Hostname: page-component-77c89778f8-rkxrd Total loading time: 0 Render date: 2024-07-19T09:36:14.410Z Has data issue: false hasContentIssue false
  • English
  • Français

Flow cytometric assessment of distinct physiological stages within Cryptosporidium parvum sporozoites post-excystation

Published online by Cambridge University Press:  24 June 2009

B. J. KING ,
D. HOEFEL ,
S. P. LIM ,
B. S. ROBINSON  and
P. T. MONIS
B. J. KING
Affiliation:
The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia 5000, Australia
D. HOEFEL
Affiliation:
The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia 5000, Australia
S. P. LIM
Affiliation:
The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia 5000, Australia
B. S. ROBINSON
Affiliation:
The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia 5000, Australia
P. T. MONIS*
Affiliation:
The Co-operative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Adelaide, South Australia 5000, Australia
*
*Corresponding author: Tel: +618 7424 2062. Fax: 618 7003 2062. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37°C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

Keywords

Cryptosporidiumsporozoitesflow cytometryapical organelle dischargeexocytosiscalcium
Type
Research Article
Information
Parasitology , Volume 136 , Issue 9 , August 2009 , pp. 953 - 966
Copyright
Copyright © Cambridge University Press 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Amor, K. B., Breeuwer, P., Verbaarschot, P., Rombouts, F. M., Akkermans, A. D., De Vos, W. M. and Abee, T. (2002). Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress. Applied and Environmental Microbiology 68, 5209–5216.CrossRefGoogle ScholarPubMed
Arrizabalaga, G. and Boothroyd, J. C. (2004). Role of calcium during Toxoplasma gondii invasion and egress. International Journal for Parasitology 34, 361–368.CrossRefGoogle ScholarPubMed
Barnes, D. A., Bonnin, A., Huang, J. X., Gousset, L., Wu, J., Gut, J., Doyle, P., Dubremetz, J. F., Ward, H. and Petersen, C. (1998). A novel multi-domain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion. Molecular and Biochemical Parasitology 96, 93–110.CrossRefGoogle ScholarPubMed
Berney, M., Weilenmann, H. U. and Egli, T. (2006). Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS). Microbiology 152, 1719–1729.CrossRefGoogle ScholarPubMed
Black, M. and McAnulty, J. (2006). The investigation of an outbreak of cryptosporidiosis in New South Wales in 2005. N S W Public Health Bulletin 17, 76–79.Google ScholarPubMed
Borowski, H., Clode, P. L. and Thompson, R. C. (2008). Active invasion and/or encapsulation? A reappraisal of host-cell parasitism by Cryptosporidium. Trends in Parasitology 24, 509–516.Google Scholar
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.CrossRefGoogle ScholarPubMed
Brumback, A. C., Lieber, J. L., Angleson, J. K. and Betz, W. J. (2004). Using FM1-43 to study neuropeptide granule dynamics and exocytosis. Methods 33, 287–294.Google Scholar
Burgoyne, R. D. and Morgan, A. (1993). Regulated exocytosis. The Biochemical Journal 293, 305–316.CrossRefGoogle ScholarPubMed
Carruthers, V. B., Giddings, O. K. and Sibley, L. D. (1999). Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cellular Microbiology 1, 225–235.CrossRefGoogle ScholarPubMed
Carruthers, V. B. and Sibley, L. D. (1999). Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Molecular Microbiology 31, 421–428.CrossRefGoogle ScholarPubMed
Cevallos, A. M., Zhang, X., Waldor, M. K., Jaison, S., Zhou, X., Tzipori, S., Neutra, M. R. and Ward, H. D. (2000). Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15. Infection and Immunity 68, 4108–4116.CrossRefGoogle ScholarPubMed
Chaturvedi, S., Qi, H., Coleman, D., Rodriguez, A., Hanson, P. I., Striepen, B., Roos, D. S. and Joiner, K. A. (1999). Constitutive calcium-independent release of Toxoplasma gondii dense granules occurs through the NSF/SNAP/SNARE/Rab machinery. Journal of Biological Chemistry 274, 2424–2431.Google Scholar
Chen, X. M., O'Hara, S. P., Huang, B. Q., Nelson, J. B., Lin, J. J., Zhu, G., Ward, H. D. and Larusso, N. F. (2004). Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton, and intracellular calcium dependent and required for host cell invasion. Infection and Immunity 72, 6806–6816.CrossRefGoogle ScholarPubMed
Clapham, D. E. (1995). Calcium signaling. Cell 80, 259–268.CrossRefGoogle ScholarPubMed
Cochilla, A. J., Angleson, J. K. and Betz, W. J. (1999). Monitoring secretory membrane with FM1-43 fluorescence. Annual Review of Neuroscience 22, 1–10.Google Scholar
Cohen, R., Schmitt, B. M. and Atlas, D. (2005). Molecular identification and reconstitution of depolarization-induced exocytosis monitored by membrane capacitance. Biophysical Journal 89, 4364–4373.Google Scholar
De Castro Junior, C. J., Pinheiro, A. C., Guatimosim, C., Cordeiro, M. N., Souza, A. H., Richardson, M., Romano-Silva, M. A., Prado, M. A. and Gomez, M. V. (2008). Tx3-4 a toxin from the venom of spider Phoneutria nigriventer blocks calcium channels associated with exocytosis. Neuroscience Letters 439, 170–172.CrossRefGoogle ScholarPubMed
Dowse, T. and Soldati, D. (2004). Host cell invasion by the apicomplexans: the significance of microneme protein proteolysis. Current Opinion in Microbiology 7, 388–396.Google Scholar
Feng, H., Nie, W., Sheoran, A., Zhang, Q. and Tzipori, S. (2006). Bile acids enhance invasiveness of Cryptosporidium spp. into cultured cells. Infection and Immunity 74, 3342–3346.Google Scholar
Gee, K. R., Brown, K. A., Chen, W. N., Bishop-Stewart, J., Gray, D. and Johnson, I. (2000). Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium 27, 97–106.Google Scholar
Harris, J. R., Adrian, M. and Petry, F. (2003). Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle. Micron 34, 65–78.Google Scholar
Harris, J. R. and Petry, F. (1999). Cryptosporidium parvum: structural components of the oocyst wall. Journal of Parasitology 85, 839–849.CrossRefGoogle ScholarPubMed
Hijjawi, N. S., Meloni, B. P., Morgan, U. M. and Thompson, R. C. (2001). Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture. International Journal for Parasitology 31, 1048–1055.Google Scholar
Hijjawi, N. S., Meloni, B. P., Ng'anzo, M., Ryan, U. M., Olson, M. E., Cox, P. T., Monis, P. T. and Thompson, R. C. (2004). Complete development of Cryptosporidium parvum in host cell-free culture. International Journal for Parasitology 34, 769–777.CrossRefGoogle ScholarPubMed
Holz, R. W., Senyshyn, J. and Bittner, M. A. (1991). Mechanisms involved in calcium-dependent exocytosis. Annals of the New York Academy of Sciences 635, 382–392.Google Scholar
Huang, B. Q., Chen, X. M. and Larusso, N. F. (2004). Cryptosporidium parvum attachment to and internalization by human biliary epithelia in vitro: a morphologic study. Journal of Parasitology 90, 212–221.CrossRefGoogle ScholarPubMed
Keegan, A. R., Fanok, S., Monis, P. T. and Saint, C. P. (2003). Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection. Applied and Environmental Microbiology 69, 2505–2511.CrossRefGoogle ScholarPubMed
King, B. J., Keegan, A. R., Monis, P. T. and Saint, C. P. (2005). Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity. Applied and Environmental Microbiology 71, 3848–3857.CrossRefGoogle ScholarPubMed
Koga, T., Kozaki, S. and Takahashi, M. (2002). Exocytotic release of alanine from cultured cerebellar neurons. Brain Research 952, 282–289.Google Scholar
Lipton, P. (1999). Ischemic cell death in brain neurons. Physiological Reviews 79, 1431–1568.Google Scholar
Lovett, J. L. and Sibley, L. D. (2003). Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells. Journal of Cell Science 116, 3009–3016.Google Scholar
Matthiesen, S. H., Shenoy, S. M., Kim, K., Singer, R. H. and Satir, B. H. (2003). Role of the parafusin orthologue, PRP1, in microneme exocytosis and cell invasion in Toxoplasma gondii. Cellular Microbiology 5, 613–624.Google Scholar
Meffert, M. K., Premack, B. A. and Schulman, H. (1994). Nitric oxide stimulates Ca(2+)-independent synaptic vesicle release. Neuron 12, 1235–1244.CrossRefGoogle ScholarPubMed
Mercier, C., Adjogble, K. D., Daubener, W. and Delauw, M. F. (2005). Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites? International Journal for Parasitology 35, 829–849.CrossRefGoogle ScholarPubMed
Moreno, S. N. and Docampo, R. (2003). Calcium regulation in protozoan parasites. Current Opinion in Microbiology 6, 359–364.CrossRefGoogle ScholarPubMed
Morgan, U. M., Constantine, C. C., Forbes, D. A. and Thompson, R. C. (1997). Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis. Journal of Parasitology 83, 825–830.CrossRefGoogle ScholarPubMed
Nagamune, K. and Sibley, L. D. (2006). Comparative genomic and phylogenetic analyses of calcium ATPases and calcium-regulated proteins in the apicomplexa. Molecular Biology and Evolution 23, 1613–1627.CrossRefGoogle ScholarPubMed
Nesterenko, M. V., Woods, K. and Upton, S. J. (1999). Receptor/ligand interactions between Cryptosporidium parvum and the surface of the host cell. Biochimica et Biophysica Acta-Molecular Basis of Disease 1454, 165–173.Google Scholar
O'Donoghue, P. J. (1995). Cryptosporidium and cryptosporidiosis in man and animals. International Journal for Parasitology 25, 139–195.CrossRefGoogle Scholar
O'Hara, S. P., Huang, B. Q., Chen, X. M., Nelson, J. and Larusso, N. F. (2005). Distribution of Cryptosporidium parvum sporozoite apical organelles during attachment to and internalization by cultured biliary epithelial cells. Journal of Parasitology 91, 995–999.Google Scholar
Sadakata, T., Mizoguchi, A., Sato, Y., Katoh-Semba, R., Fukuda, M., Mikoshiba, K. and Furuichi, T. (2004). The secretory granule-associated protein CAPS2 regulates neurotrophin release and cell survival. Journal of Neuroscience 24, 43–52.CrossRefGoogle ScholarPubMed
Schuster, C. J., Ellis, A. G., Robertson, W. J., Charron, D. F., Aramini, J. J., Marshall, B. J. and Medeiros, D. T. (2005). Infectious disease outbreaks related to drinking water in Canada, 1974–2001. Canadian Journal of Public Health 96, 254–258.Google Scholar
Semenza, J. C. and Nichols, G. (2007). Cryptosporidiosis surveillance and water-borne outbreaks in Europe. Euro Surveillance 12, E13–E14.Google Scholar
Sibley, L. D. (2004). Intracellular parasite invasion strategies. Science 304, 248–253.Google Scholar
Silverman, J. A., Qi, H., Riehl, A., Beckers, C., Nakaar, V. and Joiner, K. A. (1998). Induced activation of the Toxoplasma gondii nucleoside triphosphate hydrolase leads to depletion of host cell ATP levels and rapid exit of intracellular parasites from infected cells. Journal of Biological Chemistry 273, 12352–12359.Google Scholar
Smith, A., Reacher, M., Smerdon, W., Adak, G. K., Nichols, G. and Chalmers, R. M. (2006). Outbreaks of waterborne infectious intestinal disease in England and Wales, 1992–2003. Epidemiology and Infection 134, 1141–1149.CrossRefGoogle ScholarPubMed
Smith, H. V., Nichols, R. A. and Grimason, A. M. (2005). Cryptosporidium excystation and invasion: getting to the guts of the matter. Trends in Parasitology 21, 133–142.Google Scholar
Sollner, T. H. (2003). Regulated exocytosis and SNARE function (Review). Molecular Membrane Biology 20, 209–220.Google Scholar
Tetley, L., Brown, S. M., Mcdonald, V. and Coombs, G. H. (1998). Ultrastructural analysis of the sporozoite of Cryptosporidium parvum. Microbiology 144, 3249–3255.CrossRefGoogle ScholarPubMed
Tosini, F., Agnoli, A., Mele, R., Gomez Morales, M. A. and Pozio, E. (2004). A new modular protein of Cryptosporidium parvum, with ricin B and LCCL domains, expressed in the sporozoite invasive stage. Molecular and Biochemical Parasitology 134, 137–147.Google Scholar
Tsien, R. W. and Tsien, R. Y. (1990). Calcium channels, stores, and oscillations. Annual Review of Cell Biology 6, 715–760.CrossRefGoogle ScholarPubMed
Tzipori, S. and Griffiths, J. K. (1998). Natural history and biology of Cryptosporidium parvum. Advances in Parasitology 40, 5–36.Google Scholar
Valigurova, A., Jirku, M., Koudela, B., Gelnar, M., Modry, D. and Slapeta, J. (2008). Cryptosporidia: Epicellular parasites embraced by the host cell membrane. International Journal for Parasitology 38, 913–922.CrossRefGoogle ScholarPubMed
Vesey, G., Griffiths, K. R., Gauci, M. R., Deere, D., Williams, K. L. and Veal, D. A. (1997). Simple and rapid measurement of Cryptosporidium excystation using flow cytometry. International Journal for Parasitology 27, 1353–1359.Google Scholar
Wetzel, D. M., Schmidt, J., Kuhlenschmidt, M. S., Dubey, J. P. and Sibley, L. D. (2005). Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites. Infection and Immunity 73, 5379–5387.Google Scholar