Computational protocol: An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum

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Protocol publication

[…] Wild-type bacteria were grown in 50 ml of RPMI1640 containing HL-60 cells until > 90% of the cells were infected. Around 5 x 107 infected cells were used to obtain cell free bacteria by vortexing the infected cells with 60/90 grit silicon carbide (Lortone, Inc., Mukilteo, Washington) for 30 sec followed by filtration through a 2.0 μm pore size filter and centrifugation at 700 x g for 5 min to remove remaining cell debris. The percent of infected cells in the culture was calculated by counting the number of infected and uninfected cells in duplicates Giemsa-stained preparations from the same flask and the total number of cells was determined using a hemocytometer. Cell-free bacteria were incubated for 2 hr at 34°C with 2.5 x 105 ISE6 cells in MatTek chambers (MatTek Corp., Massachusetts) to allow binding and expression of OMT. Unbound bacteria were removed by rinsing cells twice with culture medium. Likewise, bacteria were incubated with 2.5 x 105 HL-60 cells suspended in 500 μl of culture medium for 2 hr at 37°C. Unbound bacteria were removed by washing cells twice and expression of OMT in bound bacteria was analyzed by IFA.Cell samples were fixed for 10 min in methanol, and incubated with anti-rOMT serum diluted 1:200 in PBS containing 3% bovine serum albumin (BSA) for 2.5 hr at room temperature. Bacteria were labeled with anti-A. phagocytophilum dog serum (diluted 1:1,000). The slides were washed 3 times in PBS and blocked in PBS with 3% BSA for 10 min at room temperature. OMT expressing bacteria were then labeled with anti-mouse antibodies conjugated with AlexaFluor647 (1:500 dilution) (Jackson ImmunoResearch Laboratories, Inc, Pennsylvania) for 1 hr at room temperature. All A. phagocytophilum were labeled with anti-A. phagocytophilum dog serum diluted 1:500 followed by incubation with anti-dog IgG conjugated to FITC, using the same procedure. Tick and HL-60 cell nuclei were labeled using DAPI present in the VectaShield mounting medium. Microscopic images were obtained using an Olympus BX61 disk-scanning unit confocal microscope (Olympus America, Pennsylvania) utilizing a DSU-D2 confocal disk. Confocal images were acquired with a Photometrics Quantem:512SC EMCCD camera (Photometrics, Arizona), and high resolution images were acquired with a QFire color camera (Qimaging, California). Image capture software was Metamorph (Molecular Devices, California). ImageJ (US National Institutes of Health) was used to compile z-projections and Photoshop (Adobe Systems, California) was used for cropping. [...] Phyre2 [] was used to determine the putative tertiary structure of the protein (Msp4) that was identified as being methylated by iTRAQ analysis as well as the in vitro methylation assay. The putative localization of the modified residues was determined from the protein sequence and the structure generated from Phyre2. Phobius (http://phobius.sbc.su.se/cgi-bin/predict.pl) was used to determine where the modified residues were located within the membrane of the bacteria since Msp4 is a surface protein []. […]

Pipeline specifications

Software tools Phyre, Phobius
Application MS-based targeted proteomics
Organisms Anaplasma phagocytophilum, Homo sapiens, Bacteria, Dipturus trachyderma