{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T13:43:17Z","timestamp":1790257397317,"version":"4.1.0"},"reference-count":39,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5495","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2000,11,17]]},"abstract":"<jats:p>\n            <jats:italic>Histoplasma capsulatum<\/jats:italic>\n            is an effective intracellular parasite of macrophages and causes the most prevalent fungal respiratory disease in the United States. A \u201cdimorphic\u201d fungus,\n            <jats:italic>H. capsulatum<\/jats:italic>\n            exists as a saprophytic mold in soil and converts to the parasitic yeast form after inhalation. Only the yeasts secrete a calcium-binding protein (CBP) and can grow in calcium-limiting conditions. To probe the relation between calcium limitation and intracellular parasitism, we designed a strategy to disrupt\n            <jats:italic>CBP1<\/jats:italic>\n            in\n            <jats:italic>H. capsulatum<\/jats:italic>\n            using a telomeric linear plasmid and a two-step genetic selection. The resulting\n            <jats:italic>cbp1<\/jats:italic>\n            yeasts no longer grew when deprived of calcium, and they were also unable to destroy macrophages in vitro or proliferate in a mouse model of pulmonary infection.\n          <\/jats:p>","DOI":"10.1126\/science.290.5495.1368","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:47:44Z","timestamp":1027763264000},"page":"1368-1372","source":"Crossref","is-referenced-by-count":159,"title":["Intracellular Parasitism by\n            <i>Histoplasma capsulatum<\/i>\n            : Fungal Virulence and Calcium Dependence"],"prefix":"10.1126","volume":"290","author":[{"given":"Tricia Schurtz","family":"Sebghati","sequence":"first","affiliation":[{"name":"Department of Molecular Microbiology, Campus Box 8230, Washington University School of Medicine, St. Louis, MO 63110, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacquelyn T.","family":"Engle","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology, Campus Box 8230, Washington University School of Medicine, St. Louis, MO 63110, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William E.","family":"Goldman","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology, Campus Box 8230, Washington University School of Medicine, St. Louis, MO 63110, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","first-page":"623","DOI":"10.1093\/infdis\/158.3.623","volume":"158","author":"Graybill J. R.","year":"1988","unstructured":"Graybill J. R., J. Infect. Dis. 158, 623 (1988);","journal-title":"J. Infect. Dis."},{"key":"e_1_3_1_2_3","unstructured":"; R. P. Baughman et al. Am. Rev. Respir. Dis. 134 771 (1986)."},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1128\/CMR.4.4.411","volume":"4","author":"Eissenberg L. G.","year":"1991","unstructured":"Eissenberg L. G., Goldman W. E., Clin. Microbiol. Rev. 4, 411 (1991);","journal-title":"Clin. Microbiol. Rev."},{"key":"e_1_3_1_3_3","unstructured":"; J. W. Rippon in Medical Mycology. The Pathogenic Fungi and the Pathogenic Actinomycetes M. L. W. R. Wonsiewicz Ed. (Saunders Philadelphia PA 1988) p. 381;"},{"key":"e_1_3_1_3_4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1097\/00005792-198001000-00001","volume":"59","author":"Goodwin R. A.","year":"1980","unstructured":"Goodwin R. A., Shapiro J. L., Thurman G. H., Thurman S. S., Des Perez R. M., Medicine 59, 1 (1980).","journal-title":"Medicine"},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1002\/jlb.43.6.483","volume":"43","author":"Eissenberg L. G.","year":"1988","unstructured":"Eissenberg L. G., Schlesinger P. H., Goldman W. E., J. Leukocyte Biol. 43, 483 (1988).","journal-title":"J. Leukocyte Biol."},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1084\/jem.177.6.1605","volume":"177","author":"Eissenberg L. G.","year":"1993","unstructured":"Eissenberg L. G., Goldman W. E., Schlesinger P. H., J. Exp. Med. 177, 1605 (1993).","journal-title":"J. Exp. Med."},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"1440","DOI":"10.1074\/jbc.272.3.1440","volume":"272","author":"Garc\u0131\u0301a V\u00e9scovi E.","year":"1997","unstructured":"Garc\u0131\u0301a V\u00e9scovi E., Ayala Y. M., Di Cera E., Groisman E., J. Biol. Chem. 272, 1440 (1997).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","first-page":"5257","DOI":"10.1128\/iai.65.12.5257-5261.1997","volume":"65","author":"Batanghari J. W.","year":"1997","unstructured":"Batanghari J. W., Goldman W. E., Infect. Immun. 65, 5257 (1997);","journal-title":"Infect. Immun."},{"key":"e_1_3_1_7_3","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1046\/j.1462-5822.2000.00078.x","volume":"2","author":"K\u00fcgler S.","year":"2000","unstructured":"K\u00fcgler S., Young B., Miller V. L., Goldman W. E., Cell. Microbiol. 2, 549 (2000).","journal-title":"Cell. Microbiol."},{"key":"e_1_3_1_8_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.1998.00697.x"},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/S0378-1119(96)00713-5","volume":"186","author":"Hogan L. H.","year":"1997","unstructured":"Hogan L. H., Klein B. S., Gene 186, 219 (1997);","journal-title":"Gene"},{"key":"e_1_3_1_9_3","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1128\/iai.60.3.1101-1108.1992","volume":"60","author":"Varma A.","year":"1992","unstructured":"Varma A., Edman J. C., Kwon-Chung K. J., Infect. Immun. 60, 1101 (1992).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_10_2","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1007\/BF00337734","volume":"214","author":"Worsham P. L.","year":"1988","unstructured":"Worsham P. L., Goldman W. E., Mol. Gen. Genet. 214, 348 (1988).","journal-title":"Mol. Gen. Genet."},{"key":"e_1_3_1_11_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.1992.tb01796.x"},{"key":"e_1_3_1_12_2","first-page":"5135","volume":"180","author":"Woods J. P.","year":"1998","unstructured":"Woods J. P., Retallack D. M., Heinecke E. L., Goldman W. E., Infect. Immun. 180, 5135 (1998).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.175.3.636-641.1993"},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","first-page":"1697","DOI":"10.1128\/IAI.66.4.1697-1707.1998","volume":"66","author":"Woods J. P.","year":"1998","unstructured":"Woods J. P., Heinecke E. L., Goldman W. E., Infect. Immun. 66, 1697 (1998).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_15_2","unstructured":"Supplementary data are available at Science Online at www.sciencemag.org\/cgi\/content\/full\/290\/5495\/1368\/DC1."},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/0378-1119(87)90164-8","volume":"56","author":"Punt P. J.","year":"1987","unstructured":"Punt P. J., Oliver R. P., Dingemanse M. A., Pouwels P. H., van den Hondel C. A. M. J. J., Gene 56, 117 (1987).","journal-title":"Gene"},{"key":"e_1_3_1_17_2","unstructured":"Ultraviolet (UV) light mutagenesis was performed on late exponential phase yeast cells (4 \u00d7 10 8 \/ml) grown in HMM medium (19) placed on ice for 20 min and centrifuged (110 g 60 s) to remove large aggregates. The yeast suspension was diluted with fresh ice-cold HMM to a density of 10 7 colony-forming units (CFU) per milliliter. An ultraviolet (UV) germicidal lamp was used at a distance of 64 cm to irradiate the yeasts. Yeast cells were stirred during the 40-s exposure to UV light. The cells were washed with ice-cold HMM and plated on selective medium containing 5-FOA (1 mg\/ml) and uracil (100 \u03bcg\/ml). All manipulations after exposure to UV light were performed in the dark to prevent photoreactivation repair. Plates were incubated at 37\u00b0C with 95% air 5% CO 2 in a humidified incubator. Colonies arising on HMM with 5-FOA\u2013uracil were picked 3 weeks after plating and streaked for isolation on the same medium. Single colonies were picked and replica-plated onto HMM with and without uracil. Several independent isolates exhibited uracil auxotrophy in the yeast phase and one of these isolates also exhibited auxotrophy when grown as mycelia at room temperature."},{"key":"e_1_3_1_18_2","unstructured":"Before its introduction into yeasts each plasmid (2 \u03bcg) was linearized by digestion with Pac I. The linear telomeric plasmid was purified by gel electrophoresis to separate it from the kanamycin-resistance cassette and the plasmid was concentrated by ethanol precipitation. The plasmid DNA was introduced into yeasts by electroporation (13). Briefly 5 ml of a 2-day culture was centrifuged at 300 g for 5 min. The supernatant was discarded and the yeasts were resuspended in 5 ml of warm (37\u00b0C) 10% (w\/v) mannitol. The yeasts were recovered by centrifugation as above and resuspended in 200 \u03bcl of 10% (w\/v) mannitol. The yeasts were mixed with the plasmid DNA in a 0.2-cm cuvette at room temperature. The electroporations were done at a capacitance of 25 \u03bcF a resistance of 600 ohm and a voltage of 0.75 kV. Time constants were between 10 and 12 ms. The electroporated cells were directly plated onto HMM without uracil and incubated at 37\u00b0C for 12 to 14 days."},{"key":"e_1_3_1_19_2","unstructured":"The virulent strain used in this study H. capsulatum G186AR has been described previously (27). Unless otherwise indicated H. capsulatum was grown as described for yeast cells at 37\u00b0C in the defined medium HMM (19). In some experiments uracil (100 \u03bcg\/ml) hygromycin B (100 \u03bcg\/ml) and\/or 5-FOA (1 mg\/ml) were added to solid or liquid HMM."},{"key":"e_1_3_1_20_2","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1080\/02681218880000211","volume":"26","author":"Worsham P. L.","year":"1988","unstructured":"Worsham P. L., Goldman W. E., J. Med. Vet. Mycol. 26, 137 (1988).","journal-title":"J. Med. Vet. Mycol."},{"key":"e_1_3_1_21_2","unstructured":"Total genomic DNA from Histoplasma was purified as previously described (10). Southern blotting and PCR were done according to standard procedures (21). DNA probes were labeled and signals were detected with enhanced chemiluminescence direct nucleic acid labeling and detection systems (Amersham Life Science Arlington Heights IL)."},{"key":"#cr-split#-e_1_3_1_22_2.1","unstructured":"J. Sambrook E. F. Fritsch T. Maniatis Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY ed. 2 1998)"},{"key":"#cr-split#-e_1_3_1_22_2.2","unstructured":"F. M. Ausubel et al. Eds. Current Protocols in Molecular Biology (Wiley New York 2000)."},{"key":"e_1_3_1_23_2","unstructured":"For 45 CaCl 2 blotting proteins in concentrated culture supernatants were separated by SDS\u2013polyacrylamide gel electrophoresis (PAGE) and transferred to 0.1-\u03bcm nitrocellulose. After incubation in calcium-free hybridization buffer [10 mM tris-HCl (pH 6.8) 60 mM KCl and 5 mM MgCl 2 ] the filter was probed with 45 CaCl 2 (3 \u03bcCi\/ml) for 8 hours at 37\u00b0C followed by washing for 5 min with water at pH 6.8. The filter was exposed for autoradiography. Ruthenium red staining was performed by blotting proteins as described above then incubating the nitrocellulose in Ruthenium red (25 mg\/liter)."},{"key":"e_1_3_1_24_2","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/0003-2697(90)90539-L","volume":"188","author":"Charuk J. H. M.","year":"1990","unstructured":"Charuk J. H. M., Pirraglia C. A., Reithmeier R. A. F., Anal. Biochem. 188, 123 (1990).","journal-title":"Anal. Biochem."},{"key":"e_1_3_1_25_2","doi-asserted-by":"crossref","first-page":"938","DOI":"10.1093\/infdis\/152.5.938","volume":"152","author":"Levitz S. M.","year":"1985","unstructured":"Levitz S. M., Diamond R. D., J. Infect. Dis. 152, 938 (1985).","journal-title":"J. Infect. Dis."},{"key":"e_1_3_1_26_2","unstructured":"For growth of yeast in calcium-limiting conditions H. capsulatum strains G186AR ura 5 and G186AR ura5 cbp1 :: hph -6 and -7 were inoculated at 1 \u00d7 10 6 yeast cells\/ml into 24-well plates in HMM supplemented with 150 300 or 600 \u03bcM EGTA to chelate the 300 \u03bcM Ca 2+ present in HMM. Growth was monitored spectrophotometrically at an optical density of 600 nm. Metabolic activity was measured by the reduction of MTT to purple crystals of formazan and quantitated by measuring the absorbance at 570 nm."},{"key":"e_1_3_1_27_2","unstructured":"T. Schurtz Sebghati J. T. Engle W. E. Goldman data not shown."},{"key":"e_1_3_1_28_2","doi-asserted-by":"crossref","first-page":"1639","DOI":"10.1128\/iai.59.5.1639-1646.1991","volume":"59","author":"Eissenberg L. G.","year":"1991","unstructured":"Eissenberg L. G., West J. L., Woods J. P., Goldman W. E., Infect. Immun. 59, 1639 (1991).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_29_2","unstructured":"The in vitro virulence assay uses P388D1.D2 cells a randomly selected clone from P388D1 macrophage-like cells (27) which were seeded at a 2 \u00d7 10 4 cells per 7-mm well in F-12 completed with 10% fetal bovine serum. The monolayers were inoculated in triplicate with dispersed yeasts in HMM-M completed with 10% fetal bovine serum (3) supplemented with uracil for auxotrophs with a yeast:host cell ratio of 1:5. The 96-well microtiter plates were rocked continuously on a rocking platform to facilitate coculture of these cells. The medium was replaced with fresh medium at 3 days after inoculation and every other day thereafter. Using phase-contrast microscopy we examined the wells daily to estimate when \u223c95% of the macrophage monolayer was destroyed by wild-type virulent yeasts. At this time (8 days) all medium was removed and wells were washed three times with sterile phosphate-buffered saline to remove any lysed cells or free yeast cells. The macrophages were then lysed by addition of sterile distilled water (pH 7.0) which does not lyse yeast cells. The fluorescent PicoGreen double-stranded DNA (dsDNA) quantitation reagent (Molecular Probes Sunnyvale CA) was used to measure the amount of macrophage DNA present in each well. The fluorescence intensity was measured with a FluorImager SI (Molecular Probes) at 530 nm."},{"key":"e_1_3_1_30_2","unstructured":"Male Balb\/C57BL\/6J mice were purchased from Jackson Laboratories. Mice were infected intranasally with H. capsulatum cells in a 50-\u03bcl volume. For all strains tested we inoculated three animals with each inoculum dose. After 8 days the animals were killed and the lungs were homogenized in HMM medium and serially diluted and dispensed (50 \u03bcl) into HMM pour agar plates supplemented with 10 \u03bcM FeSO 4 . Plates were incubated at 37\u00b0C and CFU were counted after 10 days. To determine actual intranasal inoculum yeasts were appropriately diluted and plated and CFU were counted."},{"key":"e_1_3_1_31_2","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1007\/BF01967211","volume":"9","author":"Deepe G. S.","year":"1990","unstructured":"Deepe G. S., Bullock W. E., Eur. J. Clin. Microbiol. Infect. Dis. 9, 567 (1990).","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"e_1_3_1_32_2","doi-asserted-by":"crossref","first-page":"1335","DOI":"10.1016\/S0960-9822(07)00565-9","volume":"8","author":"Gerasimenko J. V.","year":"1998","unstructured":"Gerasimenko J. V., Tepikin A. V., Petersen O. H., Gerasimenko O. V., Curr. Biol. 8, 1335 (1998).","journal-title":"Curr. Biol."},{"key":"e_1_3_1_33_2","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1128\/IAI.67.2.624-629.1999","volume":"67","author":"Retallack D. M.","year":"1999","unstructured":"Retallack D. M., Heinecke E. L., Gibbons R., Deepe G. S., Woods J. P., Infect. Immun. 67, 624 (1999).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_34_2","unstructured":"We thank J. West Batanghari and J. Baldus Patel for preliminary experiments on CBP1 disruption in H. capsulatum. Supported by Public Health Service grants AI25584 (to W.E.G.) and A107172 and HL07317 (to Washington University). W.E.G. is a recipient of the Burroughs Wellcome Fund Scholar Award in Molecular Pathogenic Mycology."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.290.5495.1368","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T08:55:21Z","timestamp":1705136121000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.290.5495.1368"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2000,11,17]]},"references-count":39,"journal-issue":{"issue":"5495","published-print":{"date-parts":[[2000,11,17]]}},"alternative-id":["10.1126\/science.290.5495.1368"],"URL":"https:\/\/doi.org\/10.1126\/science.290.5495.1368","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2000,11,17]]}}}