Cationic peptide of the male reproductive tract, HE2{alpha}, displays antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis

M. Liao1,2,{dagger}, P. S. Ruddock3, A. S. Rizvi1, S. H. Hall4, F. S. French4 and J. R. Dillon1–3,*

1 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, ON, Canada, K1H 8M5; 2 Centre for Research in Biopharmaceuticals and Biotechnology, University of Ottawa, ON, Canada, K1H 8M5; 3 Department of Biology, University of Ottawa, ON, Canada, K1H 8M5; 4 Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill, Campus Box 7500, Chapel Hill, NC 27599, USA


* Corresponding author. Present address. College of Arts and Science, University of Saskatchewan, Room 226, Arts Building, 9 Campus Drive, Saskatoon, SK, Canada, S7N 5A5. Tel: +1-306-9664232; Fax: +1-306-9668839; E-mail: j.dillon{at}usask.ca

Received 29 March 2005; returned 4 May 2005; revised 16 August 2005; accepted 8 September 2005


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Objectives: To analyse the in vitro antimicrobial effects of synthetic HE2{alpha} peptide against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis.

Methods: The HE2{alpha} peptide was synthesized based on the C-terminal sequence of the HE2{alpha} protein. The bacterial strains tested included two antibiotic-susceptible strains of N. gonorrhoeae and four antibiotic-resistant clinical isolates, as well as S. aureus ATCC 29213 and E. faecalis ATCC 29212. Susceptibility determinations were carried out either in 0.7% casamino acids for N. gonorrhoeae isolates or in 10 mM phosphate buffer for S. aureus and E. faecalis strains. Antibacterial effects were measured in a dose- and time-dependent manner. After exposure to the peptide in solution, the number of viable cells was determined by counting colony forming units (cfu).

Results: The HE2{alpha} peptide exhibited time- and dose-dependent antibacterial effects on all N. gonorrhoeae isolates tested. S. aureus and E. faecalis strains were also susceptible to the peptide. All strains tested were susceptible to the peptide at high concentrations (50 or 100 mg/L) and some strains were susceptible to a peptide concentration of 25 mg/L.

Conclusions: The peptide HE2{alpha}, which is derived from the male urogenital tract, exhibits antibacterial activity against both Gram-positive and Gram-negative pathogens in vitro. The peptide is active against both antibiotic-susceptible and -resistant N. gonorrhoeae isolates. Further investigation of the antimicrobial properties of the peptide is warranted.

Keywords: susceptibility tests , antimicrobial peptides , sexually transmitted diseases , pathogenic bacteria


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Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis are frequently refractory to many new and commonly used antimicrobial agents.1,2 Thus, the identification of novel agents effective in inhibiting such microorganisms has gained renewed urgency. The investigation of peptides as potential antimicrobial agents has attracted attention partly because peptides such as defensins and cathelicidins,3 or bactenecins from bovine neutrophils4 are important components of the innate host defence against infection. The human epididymis 2 (HE2) family of proteins, HE2{alpha} and HE2ß1, are cationic peptides.57 The HE2 C-termini are derived from their respective proproteins by proteolytic cleavage in vivo.5 It has been shown that both the chemosynthetic and recombinantly expressed C-terminal peptides possess bactericidal activity against Escherichia coli.5,7 However, the antibacterial activity of HE2 against other bacterial pathogens has not been determined. We chose N. gonorrhoeae, E. faecalis and S. aureus as model organisms to test their susceptibility to the HE2{alpha} peptide, primarily because of the propensity of these bacteria to develop resistance to most antibiotics used clinically.1,2 Since HE2 proteins are expressed predominantly in the human reproductive tract, the primary infection site of N. gonorrhoeae, it was of particular interest to analyse the antibacterial activity of HE2{alpha} against gonococcal strains with varying antimicrobial resistance profiles.


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Bacterial strains

Bacterial strains used in this study are listed in Table 1. Six N. gonorrhoeae strains were tested, including two antibiotic-susceptible WHO control strains and four clinical isolates. The gonococcal clinical isolates comprised various antibiotic resistance profiles including plasmid-mediated and chromosomally-mediated resistance. N. gonorrhoeae was subcultured on GC Medium Base agar (Difco) containing 1% Kellogg's supplement (GCMBK).1 Ampicillin (5 mg/L) was added to GCMBK for the subculture of penicillinase-producing N. gonorrhoeae (PPNG) isolates. Cells were incubated at 35°C for 24–30 h, in 5–7% CO2, in a humid environment. S. aureus ATCC 29213 and E. faecalis ATCC 29212 (Table 1) were subcultured on Mueller–Hinton (M-H) agar (Difco) at 37°C for 20–24 h.


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Table 1. Bacterial strains used in this study

 
Peptides

The HE2{alpha} peptide (VHISHREARGPSFRICVGFLGPRWARGCSTGN) was synthesized and purified as described previously.5 A negative control peptide (CP), based on the HE2ß1 sequence, was synthesized with cysteine residues blocked [RNTIC(Acm)RMQQGIC(Acm)RLFFC(Acm)HSGEKKRDIC(Acm)SDPWNRC(Acm)C(Acm)VSNTD], which abolished its antibacterial activity against E. coli.5 We determined that CP was not active against N. gonorrhoeae, E. faecalis or S. aureus (data not shown). The peptides were dissolved in 10 mM phosphate buffer and aliquots were stored at –70°C.

Antibacterial assays

Assays for antibacterial activity were adapted from those described previously.5 The HE2{alpha} peptide lost antimicrobial activity in PBS (100 mM NaCl), and was not active in nutrient broth (data not shown). Since N. gonorrhoeae cells lyse in 10 mM phosphate buffer (data not shown), we determined that a 0.7% casamino acids (CAA; Difco) solution permitted gonococcal cell survival and did not decrease the antibacterial activity of the peptide (data not shown). Thus, a CAA solution (0.7%) was used for N. gonorrhoeae assays, and 10 mM phosphate buffer was used for S. aureus and E. faecalis assays.

Bacterial inocula were standardized from overnight cultures in either 0.7% CAA or 10 mM phosphate buffer, using a 0.5 McFarland turbidity standard (Remel, ~1 x 108 cfu/mL). The cell concentration was adjusted to 2 x 106 cfu/mL. The peptides were serially diluted on ice to concentrations of 200, 100, 50 and 25 mg/L. Equal volumes of inoculum and peptide solution were mixed in Eppendorf tubes such that the final concentration of bacteria was ~1 x 106 cfu/mL and the final concentration of peptide was 100, 50, 25 and 12.5 mg/L. Mixtures were incubated at 37°C (for E. faecalis or S. aureus) or 35°C (for N. gonorrhoeae) for 5, 10, 15, 30 and 60 min. Two control experiments were performed; cells were incubated with 0.7% CAA or 10 mM phosphate buffer in buffer control assays, or incubated with the CP control peptide solution (100 mg/L) for 30 or 60 min in negative peptide control assays. Following incubation, the reaction mixture was serially diluted with nutrient broth (GCMBK for N. gonorrhoeae, and M-H for E. faecalis or S. aureus), and 10 µL of each bacterial suspension was spotted onto appropriate agar plates in duplicate. Inoculated plates were incubated for 24–30 h, and the resulting colonies were counted as cfu/mL. Each experiment was performed twice to obtain the average cfu/mL and standard deviation (SD), and a Student's t-test was performed.


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Antigonococcal effects of the HE2{alpha} peptide

HE2{alpha} exhibited antigonococcal activity in a dose- and time-dependent manner (Figure 1a–f). N. gonorrhoeae WHO-V (Figure 1a) exhibited a decrease in viability at peptide concentrations of 100 and 50 mg/L (P < 0.05), but no appreciable reduction in viability at lower concentrations (25 and 12.5 mg/L) was observed (P > 0.05). N. gonorrhoeae WHO-III (Figure 1b) exhibited similar survival as that observed with N. gonorrhoeae WHO-V. No viable cells were observed after exposure to HE2{alpha} at high concentrations (100 or 50 mg/L) for 1 h. At a concentration of 25 mg/L, a 2 log unit reduction in viable counts was observed after 1 h of incubation. No significant antibacterial effects were observed with HE2{alpha} at a concentration of 12.5 mg/L.



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Figure 1. Antibacterial activity of HE2{alpha} against N. gonorrhoeae isolates, as well as S. aureus ATCC 29213 and E. faecalis ATCC 29212. N. gonorrhoeae WHO-V (a), WHO-III (b), PR-4 (c), PR-7 (d), PR-9 (e) and PR-12 (f); S. aureus ATCC 29213 (g); E. faecalis ATCC 29212 (h). Peptide concentrations: 100 mg/L (filled squares), 50 mg/L (open squares), 25 mg/L (filled triangles), 12.5 mg/L (open triangles), and negative control peptide 100 mg/L (open circles). Data for buffer controls (0.7% CAA or 10 mM phosphate buffer) overlapped with data for the control peptide and were not plotted. Standard deviations are presented. Asterisks indicate that the cfu value was significantly lower than that for the control peptide (P < 0.05).

 
Two PPNG isolates (PR-4 and PR-7) were also susceptible to HE2{alpha} (Figure 1c and d, respectively). For N. gonorrhoeae PR-7, no viable cells remained after exposure to high peptide concentrations (50 and 100 mg/L) for 1 h (Figure 1c). N. gonorrhoeae PR-4 was less susceptible to HE2{alpha}, with an ~2 log unit reduction after incubation with a concentration of 100 mg/L for 1 h (Figure 1d). At lower concentrations (25 and 12.5 mg/L), no significant activity against either PPNG isolate was observed (Figure 1c and d).

N. gonorrhoeae PR-9 (PP/TRNG) was susceptible to high concentrations of peptide (50 or 100 mg/L), and complete killing was observed after exposure to HE2{alpha} at a concentration of 100 mg/L for 1 h (Figure 1e). At a concentration of 25 mg/L, moderate antibacterial activity was observed (P < 0.05), while no significant decrease in bacterial survival for N. gonorrhoeae PR-9 was noted at a concentration of 12.5 mg/L.

N. gonorrhoeae PR-12, a CMRNG isolate, was also susceptible to HE2{alpha} (Figure 1f). A 3 to 4.8 log unit reduction was observed after exposure to concentrations of 100 or 50 mg/L for 1 h. At lower concentrations (25 or 12.5 mg/L), HE2{alpha} displayed significant antibacterial activity (P < 0.05).

Antibacterial activity of HE2{alpha} peptide against S. aureus

Significant effects of HE2{alpha} on the viability of S. aureus ATCC 29213 were observed after exposure to peptide concentrations at 50 and 100 mg/L for 15 min, and the log unit reduction in viability was 3.39 after exposure to 100 mg/L for 30 min. At lower peptide concentrations (25 and 12.5 mg/L), no significant antibacterial effects were observed, even after exposure for 30 min (Figure 1g).

Antibacterial activity of the HE2{alpha} peptide against E. faecalis

HE2{alpha} exhibited rapid antibacterial activity against E. faecalis ATCC 29212 (Figure 1h), resulting in significant reduction in cfu at all concentrations tested after 30 min (P < 0.05). Significant bactericidal effects were observed after 5 min of incubation.


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Antimicrobial cationic peptides are ubiquitous in nature and are thought to be components of the first line of defence against infectious agents.3 The HE2 peptides, including HE2{alpha} and HE2ß, exhibit potent time- and dose-dependent antimicrobial activity against E. coli.5,7 In this study, we demonstrated that HE2{alpha} which is from the male reproductive tract, possesses bactericidal activity in a time- and dose-dependent manner against N. gonorrhoeae, S. aureus and E. faecalis. It is noteworthy that HE2{alpha} is not only active against antibiotic-susceptible N. gonorrhoeae isolates, but also exhibits significant antibacterial effects against gonococcal isolates with various antibiotic resistance profiles.

A few peptides have been previously shown to be active against N. gonorrhoeae. A cationic peptide from porcine leucocytes, protegrin, inactivated N. gonorrhoeae, exhibiting killing effects against both antibiotic-susceptible and -resistant strains.8 Two short synthetic peptides, derived from lysosomal cathepsin G of human neutrophils, were also active against N. gonorrhoeae and S. aureus.9 This is the first report on the antigonococcal activity of a reproductive tract-derived peptide. We observed some strain selectivity of HE2{alpha} against N. gonorrhoeae isolates. It would be interesting to measure the MIC values of the peptide against gonococci8 and to determine whether synergic effects are observed between the peptide and different antibiotics.

The study of peptides against S. aureus and E. faecalis has focused on mammalian and human antibacterial peptides, such as defensins and cathelicidins,10 as well as bactenecins from bovine neutrophils.4 Strain selectivity of human ß-defensins against several strains of S. aureus was observed.10 Our study showed that HE2{alpha} was active against the single reference strains of S. aureus and E. faecalis. This peptide should be further examined for its activity against these genera by testing larger numbers of isolates including antibiotic-resistant strains.

It is not known whether the HE2{alpha} peptide is toxic to human cells, although it did not cause haemolysis of rat red blood cells in vitro.5 The chemotactic activity of HE2{alpha} should be investigated, since it may cause inflammation and irritation of mucosal surfaces. Given that the peptide is expressed in the male reproductive tract, the activity of HE2-class peptides against other STD pathogens is of great interest.


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None to declare.


    Footnotes
 
{dagger} Present address. Room A233, 120 Veterinary Road, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E3 Back


    Acknowledgements
 
We thank Suresh Yenugu for his advice on designing peptide antibacterial assays. This study was supported by a grant from the Hospital for Sick Children Foundation, Canada (Grant# XG02-075, J. R. D.), as well as grants HD04466 (F. S. F., S. H. H.) and the CONRAD CICCR Program (F. S. F., S. H. H.).


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1. Dillon JR, Pagotto F. Importance of drug resistance in gonococci: from mechanisms to monitoring. Curr Opin Infect Dis 1999; 12: 35–40.[ISI]

2. Lowry FD. Medical progress: Staphylococcus aureus infections. New Engl J Med 1998; 339: 520–30.[Free Full Text]

3. Hancock RE. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001; 1: 156–63.[CrossRef][Medline]

4. Wu M, Hancock RE. Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J Biol Chem 1999; 274: 29–35.[Abstract/Free Full Text]

5. Yenugu S, Hamil KJ, Birse CE et al. Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli. Biochem J 2003; 372: 473–83.[CrossRef][ISI][Medline]

6. Osterhoff C, Kirchhoff C, Krull N et al. Molecular cloning and characterization of novel human sperm antigen (HE2) specifically expressed in the proximal epididymis. Biol Reprod 1994; 50: 516–25.[Abstract]

7. von Horsten HH, Derr P, Kirchhoff C. Novel antimicrobial peptide of human epididymal duct origin. Biol Reprod 2002; 67: 804–13.[Abstract/Free Full Text]

8. Qu XD, Harwig SS, Shafer WM et al. Protegrin structure and activity against Neisseria gonorrhoeae. Infect Immun 1997; 65: 636–9.[Abstract]

9. Bangalore N, Travis J, Onunka VC et al. Identification of the primary antimicrobial domains in human neutrophil cathepsin G. J Biol Chem 1990; 265: 13584–8.[Abstract/Free Full Text]

10. Ericksen B, Wu Z, Lu W et al. Antibacterial activity and specificity of the six human ß-defensins. Antimicrob Agents Chemother 2005; 49: 269–75.[Abstract/Free Full Text]

11. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA, 2003.





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