Removal of PA63 from PA17by immunoaffinity
Commercially obtained 20 kDa fragment of PA, (LIST Biological Laboratories, Inc, Campbell, CA) is now referred to as PA17. It contained trace amounts of PA63 and removal of this contamination was achieved by immunoaffinity chromatography. In this procedure the monoclonal antibody BA-PA2II-14B7-1-1 (1 ml of ascites fluid), was immobilized using an ImmunoPure™ Protein G IgG Orientation Kit (Pierce) as instructed by the manufacturer. This antibody recognizes the 63 kDa receptor binding region (C-terminus) of the PA molecule [18]. PA17 kDa N-terminal fragment (LIST Biological Laboratories, Inc.; sold as PA20), 500 μl containing 250 μg of protein, was diluted with an equal volume of PBS (pH 7.3, Sigma, St. Louis, MO). The resulting 1 ml was combined with 1 ml of immobilized antibody and incubated at room temperature on a rotator for 2 hours. The suspension was then centrifuged at 3000 × g for 5 minutes to recover PA17 in the supernatant fraction, filtered through a 0.2 μm cellulose acetate low protein binding filter (Corning, Lowell, MA) and frozen in aliquots at -70°C. Proteins were analyzed by SDS PAGE using 4–15% PhastGels (Amersham, Piscataway, NJ) and Western Blot as previously described [16].
Construction of a PA-Factor Xa recombinant strain and purification of PA 20 kDa fragment designated as rPA20
The amino-terminal domain of PA is cleaved at the consensus R164–K165–K166–R167 sequence recognized by furin-like proteases in-vitro [12] and by a plasma protease in vivo [16]. This process results in the release of a 20-kDa amino-terminal fragment (PA20) and the formation of 63-kDa carboxy-terminal fragment heptamers [19]. Lethal factor (LF) and/or edema factor (EF) then bind to the heptamers and these toxic complexes are internalized via receptor-mediated endocytosis into eukaryotic cells [20]. Limited digestion of PA with trypsin results in 63 kDa and 20 kDa fragments. These fragments were isolated and fully characterized by Christensen et al. [21]. However, prolonged digestion with trypsin results in a trypsin resistant 17 kDa amino terminal fragment. Deletion of the consensus R164–K165–K166–R167 sequence eliminates the cleavage of PA by furin-like proteases and by trypsin [22]. In-vivo proteolysis of PA results in 63 kDa and 20 kDa fragments [16]; therefore we wished to be able to produce an identical and stable 20 kDa fragment in-vitro. In this study, we performed mutagenesis of the trypsin cleavage site in PA83 to make it sensitive to cleavage by Factor Xa protease because there are no other Factor Xa sensitive sites on the PA83 sequence. We constructed a PA mutant in which a Factor Xa proteolytic recognition site (IEGR) was genetically engineered into PA [12]. The Factor Xa proteolytic site was introduced into PA at the trypsin-sensitive site by a 2-step mutagenesis procedure using a Muta-Gene Phagemid InVitro Mutagenesis kit (BioRad, Hercules, CA). A 2,044 bp HindIII/BamHI fragment encoding the carboxy-teminus of pag, including amino acid residues 164–167 which comprise the trypsin-sensitive site, was inserted into pBluescriptSK (Stratagene, La Jolla, CA.) and designated pPAHB. Oligonucleotide XaFN (5'-GTACTTCGCTTTTCTATTGAGTTCGAAG-3') was used to convert the wild-type pag gene fragment in pPAHB to an R164I/K165E double mutant designated pPAHB(XaFN). Oligonucleotide Xa2FN1 (5'-GTACTTCGCCCTTCTATTGAGTTCGAAG-3') was used to convert the pag double mutant in pPAHB(XaFN) to K166G to complete the creation of the Factor Xa site and was designated pPAHB(Xa2FN1). A 670 bp PstI/HindIII fragment from pPAHB(Xa2FN1) containing the Factor Xa site codons was ligated into pYS5 similarly digested with PstI/HindIII to remove the wild type 670 bp fragment and the resulting plasmid was designated pYS1Xa2FN1 [22]. pYS1Xa2FN1 was transformed into Bacillus subtilis WB600 for expression of the PA/Factor Xa mutant [23]. PA/Factor Xa was purified from WB600 PYS1Xa2FN1 as previously described for rPA [24].
Endotoxin assay
We carried out endotoxin assays on the LIST PA20 and the Factor Xa PA20 that we prepared. Both preparations had less than 0.1 EU/ml as determined by using the Limulus Amebocyte Lysate (LAL) QCL-1000 assay kit (Cambrex Bio Science Walkersville, MD)
Exposure of monocytes and lymphocytes to rPA20
Leukopheresis units were obtained from volunteer donors using the procedures outlined in our approved human use protocol, reviewed by the established Institutional Review Board at WRAIR. The written informed consent document was provided to the volunteers in advance of the procedure.
We obtained PBMC (4 different individuals over a period of ~6 months, collected from ~8–10 AM to minimize variability) from healthy human male volunteers who had been screened to be HIV and Hepatitis B negative and were from 19–61 years of age.
rPA20 was added to newly plated cells in flasks for the time period specified. Cells incubated in the absence and presence of rPA20 were collected by centrifugation at the specified exposure time.
Exposure of monocytes and lymphocytes to the anthrax spores
Spores were prepared from B. anthracis Ames strain (pXO1+, pXO2+). Briefly, 5% sheep blood agar (SBA) plates were inoculated with B. anthracis Ames spores and incubated overnight at 35°C. Several isolated colonies were transferred to a sterile screw capped tube containing 5 ml of sterile PBS. NSM Petri plates (New sporulation medium: per liter added Tryptone; 3 g, Yeast extract; 3 g, Agar; 2 g, Lab Lemco agar; 23 g, and 1 ml of 1% MnCl2·4H2O) were inoculated with 200 μl of the prepared cell suspension. The plates were incubated for 48 hrs at 35°C and checked for sporulation progress by microscopic examination. Continued incubation at room temperature was performed until free refractive spores constituted 90–99% of total suspension. Spores were then harvested from plates using 5 ml of sterile water. Spores were washed 4 times in sterile water and checked for purity by plating 10 μl in triplicate onto 5% SBA plates and incubating overnight @ 35°C. Enumerations of spores were calculated via CFU/ml (determination of viable spores) and also for actual spores/ml using Petroff Hauser chamber.
ELISA immunoassays
An ELISA kit for TNF-α was used to determine TNF-α levels in PBMC cells treated with rPA20 according to manufacturer's instructions (Quantikine R&D systems, Minneapolis, MN). The amount of protein was quantified using Ceres UV 900-Hdi plate reader (Bio-Tek Instruments Inc., Winooski, VT).
Extraction of RNA
Total RNA was isolated from cells using the TRIzol™ reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The RNA samples were treated with DNase-1 to remove genomic DNA and were re-precipitated with isopropanol. The quality of the RNA to be used for microarray was characterized using a 2000 BioAnalyzer (Agilent, Santa Clara, CA) to verify the presence of 18 and 28S bands, to confirm the lack of degradation. RNA quantity was determined using a Nanodrop spectrophotometer.
cDNA microarrays
Preparation of Microarray Chip: Human cDNA microarrays were generated using sequence verified PCR elements including the approximate 6900 well-characterized human genes from The Easy to Spot Human UniGEM V2.0 cDNA (Incyte Genomics, Inc). The PCR products ranging from 500–700 base pairs were deposited in 3X saline sodium citrate (SSC) at an average concentration of 165 μg/μl on CMT-GAPS II aminopropyl silane-coated slides (Corning, Corning, NY) using a VersArray microarryer (Bio-Rad, Inc). The arrays were post processed by UV-cross linking at 1200 mjoules, baking for 4 hours at 80°C, and then the positively charged amine groups on slide surface were inactivated through reacting with succinic anhydride/N-methyl-2-pyrrolidinone. Upon hybridization, the quality of each microarray, i.e. the efficiency of reverse transcription (RT) reactions, labeling competence etc. was assessed.
Microarray hybridization and image processing
Microarray labeling was performed using Micromax Tyramide Signal Amplification (TSA) Labeling and Detection Kit (Perkin Elmer, Inc., MA). The slides were hybridized for 16 h at 60°C. The GenePix Pro 4000b (Axon Instruments, Inc., CA) optical scanner was used to scan the hybridized slides and the raw intensity was recorded through the Gene Pix 4000 software package (Axon Instruments, Inc., CA). Intensity of the scanned images was digitalized through Genepix 4.0 software.
Data analysis
Assessment of the overall integrity of the microarray experiment:
Microarray images were visualized using Imagene v.6 (BioDiscovery, Inc., El Segundo, CA) and data were analyzed using GeneSpring V. 7.1 (Agilent, Santa Clara, CA) and Partek Pro. V. 5.0 (Partek, St Louis, MI). Data cleansing and normalization: Using ImaGene (BioDiscovery Inc), background and foreground pixels of each spot were segmented and the highest and lowest 2% of the probe intensity was discarded. Local background correction was applied to each individual spot. The genes that passed this filter in all given experiments were selected for further study.
Data cleansing and statistical analysis was carried out using GeneSpring® 7.1 (Agilent Tech., CA). Local background was subtracted from individual spot intensity. Genes that failed this 'background check' in any of the experiments were eliminated from further analysis. Each chip was next subjected to intra-chip normalization (LOWESS). The genes that varied most between control and treated sample sets were selected via t-test analysis. The p-value cutoff was set at 0.05.
We used the reference design, where a reference RNA sample is co-hybridized with each sample on the slide. This design allows us to normalize between slides for variations that can be due to hybridization, transcription and labeling efficiencies (technical variations).
Apoptosis study using Hoechst 33258
PBMC were treated with rPA20 for 24 h. Cells were stained with Hoechst 33258 dye for 30 min and examined by fluorescence microscopy. Cells having bright; fragmented and condensed nuclei were identified as apoptotic cells. The number of apoptotic cells was counted in 10 microscopic fields (×40) in each case.
Caspase enzymatic assay
Caspase activity in PBMC cells exposed to LIST PA17 and to rPA20 was studied using the EnzChek® Caspase-3 Assay Kit #2 (Invitrogen, Carlsbad, CA). Cells were harvested after 24 hrs of exposure to rPA20 and washed in PBS. Cells were lysed and centrifuged. The Z-DEVD-R110 substrate solution was added to each of the treated and control samples. The mixture was incubated for 30 min and the fluorescence was measured at excitation/emission ~496/520 nm.
CD38 staining of PBMC cells
Peripheral blood mononuclear cells were incubated with rPA20 for 16 hours. Cells were then washed twice with PBS and labeled with allophycocyanin (APC)-conjugated mouse anti-human CD38 monoclonal antibody (Becton Dickinson Biosciences, Franklin Lakes, NJ), followed by incubation on ice for 30 min in the dark. The cells were then washed and resuspended at 2 × 106 cells/ml in cell buffer (cell assay reagents, Agilent Technologies, Palo Alto, CA). A cell assay LabChip (Agilent Technologies) was primed with priming solution (Agilent Technologies), after which 10 μl of the cell suspension (20,000 cells) was added to one of six channels. A focusing dye was applied to another chamber, which acted as a reference for the optical detection system. The chip was then placed in an Agilent Technologies Model 2100 bioanalyzer and fluorescence from the cells was measured. Fluorescent events were plotted against the fluorescent intensity (frequency histogram).