The major bactericidal activity of human seminal plasma is zinc-dependent and derived from fragmentation of the semenogelins

Abstract

One of the major roles of seminal plasma is to provide antimicrobial protection for the spermatozoa in the female reproductive tract. We found that the bactericidal activity of seminal plasma was highest after resolution of the seminal clot and that this antibacterial activity subsequently became greatly diminished. The antibacterial activity was derived from peptides generated by fragmentation of the semenogelins while the semenogelin holoproteins displayed no antibacterial activity. After ejaculation the semenogelin-derived peptides were fragmented to smaller and smaller fragments over time and thereby lost antibacterial activity. This paralleled the loss of antibacterial activity of whole seminal plasma both in vitro and after sexual intercourse. Moreover, the antibacterial activity of the semenogelin-derived peptides generated in seminal plasma was strictly zinc-dependent both at neutral and low pH. These data provide novel roles for the resolution of seminal clots and for the high zinc concentration in human seminal plasma.

Figure 1

Seminal plasma has broad bactericidal activity. A. Seminal plasma (SP) and dialyzed seminal plasma (DSP) were run on an AU-PAGE gel and analyzed in an antibacterial gel overlay assay with E. coli. Clearing zones depict antibacterial activity. Salt causes retardation of the samples in AU gels. This can explain the difference between the SP and DSP sample. B. Seminal plasma (SP), dialyzed seminal plasma (DSP), cationic fraction (CF), anionic fraction from extraction of cationic peptides (AF) and protein free seminal fluid (Fluid) were tested in a CFU-assay in a concentration of 2% of that found in seminal plasma and displayed as bacterial survival compared to buffer control. Results are shown as average from three independent experiments. C. Dialyzed seminal plasma (DSP) and the cationic fraction (CF) from seminal plasma were tested in a CFU-assay in a concentration corresponding to 2% of the concentration in seminal plasma. Results are shown as average from three independent experiments. D. S. pyogenes M5 were incubated with or without cationic fraction (CF) from seminal plasma and visualized by transmission electron microscopy.

Figure 2

A synthetic SgII-derived peptide has antibacterial activity. Antibacterial activity of a synthetic peptide derived from SgII (H-KQEGRDHDKSKGHFHMIVIHHKGGQAHHG-OH), SgII peptide A, was tested in CFU-assay against S. pyogenes and E. coli and shown as bacterial survival compared to buffer control. Average values from three independent experiments are shown. Against S. pyogenes the peptide was only tested in neutral pH with no additional ions and against E. coli the peptides was tested in neutral pH, in pH 5.5 and with addition of ZnCl₂ in neutral pH (final concentration 12.5 mM).

Figure 3

Antibacterial activity of SgI and SgII holoproteins requires cleavage by PSA. A. Seminal plasma (SP), purified SgI, purified SgII, and PSA were run on an AU-PAGE gel and analyzed by coomassie staining or by antibacterial gel overlay assay with E. coli. B and C. SgI was cleaved by PSA for 4h (B) or for 24h (C) and run on an AU-PAGE gel and analyzed by coomassie staining or by antibacterial gel overlay assay with E. coli.

Figure 4

Antibacterial activity of seminal plasma from patients with dysfunctional seminal vesicles differs from that of normal donors and vasectomized patients. Normal seminal plasma (SP), seminal plasma from vasectomized patients (Vas) and seminal plasma from patients with dysfunctional seminal vesicles (SV def) were run on a AU-PAGE gel and either analyzed by a antibacterial gel overlay assay with E. coli and coomassie staining (A) or immunoblotting with anti-semenogelin antibodies, anti-hCAP-18-antibodies and anti-SLPI-antibodies was performed (C). After dialysis the antibacterial activity of normal seminal plasma, seminal plasma from vasectomized patients and seminal plasma from patients with dysfunctional seminal vesicles were tested in a CFU-assay against E. coli and S. pyogenes (B). The results are shown as bacterial survival compared to buffer control. Results are shown as average from three independent experiments.

Figure 5

2D gel analysis of normal seminal plasma (SP), seminal plasma from a vasectomized patient (Vas), and seminal plasma from a patient with dysfunctional seminal vesicles (SV def.). 2-dimensional gel electrophoresis was performed. Protein spots were visualized by SYPRO Ruby gel stain (Bio-Rad), excised, and stored at 4°C in 1% acetic acid until analyzed by mass spectrometry.

Figure 6

The antibacterial activity of seminal plasma decreases over time. A. The proteolytic activity in seminal plasma was stopped by urea at different time-points after ejaculation and the samples were run on an AU-PAGE gel and analyzed by antibacterial gel overlay assay with E. coli or subjected to immunoblotting with anti-semenogelin antibodies B. Seminal plasma was incubated at 37°C for 1h, 6h, 12h and 24h and at 4°C for 24h. The samples were then dialyzed and tested in a CFU-assay against E. coli and S. pyogenes. The results are shown as bacterial survival compared to buffer control. Results are shown as average from three independent experiments. C. Samples of seminal plasma (SP) and seminal plasma after incubation for 24 hours at 4 °C or 37 °C were analyzed by AU-PAGE followed by immunoblotting with anti-semenogelin and anti-hCAP-18 antibodies. D. Sample of post coital seminal plasma (PC SP) collected from the vagina 10 hours after sexual intercourse were analyzed by AU-PAGE and subjected to either immunoblotting with anti-semenogelin antibodies and anti hCAP-18 antibodies or antibacterial overlay assay.

Figure 7

The antibacterial activity of seminal plasma is zinc-dependent. EDTA was added to seminal plasma to remove divalent cations. The EDTA was removed by dialysis. Zinc, calcium or magnesium was added to the cation depleted seminal plasma and the unbound ions were removed by dialysis. Dialyzed normal seminal plasma (DSP), EDTA-treated (divalent cation-depleted) seminal plasma (EDTA) with and without addition of divalent cations were tested in a CFU-assay against E. coli and S. pyogenes. The results are shown as bacterial survival compared to buffer control. Results are shown as average from three independent experiments.

Figure 8

Antibacterial activity of semenogelin-depleted seminal plasma. Semenogelin was depleted from seminal plasma by applying divalent cation-depleted seminal plasma to nickel agarose and the semenogelin was eluted with imidazole. The imidazole was removed by dialysis and zinc was added both to the semenogelin depleted seminal plasma and to the semenogelin containing eluate. Normal seminal plasma (SP), flow through from the nickel agarose representing the semenogelin-depleted seminal plasma and the eluate representing the semenogelin-free seminal plasma were run on a AU-PAGE gel and subjected to immunoblotting with anti-semenogelin antibodies and anti-SLPI antibodies (A) or analyzed in a gel overlay assay against E. coli (B). 2D gel analysis of the cationic fraction of the semenogelin eluate (C). Proteins were identified by spot matching. I denotes SgI and II denotes SgII.