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. 1995 Oct;63(10):3840–3845. doi: 10.1128/iai.63.10.3840-3845.1995

A Cryptosporidium parvum genomic region encoding hemolytic activity.

M I Steele 1, T L Kuhls 1, K Nida 1, C S Meka 1, I M Halabi 1, D A Mosier 1, W Elliott 1, D L Crawford 1, R A Greenfield 1
PMCID: PMC173540  PMID: 7558289

Abstract

Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase-mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle.

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Selected References

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  1. Alting-Mees M. A., Short J. M. pBluescript II: gene mapping vectors. Nucleic Acids Res. 1989 Nov 25;17(22):9494–9494. doi: 10.1093/nar/17.22.9494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Andrews N. W., Abrams C. K., Slatin S. L., Griffiths G. A T. cruzi-secreted protein immunologically related to the complement component C9: evidence for membrane pore-forming activity at low pH. Cell. 1990 Jun 29;61(7):1277–1287. doi: 10.1016/0092-8674(90)90692-8. [DOI] [PubMed] [Google Scholar]
  4. Andrews N. W. The acid-active hemolysin of Trypanosoma cruzi. Exp Parasitol. 1990 Aug;71(2):241–244. doi: 10.1016/0014-4894(90)90027-a. [DOI] [PubMed] [Google Scholar]
  5. Berkelman R. L. Emerging infectious diseases in the United States, 1993. J Infect Dis. 1994 Aug;170(2):272–277. doi: 10.1093/infdis/170.2.272. [DOI] [PubMed] [Google Scholar]
  6. Bermudes D., Dubremetz J. F., Achbarou A., Joiner K. A. Cloning of a cDNA encoding the dense granule protein GRA3 from Toxoplasma gondii. Mol Biochem Parasitol. 1994 Dec;68(2):247–257. doi: 10.1016/0166-6851(94)90169-4. [DOI] [PubMed] [Google Scholar]
  7. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  8. Buchanan K. L., Murphy J. W. Regulation of cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response. Infect Immun. 1994 Jul;62(7):2930–2939. doi: 10.1128/iai.62.7.2930-2939.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Current W. L., Garcia L. S. Cryptosporidiosis. Clin Microbiol Rev. 1991 Jul;4(3):325–358. doi: 10.1128/cmr.4.3.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dykstra C. C., Blagburn B. L., Tidwell R. R. Construction of genomic libraries of Cryptosporidium parvum and identification of antigen-encoding genes. J Protozool. 1991 Nov-Dec;38(6):76S–78S. [PubMed] [Google Scholar]
  12. Gaillard J. L., Berche P., Mounier J., Richard S., Sansonetti P. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect Immun. 1987 Nov;55(11):2822–2829. doi: 10.1128/iai.55.11.2822-2829.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kim K., Goozé L., Petersen C., Gut J., Nelson R. G. Isolation, sequence and molecular karyotype analysis of the actin gene of Cryptosporidium parvum. Mol Biochem Parasitol. 1992 Jan;50(1):105–113. doi: 10.1016/0166-6851(92)90248-i. [DOI] [PubMed] [Google Scholar]
  14. King C. H., Mundayoor S., Crawford J. T., Shinnick T. M. Expression of contact-dependent cytolytic activity by Mycobacterium tuberculosis and isolation of the genomic locus that encodes the activity. Infect Immun. 1993 Jun;61(6):2708–2712. doi: 10.1128/iai.61.6.2708-2712.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuhls T. L., Greenfield R. A., Mosier D. A., Crawford D. L., Joyce W. A. Cryptosporidiosis in adult and neonatal mice with severe combined immunodeficiency. J Comp Pathol. 1992 May;106(4):399–410. doi: 10.1016/0021-9975(92)90024-o. [DOI] [PubMed] [Google Scholar]
  17. Kuhls T. L., Mosier D. A., Abrams V. L., Crawford D. L., Greenfield R. A. Inability of interferon-gamma and aminoguanidine to alter Cryptosporidium parvum infection in mice with severe combined immunodeficiency. J Parasitol. 1994 Jun;80(3):480–485. [PubMed] [Google Scholar]
  18. Kuhls T. L., Mosier D. A., Crawford D. L. Effects of carbohydrates and lectins on cryptosporidial sporozoite penetration of cultured cell monolayers. J Protozool. 1991 Nov-Dec;38(6):74S–76S. [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lally N. C., Baird G. D., McQuay S. J., Wright F., Oliver J. J. A 2359-base pair DNA fragment from Cryptosporidium parvum encoding a repetitive oocyst protein. Mol Biochem Parasitol. 1992 Nov;56(1):69–78. doi: 10.1016/0166-6851(92)90155-d. [DOI] [PubMed] [Google Scholar]
  21. Lumb R., Smith K., O'Donoghue P. J., Lanser J. A. Ultrastructure of the attachment of Cryptosporidium sporozoites to tissue culture cells. Parasitol Res. 1988;74(6):531–536. doi: 10.1007/BF00531630. [DOI] [PubMed] [Google Scholar]
  22. Mac Kenzie W. R., Hoxie N. J., Proctor M. E., Gradus M. S., Blair K. A., Peterson D. E., Kazmierczak J. J., Addiss D. G., Fox K. R., Rose J. B. A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply. N Engl J Med. 1994 Jul 21;331(3):161–167. doi: 10.1056/NEJM199407213310304. [DOI] [PubMed] [Google Scholar]
  23. Mannheimer S. B., Soave R. Protozoal infections in patients with AIDS. Cryptosporidiosis, isosporiasis, cyclosporiasis, and microsporidiosis. Infect Dis Clin North Am. 1994 Jun;8(2):483–498. [PubMed] [Google Scholar]
  24. Marcial M. A., Madara J. L. Cryptosporidium: cellular localization, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterology. 1986 Mar;90(3):583–594. doi: 10.1016/0016-5085(86)91112-1. [DOI] [PubMed] [Google Scholar]
  25. Mead J. R., Arrowood M. J., Current W. L., Sterling C. R. Field inversion gel electrophoretic separation of Cryptosporidium spp. chromosome-sized DNA. J Parasitol. 1988 Jun;74(3):366–369. [PubMed] [Google Scholar]
  26. Petersen C., Gut J., Leech J. H., Nelson R. G. Identification and initial characterization of five Cryptosporidium parvum sporozoite antigen genes. Infect Immun. 1992 Jun;60(6):2343–2348. doi: 10.1128/iai.60.6.2343-2348.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Portnoy D. A., Jacks P. S., Hinrichs D. J. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med. 1988 Apr 1;167(4):1459–1471. doi: 10.1084/jem.167.4.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ranucci L., Müller H. M., La Rosa G., Reckmann I., Morales M. A., Spano F., Pozio E., Crisanti A. Characterization and immunolocalization of a Cryptosporidium protein containing repeated amino acid motifs. Infect Immun. 1993 Jun;61(6):2347–2356. doi: 10.1128/iai.61.6.2347-2356.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ribeiro Júnior H. da C., Teichberg S., Sun T., Fagundes Neto U., Lifshitz F. Ultrastructure of human cryptosporidial infection: a review. Prog Clin Parasitol. 1989;1:143–158. [PubMed] [Google Scholar]
  30. Robson K. J., Hall J. R., Jennings M. W., Harris T. J., Marsh K., Newbold C. I., Tate V. E., Weatherall D. J. A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature. 1988 Sep 1;335(6185):79–82. doi: 10.1038/335079a0. [DOI] [PubMed] [Google Scholar]
  31. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  32. Saffer L. D., Long Krug S. A., Schwartzman J. D. The role of phospholipase in host cell penetration by Toxoplasma gondii. Am J Trop Med Hyg. 1989 Feb;40(2):145–149. doi: 10.4269/ajtmh.1989.40.145. [DOI] [PubMed] [Google Scholar]
  33. Saffer L. D., Schwartzman J. D. A soluble phospholipase of Toxoplasma gondii associated with host cell penetration. J Protozool. 1991 Sep-Oct;38(5):454–460. doi: 10.1111/j.1550-7408.1991.tb04816.x. [DOI] [PubMed] [Google Scholar]
  34. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sansonetti P. J., Ryter A., Clerc P., Maurelli A. T., Mounier J. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun. 1986 Feb;51(2):461–469. doi: 10.1128/iai.51.2.461-469.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schmidt H., Karch H., Beutin L. The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coli alpha-hemolysin family. FEMS Microbiol Lett. 1994 Apr 1;117(2):189–196. doi: 10.1111/j.1574-6968.1994.tb06763.x. [DOI] [PubMed] [Google Scholar]
  37. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  38. Stanley P., Packman L. C., Koronakis V., Hughes C. Fatty acylation of two internal lysine residues required for the toxic activity of Escherichia coli hemolysin. Science. 1994 Dec 23;266(5193):1992–1996. doi: 10.1126/science.7801126. [DOI] [PubMed] [Google Scholar]
  39. Tokunaga K., Taniguchi H., Yoda K., Shimizu M., Sakiyama S. Nucleotide sequence of a full-length cDNA for mouse cytoskeletal beta-actin mRNA. Nucleic Acids Res. 1986 Mar 25;14(6):2829–2829. doi: 10.1093/nar/14.6.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Welch R. A. Pore-forming cytolysins of gram-negative bacteria. Mol Microbiol. 1991 Mar;5(3):521–528. doi: 10.1111/j.1365-2958.1991.tb00723.x. [DOI] [PubMed] [Google Scholar]

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