Wongsakorn Kiattiburut1, Ruina Zhi1, Seung Gee Lee1, Alexander C Foo2, Duane R Hickling1,3, Jeffrey W Keillor2, Natalie K Goto2, Weihua Li4, Wayne Conlan5, Jonathan B Angel1,6,7, Guangshun Wang8, Nongnuj Tanphaichitr1,7,9. 1. Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2. Department of Chemistry and Biomolecular Sciences, Faculty of Science, University of Ottawa, Ottawa, Ontario, Canada. 3. Department of Surgery, Division of Urology, The Ottawa Hospital, Ottawa, Ontario, Canada. 4. Shanghai Institute of Planned Parenthood Research, and School of Public Health, Fudan University, Shanghai, Republic of China. 5. Human Health Therapeutics Department, National Research Council Canada, Ottawa, Ontario, Canada. 6. Department of Medicine, Division in Infectious Diseases, The Ottawa Hospital, Ottawa, Ontario, Canada. 7. Department of Biochemistry, Microbiology, Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada. 8. Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA. 9. Department of Obstetrics & Gynecology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
Abstract
STUDY QUESTION: Do the truncated LL-37 peptides, GI-20 and GF-17, have spermicidal activity and microbicidal effects on the sexually transmitted infection (STI) pathogen Neisseria gonorrhoeae with equivalent potency to LL-37? SUMMARY ANSWER: GI-20 and GF-17 exhibited spermicidal effects on both mouse and human sperm as well as microbicidal action on N. gonorrhoeae with the same efficacy as LL-37. WHAT IS KNOWN ALREADY: The antimicrobial peptide LL-37 exerts microbicidal activity against various STI pathogens as well as spermicidal effects on both mouse and human sperm. STUDY DESIGN, SIZE, DURATION: Spermicidal activities of GI-20 and GF-17 were evaluated in vitro in mouse and human sperm and in vivo in mice. Finally, in vitro antimicrobial effects of LL-37, GI-20 and GF-17 on an STI pathogen, N. gonorrhoeae were determined. All experiments were repeated three times or more. In particular, sperm samples from different males were used on each experimental day. PARTICIPANTS/MATERIALS, SETTING, METHODS: The plasma membrane integrity of peptide-treated sperm was assessed by cellular exclusion of Sytox Green, a membrane impermeable fluorescent DNA dye. Successful mouse in vitro fertilization was revealed by the presence of two pronuclei in oocytes following co-incubation with capacitated untreated/peptide-pretreated sperm. Sperm plus each peptide were transcervically injected into female mice and the success of in vivo fertilization was scored by the formation of 2-4 cell embryos 42 h afterward. Reproductive tract tissues of peptide pre-exposed females were then assessed histologically for any damage. Minimal inhibitory/bactericidal concentrations of LL-37, GI-20 and GF-17 on N. gonorrhoeae were determined by a standard method. MAIN RESULTS AND THE ROLE OF CHANCE: Like LL-37, treatment of sperm with GI-20 and GF-17 resulted in dose-dependent increases in sperm plasma membrane permeabilization, reaching the maximum at 18 and 3.6 μM for human and mouse sperm, respectively (P < 0.0001, as compared with untreated sperm). Mouse sperm treated with 3.6 μM GI-20 or GF-17 did not fertilize oocytes either in vitro or in vivo. Moreover, reproductive tract tissues of female mice pre-exposed to 3.6 μM GI-20 or GF-17 remained intact with no lesions, erosions or ulcerations. At 1.8-7.2 μM, LL-37, GI-20 and GF-17 exerted bactericidal effects on N. gonorrhoeae. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration of the inhibitory effects of GI-20 and GF-17 on human in vitro and in vivo fertilization cannot be performed due to ethical issues. WIDER IMPLICATIONS OF THE FINDINGS: Like LL-37, GI-20 and GF-17 acted as spermicides and microbicides against N. gonorrhoeae, without adverse effects on female reproductive tissues. With lower synthesis costs, GI-20 and GF-17 are attractive peptides for further development into vaginal spermicides/microbicides. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Canadian Institutes of Health Research (MOP119438 and CCI82413 to N.T.) and NIH (R01 AI105147 to G.W.). There are no competing interests to declare.
STUDY QUESTION: Do the truncated LL-37 peptides, GI-20 and GF-17, have spermicidal activity and microbicidal effects on the sexually transmitted infection (STI) pathogen Neisseria gonorrhoeae with equivalent potency to LL-37? SUMMARY ANSWER: GI-20 and GF-17 exhibited spermicidal effects on both mouse and human sperm as well as microbicidal action on N. gonorrhoeae with the same efficacy as LL-37. WHAT IS KNOWN ALREADY: The antimicrobial peptide LL-37 exerts microbicidal activity against various STI pathogens as well as spermicidal effects on both mouse and human sperm. STUDY DESIGN, SIZE, DURATION: Spermicidal activities of GI-20 and GF-17 were evaluated in vitro in mouse and human sperm and in vivo in mice. Finally, in vitro antimicrobial effects of LL-37, GI-20 and GF-17 on an STI pathogen, N. gonorrhoeae were determined. All experiments were repeated three times or more. In particular, sperm samples from different males were used on each experimental day. PARTICIPANTS/MATERIALS, SETTING, METHODS: The plasma membrane integrity of peptide-treated sperm was assessed by cellular exclusion of Sytox Green, a membrane impermeable fluorescent DNA dye. Successful mouse in vitro fertilization was revealed by the presence of two pronuclei in oocytes following co-incubation with capacitated untreated/peptide-pretreated sperm. Sperm plus each peptide were transcervically injected into female mice and the success of in vivo fertilization was scored by the formation of 2-4 cell embryos 42 h afterward. Reproductive tract tissues of peptide pre-exposed females were then assessed histologically for any damage. Minimal inhibitory/bactericidal concentrations of LL-37, GI-20 and GF-17 on N. gonorrhoeae were determined by a standard method. MAIN RESULTS AND THE ROLE OF CHANCE: Like LL-37, treatment of sperm with GI-20 and GF-17 resulted in dose-dependent increases in sperm plasma membrane permeabilization, reaching the maximum at 18 and 3.6 μM for human and mouse sperm, respectively (P < 0.0001, as compared with untreated sperm). Mouse sperm treated with 3.6 μM GI-20 or GF-17 did not fertilize oocytes either in vitro or in vivo. Moreover, reproductive tract tissues of female mice pre-exposed to 3.6 μM GI-20 or GF-17 remained intact with no lesions, erosions or ulcerations. At 1.8-7.2 μM, LL-37, GI-20 and GF-17 exerted bactericidal effects on N. gonorrhoeae. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration of the inhibitory effects of GI-20 and GF-17 on human in vitro and in vivo fertilization cannot be performed due to ethical issues. WIDER IMPLICATIONS OF THE FINDINGS: Like LL-37, GI-20 and GF-17 acted as spermicides and microbicides against N. gonorrhoeae, without adverse effects on female reproductive tissues. With lower synthesis costs, GI-20 and GF-17 are attractive peptides for further development into vaginal spermicides/microbicides. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Canadian Institutes of Health Research (MOP119438 and CCI82413 to N.T.) and NIH (R01 AI105147 to G.W.). There are no competing interests to declare.
Authors: Ole E Sørensen; Lone Gram; Anders H Johnsen; Emma Andersson; Susanne Bangsbøll; G Sandra Tjabringa; Pieter S Hiemstra; Johan Malm; Arne Egesten; Niels Borregaard Journal: J Biol Chem Date: 2003-05-20 Impact factor: 5.157
Authors: Imran Mohammed; Dalia G Said; Mario Nubile; Leonardo Mastropasqua; Harminder S Dua Journal: Front Microbiol Date: 2019-09-19 Impact factor: 5.640