ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1991, 0066-4804/91/020224-04$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 35, No. 2
p. 224-227
In Vitro Activities of Lytic Peptides against the Sporozoites of Cryptosporidium parvum MICHAEL J. ARROWOOD,l* JESSE M. JAYNES,2 AND MARK C. HEALEY' Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah 84322-5600,1 and Department of Biochemistry, Louisiana State University, Baton Rouge, Louisiana 708032 Received 12 July 1990/Accepted 14 November 1990
Cryptosporidium parvum is a protozoan parasite that causes mild to severe diarrheal disease in animals and humans. There are currently no effective chemotherapeutic agents available for the treatment of cryptosporidiosis. Recent studies have described small, naturally occurring antimicrobial lytic peptides with antiprotozoal activities. In the present study, the anticryptosporidial activities of three synthetic lytic peptides were determined in an in vitro sporozoite susceptibility assay. Sporozoite viability was assessed microscopically by the uptake of the vital dyes fluorescein diacetate and propidium iodide. Sporozoite viability was reduced by 93.5% following a 60-min exposure to 10 ,uM Hecate-1 at 37°C. Shiva-10 reduced sporozoite viability by approximately 74.0% after a 60-min exposure at 100 ,M and 37°C. The cecropin-b analog SB-37 reduced sporozoite viability by 6.0% following a 60-min exposure at 100 ,uM and 37°C. A control peptide showed no anticryptosporidial activity.
blindly evaluated samples. Peptide numbers and their respective MWs were as follows: 1, 2,537; 2, 2,537; 5, 4,103; and 6 (control peptide), 3,425. Each peptide was solubilized in phosphate-buffered saline (PBS) (81 mM Na2HPO4, 15 mM KH2PO4, 137 mM NaCl, 27 mM KCl [pH 7.2]), yielding 1 mM stock solutions. Peptide solutions were dispensed in 100-RlI aliquots into microcentrifuge tubes and stored at -200C. Cryptosporidial oocysts. The C. parvum (Iowa isolate) oocysts employed in this study were originally obtained from Harley Moon (U.S. Department of Agriculture, Ames, Iowa). Oocysts were generated in experimentally infected Holstein calves and purified from feces by using discontinuous sucrose gradients (2). Purified oocysts were stored in 2.5% potassium dichromate at 4°C. Cryptosporidial sporozoites. Purified oocysts were washed with PBS to remove potassium dichromate. Approximately 5 x 106 oocysts were incubated at 37°C in 1 ml of a PBS-based excystation solution in order to excyst (release) the sporozoites. The excystation solution contained 0.25% trypsin (catalog no. T-8253; Sigma Chemical Co., St. Louis, Mo.) and 0.75% sodium taurocholate (prepared with equal portions of catalog no. T-0750 from Sigma Chemical Co. and catalog no. 102930 from ICN Biomedicals Inc., Costa Mesa, Calif.). Sporozoites were washed free of the excystation solution by centrifugation and resuspended in 1 ml of PBS. Antisporozoite assay. Sporozoite suspensions were dispensed into microcentrifuge tubes in 200-pI aliquots (approximately 2 x 106 sporozoites each). Freshly thawed lytic or control peptide solutions were added yielding 1, 10, and 100 ,uM concentrations (total fluid volumes were equivalent). The resultant sporozoite mixtures were incubated in a shaking water bath at 37°C for 60 min. At 30 and 60 min after addition of the peptides, 100-,u aliquots were removed for assessment of parasite viability. Additional sporozoite preparations (100-,lI aliquots in microcentrifuge tubes) were immersed in a 1000C water bath for 10 s and cooled to room temperature to act as heat-killed controls for validating the vital dye-staining technique. A fluorescein diacetate (catalog no. F7378; Sigma) and propidium iodide (catalog no. P4170; Sigma) vital dye-staining technique (10) was adapted for
Cryptosporidium parvum, an enteric protozoan parasite, colonizes the mucosal epithelium of the gastrointestinal tracts of a wide range of mammals causing a mild to severe, cholera-like diarrhea. The disease is especially pronounced in neonatal and immunocompromised hosts. More than 90 chemotherapeutic agents and therapeutic regimens have been tested in humans and animals infected with C. parvum without success. The lack of effective therapies for the control of cryptosporidiosis is of grave consequence for individuals who develop chronic, severe cryptosporidial diarrhea (6, 14). The discovery of lytic peptides (cecropins) in the giant silk moth (3) and the identification of lytic peptides (magainins) in the skin of frogs (16) has opened a new area of research into antimicrobial compounds. The amphipathic proteins that constitute this new class of agents are generally very small, less than 36 amino acids (molecular weight [MW],
Vol. 35, No. 2
p. 224-227
In Vitro Activities of Lytic Peptides against the Sporozoites of Cryptosporidium parvum MICHAEL J. ARROWOOD,l* JESSE M. JAYNES,2 AND MARK C. HEALEY' Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah 84322-5600,1 and Department of Biochemistry, Louisiana State University, Baton Rouge, Louisiana 708032 Received 12 July 1990/Accepted 14 November 1990
Cryptosporidium parvum is a protozoan parasite that causes mild to severe diarrheal disease in animals and humans. There are currently no effective chemotherapeutic agents available for the treatment of cryptosporidiosis. Recent studies have described small, naturally occurring antimicrobial lytic peptides with antiprotozoal activities. In the present study, the anticryptosporidial activities of three synthetic lytic peptides were determined in an in vitro sporozoite susceptibility assay. Sporozoite viability was assessed microscopically by the uptake of the vital dyes fluorescein diacetate and propidium iodide. Sporozoite viability was reduced by 93.5% following a 60-min exposure to 10 ,uM Hecate-1 at 37°C. Shiva-10 reduced sporozoite viability by approximately 74.0% after a 60-min exposure at 100 ,M and 37°C. The cecropin-b analog SB-37 reduced sporozoite viability by 6.0% following a 60-min exposure at 100 ,uM and 37°C. A control peptide showed no anticryptosporidial activity.
blindly evaluated samples. Peptide numbers and their respective MWs were as follows: 1, 2,537; 2, 2,537; 5, 4,103; and 6 (control peptide), 3,425. Each peptide was solubilized in phosphate-buffered saline (PBS) (81 mM Na2HPO4, 15 mM KH2PO4, 137 mM NaCl, 27 mM KCl [pH 7.2]), yielding 1 mM stock solutions. Peptide solutions were dispensed in 100-RlI aliquots into microcentrifuge tubes and stored at -200C. Cryptosporidial oocysts. The C. parvum (Iowa isolate) oocysts employed in this study were originally obtained from Harley Moon (U.S. Department of Agriculture, Ames, Iowa). Oocysts were generated in experimentally infected Holstein calves and purified from feces by using discontinuous sucrose gradients (2). Purified oocysts were stored in 2.5% potassium dichromate at 4°C. Cryptosporidial sporozoites. Purified oocysts were washed with PBS to remove potassium dichromate. Approximately 5 x 106 oocysts were incubated at 37°C in 1 ml of a PBS-based excystation solution in order to excyst (release) the sporozoites. The excystation solution contained 0.25% trypsin (catalog no. T-8253; Sigma Chemical Co., St. Louis, Mo.) and 0.75% sodium taurocholate (prepared with equal portions of catalog no. T-0750 from Sigma Chemical Co. and catalog no. 102930 from ICN Biomedicals Inc., Costa Mesa, Calif.). Sporozoites were washed free of the excystation solution by centrifugation and resuspended in 1 ml of PBS. Antisporozoite assay. Sporozoite suspensions were dispensed into microcentrifuge tubes in 200-pI aliquots (approximately 2 x 106 sporozoites each). Freshly thawed lytic or control peptide solutions were added yielding 1, 10, and 100 ,uM concentrations (total fluid volumes were equivalent). The resultant sporozoite mixtures were incubated in a shaking water bath at 37°C for 60 min. At 30 and 60 min after addition of the peptides, 100-,u aliquots were removed for assessment of parasite viability. Additional sporozoite preparations (100-,lI aliquots in microcentrifuge tubes) were immersed in a 1000C water bath for 10 s and cooled to room temperature to act as heat-killed controls for validating the vital dye-staining technique. A fluorescein diacetate (catalog no. F7378; Sigma) and propidium iodide (catalog no. P4170; Sigma) vital dye-staining technique (10) was adapted for
Cryptosporidium parvum, an enteric protozoan parasite, colonizes the mucosal epithelium of the gastrointestinal tracts of a wide range of mammals causing a mild to severe, cholera-like diarrhea. The disease is especially pronounced in neonatal and immunocompromised hosts. More than 90 chemotherapeutic agents and therapeutic regimens have been tested in humans and animals infected with C. parvum without success. The lack of effective therapies for the control of cryptosporidiosis is of grave consequence for individuals who develop chronic, severe cryptosporidial diarrhea (6, 14). The discovery of lytic peptides (cecropins) in the giant silk moth (3) and the identification of lytic peptides (magainins) in the skin of frogs (16) has opened a new area of research into antimicrobial compounds. The amphipathic proteins that constitute this new class of agents are generally very small, less than 36 amino acids (molecular weight [MW],
