Antimicrobial peptide LL-37 and its truncated forms, GI-20 and GF-17, exert spermicidal effects and microbicidal activity against Neisseria gonorrhoeae

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.

Figure 1

Effects of LL-37, GI-20 and GF-17 on surface membrane permeabilization on mouse (A) and human (B) sperm. P-CAP and F-CAP sperm untreated (control) or treated with the peptide (0.36–3.6 μM for mouse sperm and 3.6–18 μM for human sperm) were evaluated for surface membrane permeabilization by Sytox Green staining. Percentages of sperm with permeabilized membranes in total sperm population were expressed as means ± SDs of three experiments using different sperm samples. The symbols *, **, *** and **** denote P < 0.05, 0.01, 0.001 and 0.0001, respectively, for the significant differences among the three peptides. (C) Zero and minimal adverse effects of GF-17D3 on mouse and human sperm surface membranes, respectively. F-CAP sperm were treated with GF-17 or GF-17D3 at 3.6 μM for mouse sperm and at 18 μM for human sperm. Expression of data from three different sperm samples and statistical analyses for significant differences of data from GF-17- and GF-17D3-treated samples vs those of control-untreated samples followed the same approaches as in A and B.

Figure 2

(A) Decreased ability of GI-20- and GF-17-treated mouse sperm to fertilize mature oocytes in vitro. F-CAP mouse sperm, untreated or pretreated with various concentrations of GI-20 or GF-17, were co-incubated with mature oocytes. F-CAP sperm pretreated with 3.6 μM LL-37 and 3.6 μM scrambled LL-37 were also used for oocyte co-incubation. More than 80% of oocytes co-incubated with control sperm were fertilized and designated as 100% control. Fertilization data of peptide-treated sperm were expressed as means ± SDs of percent controls from three or more replicate experiments performed on different days. (B) Inefficiency of the mutant GF-17D3 peptide in inhibiting mouse in vitro fertilization. F-CAP mouse sperm untreated (control) or pretreated with 3.6 μM GF-17 or GF-17D3 were co-incubated with mature oocytes. Data from three replicate experiments performed on different days were expressed as means ± SDs of percent fertilized oocytes of the total oocytes in the gamete co-incubates. In both A and B, n = total number of inseminated oocytes from all experimental days. The symbols *, **, *** and **** indicate significant differences with P < 0.05, 0.01, 0.001 and 0.0001, respectively, as analyzed by one-way ANOVA.

Figure 3

Effects of GI-20 and GF-17 on surface membrane permeabilization on human swim-up sperm. Swim-up sperm treated with the peptide (3.6–18 μM) were assessed for percent of total sperm with Sytox Green staining. Untreated swim-up sperm served as negative controls. Data were expressed as means ± SDs of results from the three donors. Significant differences of the effects between the two peptides at each tested concentration were analyzed by two-way ANOVA followed by Tukey’s multiple comparison. The symbols * and ** denote P < 0.05 and 0.01, respectively.

Figure 4

Lack of histological damages to the reproductive tract tissues of the female mice inseminated with GI-20 or GF-17. The vagina (top panels), cervix (middle panels) and uterus (bottom panels) were collected from the female mice 24 h post-insemination for paraffin block preparation. Sections were stained with hematoxylin/eosin. Control tissues were those from female mice transcervically injected with sperm alone. Bar = 100 μm. Shown here are representative results from one of the three females, transcervically injected with sperm alone (panels a, d and g), sperm + GI-20 (panels b, e and h) or sperm + GF-17 (panels c, f and i).