Reduced Expression of CD45 Protein-tyrosine Phosphatase Provides Protection against Anthrax Pathogenesis
2009; Elsevier BV; Volume: 284; Issue: 19 Linguagem: Inglês
10.1074/jbc.m809633200
ISSN1083-351X
AutoresRekha G. Panchal, Ricky L. Ulrich, Steven B. Bradfute, Douglas Lane, Gordon Ruthel, Tara Kenny, Patrick L. Iversen, Arthur O. Anderson, Rick Gussio, William C. Raschke, Sina Bavari,
Tópico(s)Poxvirus research and outbreaks
ResumoThe modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis. The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis. Interactions between microbes and immune cells play a critical role in microbial pathogenesis. Many pathogenic organisms exploit the host immune machinery and subsequently modulate cell function, signaling, migration, and cytoskeleton rearrangement. Hence, identifying host cellular components with which microbes interact will allow for a more comprehensive understanding of microbial pathogenesis, define common strategies used by multiple pathogens, and elucidate unique tactics evolved by individual species to help establish infections or evade host innate responses. Another interesting aspect of infection is that diverse pathogens seem to target common cellular pathways (1Bhavsar A.P. Guttman J.A. Finlay B.B. Nature. 2007; 449: 827-834Crossref PubMed Scopus (400) Google Scholar, 2Finlay B.B. Cossart P. Science. 1997; 276: 718-725Crossref PubMed Scopus (632) Google Scholar). Thus, identifying host targets exploited by multiple pathogens will be useful in the development of broad-spectrum host-oriented therapeutics and vaccines.Protein kinases and phosphatases regulate a range of cellular responses to external and internal stimuli, including cell proliferation, metabolism, and apoptosis. Aberrant kinase and/or phosphatase activities underlie many different types of pathological conditions from cancer to infectious diseases. Protein kinases have been extensively investigated as targets for drug discovery. In addition, phosphatases are now being recognized as important regulators of many biological processes. In particular, there is an increasing interest in protein-tyrosine phosphatases (PTPs) 3The abbreviations used are: PTP, protein-tyrosine phosphatase; PMO, phosphorodiamidate morpholino oligomers; LF, lethal factor; MAPKK/MEK, mitogen-activated protein kinase kinase; LT, lethal toxin; DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; PA, protective antigen; CFU, colony-forming units; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline. 3The abbreviations used are: PTP, protein-tyrosine phosphatase; PMO, phosphorodiamidate morpholino oligomers; LF, lethal factor; MAPKK/MEK, mitogen-activated protein kinase kinase; LT, lethal toxin; DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; PA, protective antigen; CFU, colony-forming units; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline. as drug targets (3Ducruet A.P. Vogt A. Wipf P. Lazo J.S. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 725-750Crossref PubMed Scopus (84) Google Scholar, 4Pei Z. Liu G. 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Friedberg I. Friedberg I. Osterman A. Godzik A. Hunter T. Dixon J. Mustelin T. Cell. 2004; 117: 699-711Abstract Full Text Full Text PDF PubMed Scopus (1502) Google Scholar, 10Andersen J.N. Jansen P.G. Echwald S.M. Mortensen O.H. Fukada T. Del Vecchio R. Tonks N.K. Moller N.P. FASEB J. 2004; 18: 8-30Crossref PubMed Scopus (270) Google Scholar, 11Bottini N. Bottini E. Gloria-Bottini F. Mustelin T. Arch. Immunol. Ther. Exp. (Warsz). 2002; 50: 95-104PubMed Google Scholar). The involvement of cellular and bacterial PTPs during intracellular microbial pathogenesis has been a topic of significant interest (2Finlay B.B. Cossart P. Science. 1997; 276: 718-725Crossref PubMed Scopus (632) Google Scholar, 12Higashi H. Tsutsumi R. Muto S. Sugiyama T. Azuma T. Asaka M. Hatakeyama M. Science. 2002; 295: 683-686Crossref PubMed Scopus (842) Google Scholar, 13Nandan D. Knutson K.L. Lo R. Reiner N.E. J. Leukocyte Biol. 2000; 67: 464-470Crossref PubMed Scopus (39) Google Scholar). The bacterial PTP YopH, secreted by Yersinia pestis, interferes with the host adhesion-regulated signaling pathway via dephosphorylation of selective tyrosine-phosphorylated proteins (14Black D.S. Marie-Cardine A. Schraven B. Bliska J.B. Cell. Microbiol. 2000; 2: 401-414Crossref PubMed Scopus (111) Google Scholar). Activation of host PTPs after infection with bacteria or their virulence factors has been demonstrated for a diverse group of microorganisms such as Mycobacterium tuberculosis and Leishmania donovani (13Nandan D. Knutson K.L. Lo R. Reiner N.E. J. Leukocyte Biol. 2000; 67: 464-470Crossref PubMed Scopus (39) Google Scholar). Specific mechanistic models of how PTPs contribute to the development of infection and disease progression by highly lethal organisms still remain unclear.Bacillus anthracis, a Gram-positive spore-forming bacterium, is the etiologic agent of anthrax. The lethal toxin (LT) produced by B. anthracis can cleave host cell mitogen-activated protein kinase kinases (MAPKK), thereby affecting the immune response and the host ability to fight the infection (15Agrawal A. Pulendran B. Cell. Mol. Life Sci. 2004; 61: 2859-2865Crossref PubMed Scopus (44) Google Scholar, 16Duesbery N.S. Webb C.P. Leppla S.H. Gordon V.M. Klimpel K.R. Copeland T.D. Ahn N.G. Oskarsson M.K. Fukasawa K. Paull K.D. Vande Woude G.F. Science. 1998; 280: 734-737Crossref PubMed Scopus (884) Google Scholar). Macrophages are the primary targets of anthrax LT. However, macrophages from only certain strains of mice are susceptible to LT-mediated cell death (17Friedlander A.M. Bhatnagar R. Leppla S.H. Johnson L. Singh Y. Infect. Immun. 1993; 61: 245-252Crossref PubMed Google Scholar, 18Singh Y. Leppla S.H. Bhatnagar R. Friedlander A.M. J. Biol. Chem. 1989; 264: 11099-11102Abstract Full Text PDF PubMed Google Scholar). To date, there is no known direct relation between MAPKK cleavage and LT-induced macrophage cell death, as LT-resistant macrophages exhibit MAPKK cleavage (19Kim S.O. Jing Q. Hoebe K. Beutler B. Duesbery N.S. Han J. J. Biol. Chem. 2003; 278: 7413-7421Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 20Pellizzari R. Guidi-Rontani C. Vitale G. Mock M. Montecucco C. FEBS Lett. 1999; 462: 199-204Crossref PubMed Scopus (254) Google Scholar, 21Ribot W.J. Panchal R.G. Brittingham K.C. Ruthel G. Kenny T.A. Lane D. Curry B. Hoover T.A. Friedlander A.M. Bavari S. Infect. Immun. 2006; 74: 5029-5034Crossref PubMed Scopus (57) Google Scholar). This suggests that another cellular target(s) may play a role in anthrax pathogenesis.Previously, using a chemical genetic approach, we identified a class of Cdc25 inhibitors that protected macrophages from cell death induced by anthrax LT (22Panchal R.G. Ruthel G. Brittingham K.C. Lane D. Kenny T.A. Gussio R. Lazo J.S. Bavari S. Chem. Biol. 2007; 14: 245-255Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Although Cdc25 was not the cellular target, induction of anti-apoptotic responses by the compounds via either the MAPK-dependent or -independent pathways was responsible for the protective phenotype.In the present study we investigated if the previously identified phosphatase inhibitors (22Panchal R.G. Ruthel G. Brittingham K.C. Lane D. Kenny T.A. Gussio R. Lazo J.S. Bavari S. Chem. Biol. 2007; 14: 245-255Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) and their analogs produced any phenotypic changes in the B. anthracis infection model. Two compounds that previously protected LT-treated macrophages (22Panchal R.G. Ruthel G. Brittingham K.C. Lane D. Kenny T.A. Gussio R. Lazo J.S. Bavari S. Chem. Biol. 2007; 14: 245-255Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) also protect B. anthracis-infected macrophages. Subsequent in vitro phosphatase profiling studies identified CD45, a previously unknown target of one of the small molecules, as the most sensitive enzyme to the inhibitor. We then investigated the effect of CD45 reduction in anthrax pathogenesis both in cells and in vivo by using antisense phosphorodiamidate morpholino oligomers and mice engineered to express reduced levels of CD45.EXPERIMENTAL PROCEDURESHeterozygous and Transgenic Mice—All mice in this study are of the C57BL/6 genetic background. C57BL/6 (CD45100%) wild type mice and exon-9-disrupted CD45 knock-out mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). The heterozygous CD4562% mice have one exon 9 knock-out allele and one wild type allele. Transgenic mice containing a point mutation (C817S) in the membrane proximal phosphatase domain of the CD45 minigene were produced using a CD45 minigene construct containing cDNA for exons 1b-3, the genomic sequence from exon 3 to exon 9, which includes the variably spliced exons and surrounding introns, and cDNA from exon 9 through the polyadenylation signal region in exon 33, as described previously (23Virts E.L. Raschke W.C. J. Biol. Chem. 2001; 276: 19913-19920Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 24Virts E.L. Diago O. Raschke W.C. Blood. 2003; 101: 849-855Crossref PubMed Scopus (15) Google Scholar). The minigene expresses CD45 in transgenic mice in the same leukocyte-restricted manner and with the same isoform regulation in leukocyte subsets as the endogenous gene (24Virts E.L. Diago O. Raschke W.C. Blood. 2003; 101: 849-855Crossref PubMed Scopus (15) Google Scholar). The C817S mutation eliminates the catalytic site of the membrane proximal PTP domain and has been shown to abolish CD45 PTP activity in vitro (25Desai D.M. Sap J. Silvennoinen O. Schlessinger J. Weiss A. EMBO J. 1994; 13: 4002-4010Crossref PubMed Scopus (90) Google Scholar), which has been confirmed in ex vivo studies. 4E. L. Virts, N. Raschke, R. G. Panchal, S. Bavari and W. C. Raschke, manuscript in preparation. The CSV10 transgenic founder generated using the C817S mutant minigene was bred onto the exon-9-disrupted CD45 knock-out strain for seven generations to place the transgene on the CD45 knock-out and C57BL/6 backgrounds.The CD45 cell surface expression levels of heterozygous and CSV10+/- mice were determined with flow cytometry by comparison of the mean fluorescence intensities of the lymphocyte populations to those of CD45100% wild type and CD450% knock-out mice (24Virts E.L. Diago O. Raschke W.C. Blood. 2003; 101: 849-855Crossref PubMed Scopus (15) Google Scholar). The expression level of CSV10 transgenic mice containing one copy of the transgene insertion locus (CSV10+/-) is 62–65%, a level similar to that expressed by CD4562% heterozygous mice.Chemical Library—A focused library of known phosphatase inhibitors and related napthoquinone- and dione-containing derivatives was used for screening (supplemental Table 1). These compounds were obtained from the NCI Open Chemical Repository, and their structures are available on the following web site, pubchem.ncbi.nlm.nih.gov. The CD45 inhibitor (N-(9,10-dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl propionamide) was obtained from Calbiochem, whereas its related analogs (phenanthrene-9,10 dione) and (4-nitrophenanthrene-9,10 dione) were obtained from Sigma.Phosphorodiamidate Morpholino Oligomer (PMO) Design and Synthesis—The sequence of the CD45 PMO targeting the translational start site is 5′-CCACAAACCCATGGTCATATC-3′. The scrambled PMO (5′-CGGACACACAAAAAGAAAGAAG-3′) was used as a nonbacterial negative control. For efficient delivery of PMOs into cells, an Arg-rich peptide (CH3CONH-(RAhxR)4-Ahx-βAla, designated P007; in which R stands for arginine, Ahx stands for 6-aminohexanoic acid, and βAla stands for β-alanine) was covalently conjugated to the 5′ end of the PMOs through a noncleavable piperazine linker. The methods for the syntheses of PMOs, the conjugation of P007, and the purification and analyses of P007-PMOs have all been described previously (26Moulton H.M. Nelson M.H. Hatlevig S.A. Reddy M.T. Iversen P.L. Bioconjugate Chem. 2004; 15: 290-299Crossref PubMed Scopus (169) Google Scholar, 27Summerton J. Weller D. Antisense Nucleic Acid Drug Dev. 1997; 7: 187-195Crossref PubMed Scopus (866) Google Scholar).Phosphatase Activity Assay—Protein phosphatases were purchased from Upstate Biotechnology (Lake Placid, NY). A generic substrate, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate), was purchased from Invitrogen. All assays were performed in 50 mm HEPES containing 1 mm dithiothreitol and 0.1% bovine serum albumin, pH 7.4, with the following modifications or additions: SHP1, PTPMEG-2, and PTPβ (10 mm MgCl2); PP1α, PP1β, and PP2A (10 mm MnCl2); HePTP, VHR, CD45, TC-PTP, SHP-2, LMPTPA (pH 4.5), and LMPTPB (pH 4.5); PTPMEG-1 (4.8 mm MgCl2 and 3.2 mm MnCl2); PTP-1B (25 mm HEPES, 50 mm NaCl, 5 mm dithiothreitol, and 2.5 mm EDTA). Compound (10 μm) was added to 15 μl of enzyme and incubated for 10 min followed by 10 μl of DiFMUP at a final concentration of 100 μm. The 384-well plate was incubated at room temperature for 60 min and then read in an Analyst (MDC using excitation 360 nm; emission 450 nm). The effect of the compound was compared with control wells containing DMSO (1%). The different phosphatase enzyme characteristics and their concentrations used in this assay have been detailed in supplemental Table 2.Cdc25B phosphatase activity was measured as described previously (22Panchal R.G. Ruthel G. Brittingham K.C. Lane D. Kenny T.A. Gussio R. Lazo J.S. Bavari S. Chem. Biol. 2007; 14: 245-255Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 28Lazo J.S. Nemoto K. Pestell K.E. Cooley K. Southwick E.C. Mitchell D.A. Furey W. Gussio R. Zaharevitz D.W. Joo B. Wipf P. Mol. Pharmacol. 2002; 61: 720-728Crossref PubMed Scopus (172) Google Scholar). Briefly, an assay mixture containing 30 mm Tris, pH 8.0, 75 mm NaCl, 1 mm EDTA, 0.033% bovine serum albumin, I mm dithiothreitol, 40 μm 3-O-methyl fluorescein phosphate, and 0.7 μg/ml His-Cdc25B catalytic domain (a kind gift from Dr. John Lazo, University of Pittsburgh) was incubated with NSC 95397 in DMSO or DMSO (control) for 1 h at room temperature. The reaction was quenched by adding 100 mm NaOH, and the increase in fluorescence was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm.To measure CD45 phosphatase activity in protein lysates, equal concentrations of total protein (200 μg) from untreated or PMO-treated macrophages (8 μm, 72 h treatment) were first precleared with protein G-Sepharose beads and then immunoprecipitated overnight with either the nonspecific monoclonal antibody or CD45-specific (clone 30-F11, BD Biosciences) antibody in the presence of protein G beads. After washing, the beads were incubated with 100 μm DiFMUP substrate in 100 μl of assay buffer for 1 h. Supernatant was transferred into 96-well plates, and fluorescence intensity was measured at excitation 358 nm and emission 455 nm. The experiments were repeated independently at least three times. The results are given as averages with S.D.Flow Cytometry—Antibodies used for fluorescence-activated cell sorter analysis were purchased from BD Pharmingen unless otherwise noted. Antibodies used were directly conjugated to fluorescein isothiocyanate, phycoerythrin (PE), allophycocyanin (APC), peridinin chlorophyll protein (PerCP), or PECy5. Clones used in these studies included CD45 (30-F11), CD3 (17A2), CD4 (RM4–5), CD8 (53-6.7), CD11b (M1/70), CD11c (N418, eBioscience), CD19 (1D3), NK1.1 (PK136, eBioscience), major histocompatibility complex (MHC) I (28-14-8), MHC II (M5/114.15.2), CD44 (IM7), and Ly6G (1A8). Cells (1 × 106) were resuspended in Fc block (anti CD16/CD32 antibody diluted in RPMI medium containing 10% fetal bovine serum), incubated on ice for 30 min, centrifuged, and stained with appropriate combinations of labeled antibodies. After incubation on ice for 60 min, cells were washed and resuspended in 10% formaldehyde. Fluorescence-activated cell sorter analysis was performed using a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using FlowJo software.Cell Viability Assay of B. anthracis-infected Macrophages— To test the effects of compounds on cell viability after B. anthracis infection, J774A.1 cells (6 × 105) were pretreated with DMSO (1%) control or compound (10 μm). After 1 h cells were infected with Sterne spores of B. anthracis (5 m.o.i.). After 4 h of incubation at 37 °C, bacterial growth was inhibited by the addition of the antibiotics penicillin (100 IU) and streptomycin (100 μg/ml). To determine cell viability sytox green nucleic acid stain (1 μm, Molecular Probes), which is impermeant to live cells, was added and incubated for 15 min at 37 °C. The cells were centrifuged at 2000 rpm for 2 min and then washed 2 times with complete medium containing antibiotics. The cells were fixed with 1% formaldehyde for 15 min and then analyzed by flow cytometry.To test the effects of CD45 knock-down on cell viability after B. anthracis infection, J774A.1 cells (6 × 105) were either left untreated or treated with CD45 or SC PMOs. After 72 h cells were harvested and infected with the Sterne spores (5 m.o.i.). After 4 h of incubation at 37 °C, cell viability was measured by the uptake of sytox green dye (as described above).Immunoblot Analysis—J774A.1 cells (∼1 × 106) seeded in a 6-well plate were either left untreated or incubated with CD45 or scrambled PMO for 72 h. Cells were harvested and lysed in buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, 1% Triton X-100, and protease inhibitor mixture (Sigma). The cell lysates were incubated for 30 min on ice and then centrifuged for 30 min at 14,000 rpm. Cell extracts (30 μg) were electrophoresed on SDS-PAGE and then subjected to Western blotting. A CD45-specific mouse monoclonal antibody (clone 69, BD Pharmingen) was used to detect the immunoreactive proteins that were visualized by Enhanced Chemiluminescence (ECL).To determine the MEK cleavage pattern, J774A.1 cells were treated with the CD45 or scrambled PMOs (8 μm) for 72 h. Cells were harvested and then treated with anthrax lethal toxin (100 ng/ml Protective antigen (PA83) and 20 ng/ml of lethal factor (LF)) or infected with B. anthracis Sterne spores (5 m.o.i.). After a 4-h incubation time, cells were washed with phosphate-buffered saline (PBS), lysed, and electrophoresed as described above. Western blots were probed with MEK1"NT" antibody (Upstate Biotechnology) or glyceraldehyde-3-phosphate dehydrogenase for uniform protein loading and visualized by ECL.Animal Studies— 8–10-Week-old mice were used in this study and included both males and females. For in vivo B. anthracis studies, C57BL/6 wild type control (CD45100%), CD4562%, CD4511%, CD4536%, CD450%, and CSV10+/- mice were challenged via an intraperitoneal route with ∼300 colonyforming units (CFU) of Ames strain of B. anthracis. The mice were monitored for 1 month post-challenge.To test the efficacy of PMOs in vivo, BALB/c mice (6–8 weeks old, n = 6) were pretreated via subcutaneous route with PBS or CD45 PMO or scrambled PMO for 2 days (days -2 and -1). On the third day (day 0), the mice were treated with the PMOs and infected via intraperitoneal route with Ames spores (∼750 CFU). An additional PBS or PMO treatment was given the day after challenge (day 1). Non-tagged PMOs were used for in vivo studies and injected at a dose of 100 mg/kg/day. The mice were monitored for 1 month post-challenge. B. anthracis Ames spores from the same batch were used for all the in vivo mouse studies described in Fig. 4 and 5.FIGURE 5Mice expressing intermediate CD45 levels survive B. anthracis infection. A, mice with intermediate CD45 expression levels (62%) challenged with Ames B. anthracis spores showed a 65% survival rate. In contrast, CD45100%, CD450%, CD4511%, CD4536%, or CSV10+/- (62%) mice with inactive CD45 phosphatase activity showed little to no protection after B. anthracis challenge. B, immunohistochemical staining of spleen with anti-capsule antibody did not show any bacterial load in the CD4562% mice surviving B. anthracis challenge (∼48 h) (right panel) versus moribund CD45100% mice (48 h) (left panel). Scale bar = 100 μm, 20× magnification. RP, red pulp; MZ, marginal zone; PALS, periarteriolar lymphoid sheath.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the humoral responses, CD45100% and CD4562% mice were vaccinated 2 times at 2-week intervals via intraperitoneal injection with 100 μl of anthrax vaccine adsorbed mixed with 10 μg of QS-21 diluted in endotoxin-free PBS. After 35 days, mice were euthanized, blood was collected, and protective antigen (PA)-specific antibodies were measured by enzyme-linked immunosorbent assay.All research was conducted under an approved protocol and in compliance with the Animal Welfare Act and other federal statutes and regulations related to animals and experiments involving animals and adhered to principles stated in the "Guide for the Care and Use of Laboratory Animals," National Research Council 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of laboratory Animal Care International.Phagocytosis and Spore Viability—To enumerate the spores ingested by macrophages, thioglycolate-elicited peritoneal macrophages from CD45 100, 62, and 0% mice were infected with 5 m.o.i. of green fluorescent protein-Sterne spores and plate-centrifuged to synchronize the infection. After 30 min non-permeabilized cells were incubated with a mix of antibodies specific for B. anthracis spore exosporium (to label extracellular spores) and Bacillus polysaccharide (to label extracellular vegetative bacilli) (kindly provided by T. Abshire and J. Ezzel, United States Army Medical Research Institute of Infectious Diseases) followed by a secondary incubation with antibody conjugated to Alexa-594-nm fluorophore. This method labels only those spores adhered to the outside surface of the macrophages. After fixation with formaldehyde, cells were stained with Hoechst dyes, and images from nine sites/well were collected and analyzed using the Discovery-1 high content screening system (Molecular Devices, Downington, PA). Images were analyzed using the cell health module of MataXpress imaging analysis software. Total cell count was based on the number of Hoechst-stained cell nuclei, whereas co-localization of red (anti-spore and anti-bacterial antibody) and green (green fluorescent protein-Sterne spores) fluorescence was scored as spores being on the outside of the cell and with green-only fluorescence being scored as ingested spores.To measure spore viability, thioglycolate-elicited peritoneal macrophages purified by plastic adherence were infected with Sterne spores at an m.o.i. of 5. After 30 min cell pellets collected by centrifugation were lysed in sterile water and serially diluted, and aliquots were plated onto solid LB agar medium plates, which were then incubated at 37 °C for 16 h. CFU were counted, and data are represented as CFU/ml. Experiments were performed in duplicate and repeated three independent times.Macrophage Apoptosis—Thioglycolate elicited peritoneal macrophages from 100 or 62% CD45 expressor mice were either left untreated or infected with Sterne B. anthracis spores (10 m.o.i.) or staurosporine (2 μm) as a positive control. After 6 h, macrophage apoptosis was measured using the Apo-One Homogeneous Caspase 3/7 kit (Promega), as per the manufacturer instructions.Cytotoxicity Assay—J774A.1 macrophages (5 × 104) were either left untreated or treated with scrambled control or CD45 PMO (5 μm). After 72 h cells were treated with 100 ng/ml PA83 and 20 ng/ml LF, a toxin concentration that results in 80–90% killing of macrophages. After 4 h 25 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (1 mg/ml) dye was added, and cells were further incubated for 2 h. The reaction was stopped by adding equal volume of lysis buffer (50% N,N-dimethylformamide and 20% SDS, pH 4.7). Plates were incubated overnight at 37 °C, and cell viability was determined by measuring the absorbance at 570 nm in a multiwell plate reader.Antibacterial Growth Inhibition Assay—B. anthracis Sterne spores (5 × 105 CFU/ml) diluted in Mueller-Hinton broth were treated with either DMSO control or compounds NSC 95397 (10 μm) or NSC 270012 (10 μm). At time intervals of 2, 5, 7, and 24 h, absorbance at 600 nm was measured. Experiments were done in duplicate and repeated at least two independent times.Immunohistochemical Staining—To detect the presence of bacilli in infected tissues, the EnVision system (Dako) was used. Briefly, tissue sections were deparaffinized, blocked in methanol/H2O2 solution for 30 min at room temperature, rinsed with water, pretreated with Tris-EDTA, pH 9.0, at 97 °C for 30 min, and then blocked with mouse IgG blocking buffer (Vector Laboratories, 1:20). The tissues were then incubated with mouse anti-capsule antibody (#593) or mouse IgG as negative control serum for 30 min at room temperature. After rinsing 3 times with PBS, peroxidase-labeled polymer conjugated to goat anti-mouse immunoglobulins was added and incubated for 30 min. After rinsing with PBS, substrate-chromogen solution was added and incubated for 5 min. Tissues were then rinsed with PBS, stained with hematoxylin, dehydrated, and mounted with Permount.RESULTSChemical Genetic Screening and Phosphatase Profiling—To assess whether inhibition of host phosphatase function would elicit protection against B. anthracis-induced cell death, a chemical genetic approach was used wherein a focused library of known Cdc25 phosphatase inhibitors (22Panchal R.G. Ruthel G. Brittingham K.C. Lane D. Kenny T.A. Gussio R. Lazo J.S. Bavari S. Chem. Biol. 2007; 14: 245-255Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) and their analogs (supplemental Table 1) were screened in in
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