in vitro infection assessment (
[0064] In particular,
[0065] We investigated if protein synthesis by the cell-free Ehrlichia in the axenic media has any pH preference and secondly if an altered pH may promote biosynthesis by the DC form (
[0066] We then assessed the impact of different energy sources for cell-free activity; the axenic medium supplemented with G6P, alpha ketoglutarate or sodium acetate supported the protein biosynthesis similarly for RC form of E. chaffeensis recovered from DH82 cultures although G6P appeared to be more favored but not significantly different as the carbon source (
[0067] Based on the incorporation data in the above-described experiments, we reasoned that only moderate protein biosysthesis has occurred by RCs. To validate these data, we compared protein profiles of total proteins resolved on an acrylamide gel before and after assessing by Western blot analysis using a murine polyclonal serum collected from E. chaffeensis infected mice (
[0068] We then tested if the axenic medium also promoted DNA synthesis (
[0069] Discussion
[0070] Two major limitations of carrying out research on obligate bacterial pathogens, particularly on rickettsials belonging to the Anaplasmataceae family, are the lack of fully established methods for targeted mutagenesis and the inability to grow the bacteria in the absence of a host cell. While targeted mutagenesis methods aid in understanding the contributions of various genes involved in pathogenesis and in defining the genes critical for the pathogens' vector and vertebrate host cell-specific growth, the ability to grow the pathogens in a cell-free media can facilitate greatly in studies focused on understanding functions of various bacterial proteins without the influence of a host cell. Further, growth in cell free medium will aid in rapidly recovering mutant organisms and also to clonally purify mutants. Indeed, recent studies on C. burnetii demonstrated that significant progress could be made with the advent of fully established methods of mutagenesis and axenic growth. In an effort to address these two major deficiencies for the field of research on Anaplasmataceae family pathogens, such as those belonging to the genera Ehrlichia and Anaplasma, we recently described methods for creating stable targeted mutations to both disrupt and also restore the function of a disrupted gene in E. chaffeensis. In the present study, we focused on the second major challenge for the field; the development of axenic culture medium for E. chaffeensis. We present the first evidence in the axenic media that the protein biosynthesis and DNA synthesis are possible in a host cell-free culture media for E. chaffeensis organisms. We believe that the data described here are critical in moving the field forward in various fronts; 1) making the improvements to the axenic media growth method in promoting transition of replicating form to infectious form in E. chaffeensis and in other related Anaplasmataceae pathogens, 2) to aid in identifying and characterizing effector proteins involved in influencing the host, 3) in studying the potential interactions of the bacterial phagosome with mitochondria, host cytoplasmic proteins and nucleus, and 4) in facilitating the clonal purification of mutated organisms following creating insertion mutations by random and targeted mutagenesis methods.
[0071] Firstly, we presented a method for purification of E. chaffeensis DCs and RCs from host cells by employing renografin density gradient centrifugation. We discovered that the DC form of the bacterium fractioned at a lower concentration of renografin compared to the RC form. The presence of DCs and RCs within the gradient fractions was confirmed by three independent methods; ability to infect naive host cells, morphology, and by protein expression. Our studies demonstrate that axenic media supported protein synthesis only in the RCs of E. chaffeensis. Similarly, we presented evidence that the cell-free media supported the DNA synthesis of RCs. Axenic media-specific protein synthesis was further confirmed by inclusion of inhibitors; chloramphenicol or rifampin in the cell-free media.
[0072] Axenic media-specific protein synthesis in E. chaffeensis is similar to a prior study demonstrating the cell-free protein biosynthesis for C. trachomatis. While the C. trachomatis study doesn't address if the axenic media also supports the gross DNA synthesis, we reasoned that the media for this intra-cellular pathogen might have similarly supported a limited DNA synthesis. In particular, in the current study we presented evidence that the axenic media also supports the bacterial DNA synthesis. Despite protein and DNA synthesis shown in the absence of a host cell for RCs, our data suggest that the abundance of proteins and DNA made is limited. Total bacterial proteins resolved on a polyacrylamide gel and followed by staining with silver nitrate and Western blot analysis suggested that the bacterial replication was limited in the axenic media incubation experiments. We further investigated if variations in the pH of the media and varying energy sources may improve the protein biosynthesis. Despite G6P appears as the best energy source, we did not note any significant variations in the protein synthesis in the axenic media and nor did the altered media pH and carbon sources promoted protein synthesis in DC fraction of E. chaffeensis.
[0073] Two important goals to improve the axenic media for E. chaffeensis are; 1) to modify the media conditions, such as adding thymidine in the media cocktail, to promote the increased DNA and protein synthesis resulting the continued replication of the RC form and 2) to transform the RCs to DCs under axenic media conditions. These improvements may be possible if the axenic media growth is assessed with host cell derived purified E. chaffeensis RC-containing phagosomes in place of purified RCs (
EXAMPLE 2
[0074] This example uses the medium previously described for C. trachomatis, referred to as CIP-120, to evaluate the possibility of developing axenic culture methods for both E. chaffeensis RC-containing phagosomes and A. phagocytophilum RC-containing phagosomes in the absence of host cells.
[0075] Materials and Methods
[0076] Cell Lines and Cultivation of A. Phagocytophilum and E. Chaffeensis
[0077] The human promyelocytic cell lines HL-60 (ATCC CCL-240, Manassas, Va.) and A. phagocytophilum strain NCH-1-infected HL-60 was cultured in complete RPMI 1640 medium (Gibco/ThermoFisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen/ThermoFisher Scientific, Waltham, Mass.) and 2 mM L-glutamine (Mediatech, Manassas, Va.), by following the protocols described elsewhere for Anaplasma phagocytophilum strain NCH-1. Cultivation of E. chaffeensis in HL60 cells in complete RPMI 1640 medium (Gibco/ThermoFisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen/ThermoFisher Scientific, Waltham, Mass.) and 2 mM L-glutamine (Mediatech, Manassas, Va.) was performed by following the protocols described for Anaplasma phagocytophilum strain NCH-1. To prepare cell-free Anaplasmainocula, about 80-100% A. phagocytophilum-infected HL-60 cells in a T25 flask were harvested by centrifugation at 400g for 10 minutes 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 seconds. The cell debris and unbroken cells were removed by centrifugation at 200g for 10 minutes at 4 C. The supernatant was passed through a 2.7 m pore-size syringe filter (Whatman, Pittsburgh, Pa.). HL60 cells were incubated with host cell-free A. phagocytophilum (multiplicity of infection of 100:1, bacteria to host cell) for 120 minutes to allow for internalization. Non-ingested A. phagocytophilum were removed by washing with PBS, and the cells were incubated for 36 to 48 hours in a T150 flask. Similar infection protocol is followed when infecting by E. chaffeensis. The infected host cell cultures were harvested by centrifugation at 600g for 5 minutes at 4 C. and used for purifying the host cell-free phagosomes, as outlined below.
[0078] Purification of Phagosomes
[0079] Purification of phagosomes from the A. phagocytophilum-infected HL-60 and E. chaffeensis-infected HL-60 was performed by subjecting to sugar density gradient centrifugation in combination with magnet assisted cell sorting (MACS) as described previously with some minor modifications. In brief, the infected HL60 cells were pelleted at 4 C. for 5 minutes at 350g. The cells were washed twice with PBS and once with homogenization buffer (250 mM sucrose, 0.5 mM EGTA, 20 mM HEPES/KOH, pH 7.2). Cells were then resuspended in homogenization buffer with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo., USA). The cells were then homogenized at 4 C. using a 10 ml syringe with a 23.sup.-G needle; typically 10-15 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. Whole cells and nuclei were then pelleted in a 15 mL tube at 4 C. for 5 minutes at 300g.
[0080] The resulting supernatant contained phagosomes, which was designated as the post-nuclear supernatant (PNS). The PNS was brought to a final concentration of 39% sucrose. The sucrose gradient was made by layering 1 mL of the PNS (39% sucrose) onto 2 mL of 55% sucrose layered onto 1 mL of 65% sucrose. We then layered 2 mL of 10% sucrose onto 2 mL of 25% sucrose solution onto the PNS. The phagosomes were isolated from the 55-65% interface using a 16g needle and not disturbing any other fraction. For further MACS separation, the crude phagosomes were incubated with rabbit Rab5 (1:1000) antibody for 1.5 hours, followed by incubation with MACS secondary goat anti-rabbit antibody (1:100, Miltenyi) for another 1.5 hours, and then loaded on a MACS LS separation column (Miltenyi) column in steps of 2 mL and washed with three times the input volume of HSMG buffer. The phagosomes were then eluted with 3 mL HSMG buffer HSMG buffer (20 mM HEPES, 250mM sucrose, 1.5mM MgCl2, 0.5 mM EGTA, pH 7.4) after removal of the magnet, aided by gentle pushing using the supplied plunger. The elution was placed into 10 mL of PBS (4 C.) and centrifuged at 40,000 g for 30 minutes at 4 C. The final purified pellets were resuspended in PBS for use in the cell free activity experiments.
[0081] Confocal Microscopy Analysis
[0082] The final purified phagosomes were plated onto 8-well culture chamber slide to adhere for 1 hour, and then incubated with Alexa Flour goat antirabbit 488 for 1 hour. The slides were washed with PBS and mounted with the mounting media containing DAPI. For infected HL60 cells staining, the infected HL60 cells were plated onto 8-well culture chamber slide and allowed to adhere for 1 hour in the 37 C. incubator. The cells were fixed with 4% formadehyde for 10 minutes at room temperature and permeabilized for 10 minutes. Subsequently, the cells were stained with rabbit Rab5 (1:1000) antibody overnight at 4 C. The antigen slides were washed with PBS to remove unbound primary antibody, and incubated with second antibody (Alexa Flour goat antirabbit 488) for 1 hour. The slides were washed with PBS and mounted with the mounting media containing DAPI. The slides were examined with a Zeiss LSM 700 laser scanning confocal microscopy (Carl Zeiss Optronics GmbH, Oberkochen, Germany).
[0083] Preparation of axenic medium: The axenic medium was prepared according to the previous study on C. trachomatis cultured in axenic medium, and the compositions and concentrations of each component in the axenic medium were listed in Table 1. Depending on the experiment carried out, the medium contained or excluded G6P or ATP to serve as carbon sources. Similarly, pH of the media is modified as per the experimental need.
[0084] Protein synthesis by .sup.35S-cysteine-methionine incorporation: Protein synthesis in cell-free purified phagosomes of A. phagocytophilum or E. chaffeensis was measured by incorporation of .sup.355-Cys-Met (Perkin Elmer, Waltham, Mass.) as described previously. For normalization of bacterial total protein content, the suspension of the cell-free phagosomes were lysed in 1% (wt/vol) SDS for 5 minutes at 100 C. and the total protein concentration was determined using Protein Assay kit (Bio-Rad, Hercules, Calif.). Subsequently, the suspensions of cell-free phagosomes were equally split into micro-centrifuge tubes at the amount of 30 g total protein. Partially opened micro-centrifuge tubes containing 500 L of medium supplemented with 70 Ci of .sup.35S-Cys-Met were incubated 24 hours at 37 C. in a tri-gas incubator set to maintain 2.5% O.sub.2. The cell-free organisms were pelleted at the end of incubation by centrifugation at 15,000g for 15 minutes at 4 C., washed with K-36 buffer (0.05M K.sub.2HPO.sub.4, 0.05M KH.sub.2PO.sub.4, 0.1M KCl, 0.15M NaCl, pH7.0) twice, and disrupted by adding 30 L of 2SDS-PAGE sample buffer and by boiling for 5 minutes. Ten L of lysate each was then transferred to a tube containing 5 mL of biosafe liquid II and used for quantification of .sup.35S-Cys-Met incorporation using the protocol 4 (.sup.35S) in a liquid scintillation counter (TRI-CARB 2100TR, PerkinElmer, Waltham, Mass.). For visualizing the of radiolabel incorporation into phagosomes proteins, equal volumes of sample lysates were also separated in an 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 added .sup.35S-Cys-Met, resolved on an SDS-PAGE gel and stained using silver nitrate staining kit (Pierce/ThermoFisher Scientific) as per the manufacturer's recommendations.
[0085] DNA synthesis by .sup.3H-thymidine incorporation: Purified cell-free phagosomes were also assessed for incorporation of .sup.3H-thymidine (Perkin Elmer, Waltham, Mass.) into the bacterial DNA simultaneously with the incorporation .sup.355-Cys-Met into proteins. Briefly, A. phagocytophilum or E. chaffeensis cell-free phagosomes were incubated for 48 hours at 37 C. with 2.5% O.sub.2 in micro-centrifuge tubes containing 500 L of medium supplemented with 20 Ci of .sup.3H-thymidine and 70 Ci of .sup.355-Cys-Met. The phagosomes were pelleted at 15,000g for 15 minutes at 4 C., washed with K-36 twice, and lysed in 30 L of 2 SDS-PAGE sample buffer boiling for 5 minutes. 10 L of lysate each was added into 5 mL of Biosafe liquid II (Grainger, Hartford, Conn.) and used for quantification of .sup.3H-thymidine incorporation using the protocol 10 (.sup.3H) and .sup.35S-Cys-Met incorporation (the protocol 4, .sup.35S) by liquid scintillation counting (TRI-CARB 2100TR, PerkinElmer, Waltham, Mass.), respectively.
[0086] RNA Synthesis Assessed by Quantitative Real-Time RT-PCR
[0087] Quantitative real-time RT-PCR was employed to measure A. phagocytophilum or E. chaffeensis 16S rRNA expression. In brief, cultures of A. phagocytophilum or E. chaffeensis grown in several T150 flasks were used in recovering cell-free phagosomes form. The phagosomes organisms in triplicate microcentrifuge tubes were incubated for 0, 2, 6, 12, and 24 hours with 500 L of axenic medium containing G6P and ATP at 37 C. with 2.5% O2. At the end of the specified incubation times, cells were recovered by centrifugation at 15,000g for 10 minutes at 4 C. The bacterial pellets were then inactivated immediately in the TRI reagent solution, and then used to isolate total RNA by TRI reagent protocol (Sigma-Aldrich, St. Louis, Mo.). Final recovered RNA from each tube was resuspended in 25 l of nuclease-free water, then DNase treated to remove residual genomic DNAs using RQ1 DNase (Thermo Fisher Scientific, Waltham, Mass.). RNA from each tube was diluted 1:1000 in nuclease-free water and 2 l each was used in 25 l reaction in performing Taq-Man probe-based real-time RT-PCR targeted to the E. chaffeensis 16S RNA as previously described. The RNA levels in each sample were expressed by Ct values. Variation among triplicates for each time point was calculated and presented with the respective standard deviations observed. Fold changes were calculated relative to the Ct values observed for the RNA recovered before incubation (0 hours) compared to different incubation times. The data were then assessed for statistical significance.
[0088] Sodium dodecyl sulfate polyacrylamide gel electrophoresis: Five L of NuPAGE LDS sample buffer and 2 L of NuPAGE reducing agent (Invitrogen/ThermoFisher Scientific) were added to each of 10 L of sample solution following cell-free incubation experiments in the axenic medium, boiled for 5 minutes, and then loaded onto a Mini-PROTEAN Precast Bis-Tris 4% to 14% gels (Bio-rad, Hercules, Calif.) and subjected to electrophoresis (100 mA/gel for 60 minutes). The gels were then stained with Silver staining kit (Pierce/ThermoFisher Scientific) according to the manufacturer's recommendations.
[0089] Western blot analysis to assess protein synthesis: For detection of the DnaK and P28 proteins of E. chaffeensis or the Dnak of A. phagocytophilum, the above described electrophoresed proteins were transferred onto a nitrocellulose membrane (Thermo Fisher Scientific, Waltham, Mass.) by subjecting to electro-blotting using an electrophoretic transfer unit (Bio-Rad). Protein transfer buffer was prepared as per the manufacturer's instructions and used in the protein transfer protocols. Subsequently, E. chaffeensis DnaK and P28 expressions were assessed using the polyclonal rabbit antisera raised against a recombinant E. chaffeensis proteins for DnaK and P28, respectively. Secondary anti-rabbit antibody conjugated with horseradish peroxidase (Sigma-Aldrich, St. Louis, Mo., USA) and Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Mass., USA) were used for the signal detection, respectively.
[0090] Preparation of E. chaffeensis cultures for use in transmission electron microscopy: Purified E. chaffeensis phagosomes were resuspended in PBS and used in transmission electron microscopy analysis by following the methods described previously. Briefly, all centrifugation steps used in preparing the TEM samples were performed at 4 C. for 5 minutes at 200g, unless otherwise specified. The cultures in PBS were fixed with 1 ml of Karnovsky's fixative containing 2% paraformaldehyde, 2.5% gluteraldehyde in 0.1 M cacodylate buffer (pH7.4) overnight. The cell-free E. chaffeensis organisms were then washed three times with 1 ml of 0.1 M cacodylate buffer and were incubated in 1 ml of 1% osmium tetraoxide in 0.1 M cacodylate buffer for one hour, washed three times using deionized water and then resuspended in 2% trypsin soy agar solution. Each sample was diced with a teflon coated razor blade and placed in a wheaton glass vials with 50% ethanol at room temperature for 15 minutes, then stained with 70% ethanol/uranyl acetate in the dark for one hour at room temperature. The bacterial suspension in soy agar was then subjected to dehydration process with an ethanol gradient of increased concentrations from 50% to 100%. All samples embedded in the soy agar-gluteraldehyde resin were subsequently transferred to silicon molds to allow for polymerization to be completed. All blocks were examined under a dissecting scope to identify a sample that was flush to the end of the block using an Ultracut E-Reichert-Jung ultramicrotome. Sections of 0.5 m size were cut in the range of 75-90 nm, and transferred to Athene Thin Bar copper grids (Ted Pella, Redding, Calif.). The grids were stained with uranyl acetate in 70% ethanol followed by Reynold's lead citrate. The stained grids were examined under a Hitachi H-300 electron microscope (Hitachi High-Tech, San Jose, Calif.).
[0091] Results
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