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Endocrinology, doi:10.1210/en.2003-1698
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Endocrinology Vol. 145, No. 7 3165-3173
Copyright © 2004 by The Endocrine Society

The Androgen-Regulated Epididymal Sperm-Binding Protein, Human ß-Defensin 118 (DEFB118) (Formerly ESC42), Is an Antimicrobial ß-Defensin

Suresh Yenugu, Katherine G. Hamil, Yashwanth Radhakrishnan, Frank S. French and Susan H. Hall

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Dr. Susan H. Hall, Laboratories for Reproductive Biology, CB 7500, Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599-7500. E-mail: shh{at}med.unc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Spermatozoa bind a variety of proteins as they pass through the proximal regions of the epididymis, where they acquire forward motility and fertilizing ability. Recent evidence indicates that certain epididymis-specific secretory proteins that bind sperm have antibacterial activity and may function as part of the innate immune system. We reported earlier that ESC42, now designated human ß-defensin 118 (DEFB118), is a sperm-binding protein. In this study, we demonstrate that DEFB118 has potent antibacterial activity that is dose, time, and structure dependent. Incubation of Escherichia coli for 60 min with 10 µg/ml DEFB118 reduced bacterial survival to 20% of the control, and 25 µg/ml reduced survival to 5% of the control. DEFB118 concentrations of 50 and 100 µg/ml further reduced survival to less than 2 and 1%, respectively. A biphasic effect of salt concentration on the antibacterial activity of DEFB118 was observed. Reduction of disulfide bonds and alkylation of cysteines resulted in the complete loss of antibacterial activity. DEFB118 caused rapid permeabilization of both outer and inner membranes of E. coli and striking morphological alterations in the bacterial surfaces visible by scanning electron microscopy consistent with a membrane-disruptive mechanism of bacterial killing. In contrast, eukaryotic cell membranes were not permeabilized by DEFB118, as indicated by the rat erythrocyte hemolytic assay. Studies on DEFB118 inhibition of macromolecular synthesis and membrane permeability in E. coli were consistent with a primary effect at the cell membrane level. DEFB118 may contribute to epididymal innate immunity and protect the sperm against attack by microorganisms in the male and female reproductive tracts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPERM MATURATION IS a complex, sequentially ordered process that occurs as the spermatozoa pass through the epididymis, where they acquire forward motility and fertilizing ability. Numerous secreted epididymal proteins bind to the sperm during passage, but their functions are not well understood (1, 2, 3). In addition to earlier evidence that sperm binding proteins are involved primarily in preparing sperm for fertilization, recent evidence suggests a function in male reproductive tract immunity. We showed that the human sperm binding proteins and peptides of the defensin-like HE2 family are antibacterial by a membrane permeabilization mechanism and are thereby of potential importance to male reproductive tract immunity (4). Similarly, antibacterial activities of the human sperm binding proteins such as cystatin (CST) 11 (5, 6), lactoferrin (7), and human cathelicidin antimicrobial peptide (hCAP18) (8) are established. Additional antimicrobial proteins reported in the lumen of the male reproductive tract include bovine seminal plasmin (9), cathelicidins (10), Bin1b (11), and members of the ß-defensin family (12, 13, 14, 15).

A wide variety of antimicrobial proteins belonging to different classes have been identified in the animal kingdom, from insects to humans (16). In humans, the best characterized and most abundant antibiotic proteins are the defensins. Defensins are generally broad-spectrum cationic antimicrobial proteins. Based on disulfide bonding, genomic organization, and tissue distribution, two classes of defensins are identified, {alpha} and ß. The {alpha}-defensins are expressed in neutrophils and paneth cells, whereas the ß-defensins are expressed in the epithelia. Among the more than 30 ß-defensins known in humans, several exhibit broad-spectrum antimicrobial activity that is salt sensitive and structure dependent. The mechanism of antibacterial action is thought to involve damage of membranes by thinning or destabilizing the bilayer, resulting in formation of pores and dissipation of ion gradients across the disrupted membrane (17, 18, 19). In addition, some antibacterial peptides interact with critical factors inside the cells, thereby disrupting normal cellular function (20, 21).

The human ß-defensin 118 (DEFB118) gene [described previously as ESC42 (22)] is located in the ß-defensin gene cluster on chromosome 20q11, one of five clusters discovered using a computational genomic search strategy (23). Nomenclature changes subsequent to Ref. 23 were published in an addendum containing a final list of official names. The addendum is available online (http://www.pnas.org/cgi/content/full/222517899/DC1). DEFB118 expression is restricted to the epididymis and is regulated by androgen. The protein is present in epithelial cells of efferent ducts and most abundant in the caput epithelium, where it is present in the lumen and located on sperm (22). Functional analysis of the DEFB118 protein has not been reported. In this paper, we establish that DEFB118 exhibits antibacterial activity that is structure dependent and salt tolerant. It kills bacteria by a mechanism involving permeabilization of membranes resulting in release of cell contents and inhibition of macromolecular synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant protein preparation
Recombinant DEFB118 protein was prepared as described (4). In brief, E. coli strain M15 (pREP4) was transformed with pQE30 vector (Qiagen, Valencia, CA) containing the DEFB118 cDNA according to the Qiagen protocol. Fusion protein expression was induced with 1 mM isopropyl-1-thio-ß-D-galactoside for 2 h at 37 C, and the recombinant protein was purified with nickel-nitrilotriacetic acid-agarose (Qiagen) and dialyzed against 10 mM sodium phosphate buffer (pH 7.4). The His-tag (MRGSHHHHHHGS) was removed using the Tagzyme system (Qiagen) according to the manufacturer’s protocol.

DEFB118 was reduced and alkylated according to the protocol described at Massachusetts Institute of Technology Center for Cancer Research, Howard Hughes Medical Institute Biopolymers Laboratory web site (http://web.mit.edu/biopolymers/www/protein_sequencing.html), as described previously (4). A control containing sodium phosphate buffer without protein was treated similarly to determine whether residual ß-mercaptoethanol and iodoacetamide affected bacterial growth in the assays.

Antibacterial activity
The antibacterial activity of DEFB118 was tested against E. coli XL-1 blue (Stratagene, La Jolla, CA) by using the colony-forming unit (CFU) assay as described earlier (4). Briefly, mid-log phase E. coli diluted to approximately 2 x 106 CFU/ml in 10 mM sodium phosphate (pH 7.4) were incubated with varying concentrations of DEFB118. The assay mixtures were serially diluted, spread on Luria Bertani agar plates, and incubated overnight at 37 C to allow full colony development. The resulting colonies were counted, and antibacterial activity was expressed as percentage of survival using the following formula: percentage of survival = (number of colonies surviving after treatment with the antibacterial protein/number of colonies surviving in the absence of antibacterial protein) x 100. As negative controls, the epididymis-specific lipocalin (LCN6) (24) and BSA were included in the assays. In some antibacterial assays, E. coli and DEFB118 were incubated in 10 mM sodium phosphate (pH 7.4) containing 25–300 mM NaCl to test the effect of salt on the antibacterial activity.

Outer and inner membrane permeabilization assays
The outer membrane permeabilization ability of DEFB118 was determined by the N-phenyl-1-napthylamine (NPN, Molecular Probes, Eugene, OR) assay (25). NPN fluoresces weakly in an aqueous environment but strongly in the hydrophobic interior of cell membranes. Upon destabilization of the outer bacterial membrane by antimicrobial agents, the dye enters the damaged membrane, where it emits stronger fluorescence. Mid-log phase E. coli washed and suspended in 5 mM HEPES buffer (pH 7.4) containing 5 mM glucose were used for the permeabilization studies. For the outer membrane assay, varying concentrations of DEFB118 were added to E. coli suspension containing 10 µM NPN, and the increase in fluorescence was measured. Inner membrane permeabilization was measured as described earlier (26) using the cyanine dye 3,5-dipropylthiadicarbocyanine iodide (diSC3–5) (Molecular Probes), which distributes between the cells and medium, depending on the membrane potential. Once the dye enters the cells, it forms self-quenching aggregates, resulting in a decrease in fluorescence. Upon permeabilization of the inner membrane by an antimicrobial agent, the dye is released back into the medium, and an increase in fluorescence is measured. E. coli were incubated with 0.4 µM diSC3–5 until the uptake was maximal, as indicated by a stable reduction in fluorescence. Varying concentrations of DEFB118 were added to this suspension, and the increase in fluorescence was monitored.

Hemolytic activity
Hemolytic activity of DEFB118 was determined as described earlier (4). In brief, erythrocytes from heparinized rat blood were washed thrice with 0.9% saline and resuspended to a concentration of 5% in saline. Erythrocytes were treated with different concentrations of DEFB118 (10–100 µg/ml) in a 96-well plate and incubated at 37 C for 1 h. The plate was centrifuged at 1000 x g for 10 min, and supernatants were transferred to a fresh plate. Absorbance at 560 nm of saline and 1% Triton X-100-treated erythrocytes served as 0 and 100% hemolysis controls, respectively.

Scanning electron microscopy
Approximately 108 CFU/ml of E. coli resuspended in 10 mM sodium phosphate buffer (pH 7.4) were treated with 50 µg/ml DEFB118 and incubated for 30, 60, and 120 min at 37 C. After incubation, cells were washed and resuspended in 10 mM sodium phosphate buffer (pH 7.4) and were fixed with an equal volume of 4% glutaraldehyde. The fixed samples were stored overnight to several days at 4 C in the fixative solution. Using a microanalysis vacuum filter holder (Fisher Scientific, Suwanee, GA) and a 0.1-µm polycarbonate membrane filter (Poretics Corporation, Livermore, CA), the suspended fixed cells were vacuum-filtered onto the membrane substrate, rinsed with 0.15 M sodium phosphate buffer, and dehydrated through a graded series of ethanol (30, 50, 75, and 100%). During the entire filtration, rinsing, and dehydration process, the cells were kept covered with fluid to prevent air-drying. The filters were transferred in 100% ethanol to a critical point dryer (Balzers CPD-020, Bal-Tec AG, Vaduz, Liechtenstein) and dried using carbon dioxide as the transition solvent. The filters were mounted on aluminum specimen supports with carbon adhesive tabs and coated with a 15-nm thickness of gold-palladium metal (60:40 alloy) using a Hummer X sputter coater (Anatech, Ltd., Alexandria, VA). Samples were examined on a Cambridge Stereoscan 200 scanning electron microscope (LEO Electron Microscopy, Inc., Thornwood, NY) using an accelerating voltage of 20 kV.

Macromolecular synthesis
The effect of DEFB118 on the incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine (PerkinElmer, Boston, MA) into bacterial DNA, RNA, and proteins, respectively, was studied using E. coli. In brief, mid-log phase bacteria were resuspended in 10 mM sodium phosphate buffer (pH 7.4) at a density of 1 x 106 CFU/ml. After a preincubation at 37 C for 10 min, bacteria were incubated with DEFB118 and 2.5 µl/ml of either [methyl-3H]thymidine (20 Ci/mmol), [5-3H]uridine (25.5 Ci/mmol), or L-[4,5-3H(N)]leucine (59.5 Ci/mmol) for different time points. As positive controls, the DNA, RNA, and protein synthesis inhibitors, namely ciprofloxacin (1 µg/ml), rifampicin (2 µg/ml), and tetracycline (2 µg/ml) (Sigma, St. Louis, MO), were included in the assays. After the incubation, bacterial suspensions were added to ice-cold 10% trichloroacetic acid, mixed well, and allowed to stand on ice for 40 min. Samples were then collected over vacuum on Fisherbrand 2.4 cm GF/C glass microfiber filters (Fisher Scientific, Pittsburgh, PA) and washed thoroughly with 5% trichloroacetic acid and 70% ethanol. The filters were then dried and placed in 5 ml of EcoScint scintillation cocktail (National Diagnostics, Atlanta, GA), and counts were obtained in a LKB 1214 Rackbeta liquid scintillation counter (LKB Wallace, Turku, Finland) for 1 min for each filter.

Statistical analyses using Student’s t test were performed using Sigma Plot software (SPSS Inc., Chicago, IL). Values shown are mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antibacterial activity
A feature of the DEFB118 protein, unusual among the typically cationic ß-defensins, is the acidic C-terminal domain extending 68 amino acids beyond the six-cysteine array (Fig. 1Go). The calculated theoretical isoelectric point (pI) of this domain is 4.7 (27). The typical cationic property of the six-cysteine array is conserved (calculated pI = 9.1), and, as a whole, the protein is slightly acidic (calculated pI = 6.5). Because the mechanism of action of the ß-defensins is thought to depend on interaction of basic amino acids with negatively charged bacterial surfaces, the acidic C-terminal half of DEFB118 would have the potential to interfere with the antibacterial activity of this defensin. To determine whether DEFB118 can kill bacteria, E. coli were incubated with different concentrations of the recombinant protein. When incubated with 10 µg/ml DEFB118 for 15 min or longer, 20% of bacteria survived (Fig. 2Go), whereas survival was reduced to less than 10% after treatment with 25 µg/ml DEFB118 and further reduced to less than 2 and 1% in the presence of 50 and 100 µg/ml, respectively. Increasing treatment time from 15 to 120 min did not further decrease bacterial survival. The negative control, recombinant His-tagged LCN6, did not exhibit any antibacterial activity when incubated for 2 h, even at the highest concentration tested, 100 µg/ml (Fig. 2Go, inset). Similarly, BSA showed no detectable antibacterial activity (data not shown). Because the His-tag might influence the structure and function of the recombinant protein, the His-tag was removed, and the antibacterial potency of the protein was tested. Treatment of E. coli for 120 min with 100 µg/ml His-tag-removed DEFB118 reduced survival to 5%, showing that the His-tag-removed protein maintained most of its antimicrobial activity (Fig. 2Go, inset). Previous reports indicated that the structural integrity conferred by sulfhydryl linkages is essential for the antimicrobial activity of antibiotic proteins and peptides (28). To determine whether disulfide bonds in the six-cysteine motif of DEFB118 are essential for its activity, the cysteines were reduced and alkylated. Denatured DEFB118 failed to kill bacteria at a concentration of 100 µg/ml, even when incubated for 2 h, suggesting the importance of the disulfide bonds to antibacterial activity (Fig. 3AGo).



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  		FIG. 1. Amino acid sequence alignment of ß-defensins 1 and 2 and DEFB118. Amino acid sequences of ß-defensins 1 and 2 and DEFB118, signal peptides removed, are shown with their six-cysteine arrays aligned. The cysteines are underlined.

 


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  		FIG. 2. Antibacterial activity of DEFB118. E. coli were incubated for 15–120 min with 10 µg/ml ({blacksquare}); 25 µg/ml ({blacktriangleup}); 50 µg/ml ({blacktriangledown}); and 100 µg/ml ({diamondsuit}) DEFB118. Inset shows the antibacterial activity of LCN6 ({blacktriangledown}) and DEFB118 with ({diamondsuit}) and without ({diamond}) His-tag for 120 min. Values shown are mean ± SD.

 


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  		FIG. 3. Structural dependence and salt sensitivity of DEFB118. A, Antibacterial activity of disulfide bond reduced and alkylated DEFB118. E. coli were incubated with 100 µg/ml of reduced and alkylated DEFB118 for 2 h. NaPO4 indicates bacteria incubated with only 10 mM sodium phosphate buffer (pH 7.4); control indicates sodium phosphate buffer without DEFB118 but containing the reagents used to reduce and alkylate DEFB118. Values shown are mean ± SD. B, Effect of NaCl on the antibacterial activity of DEFB118. E. coli were incubated for 2 h with 100 µg/ml DEFB118 with () and without His-tag () in sodium phosphate buffer containing 25–300 mM NaCl. Values shown are mean ± SD. *, P ≤ 0.01 compared with 0 mM NaCl + His-tagged DEFB118; +, P ≤ 0.01 compared with 0 mM NaCl + His-tag-removed DEFB118.

 
Antimicrobial proteins and peptides, including ß-defensins, have been shown to lose their activity in high salt concentrations (29, 30). To determine whether DEFB118 activity is similarly affected, bacteria were incubated with 100 µg/ml DEFB118 for 2 h in increasing concentrations of NaCl (Fig. 3BGo). In the absence of NaCl, DEFB118 permitted less than 1% bacterial survival. In the presence or absence of the His-tag, DEFB118 displayed maximal antibacterial potency in both 0- and 300-mM NaCl treatments. Over this range of concentrations, minimal antibacterial potency of DEFB118 occurred in 25 mM NaCl. This biphasic response to NaCl suggests a dual mechanism of bacterial killing, possibly involving different domains of DEFB118 or a conformational change in increasing NaCl.

Membrane permeabilization
E. coli outer membrane permeabilization by DEFB118 was investigated using NPN dye. DEFB118 caused a rapid, dose-dependent increase in fluorescence, indicating that outer membrane permeabilization was maximal within 1 min after the addition of DEFB118 (Fig. 4AGo). The inner membrane permeabilization capacity of DEFB118 was studied using the cyanine dye diSC3–5, which distributes between the cells and medium, depending on the membrane potential. Upon addition of DEFB118, there was an increase in fluorescence (within 2 min), indicating release of the dye due to permeabilization of the inner membrane (Fig. 4BGo). By 5 min, the release of the dye was maximal. The importance of structural integrity conferred by disulfide bonds to membrane permeabilizing activity was investigated by performing the assays with disulfide bond reduced and alkylated DEFB118. Disulfide bond reduction and alkylation completely abolished the membrane permeabilizing activity of DEFB118 (Fig. 4Go, A and B). The outer and inner membrane permeabilization ability of His-tag-removed DEFB118 was similar to that of its His-tagged counterpart (Fig. 4Go, A and B).



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  		FIG. 4. Permeabilization of E. coli membranes by DEFB118. A, Outer membrane permeabilization. B, Inner membrane permeabilization. {blacksquare}, 10 µg/ml; {blacktriangleup}, 25 µg/ml; {blacktriangledown}, 50 µg/ml; {diamondsuit}, 100 µg/ml; {bullet}, 100 µg/ml reduced and alkylated DEFB118; {triangleup}, 25 µg/ml DEFB118 without His-tag; and {triangledown}, 50 µg/ml DEFB118 without His-tag.

 
Hemolytic activity
The effect of DEFB118 on the membrane integrity of eukaryotic membranes was tested in the rat erythrocyte assay. DEFB118 (10–100 µg/ml) did not induce any hemolysis of rat erythrocytes when incubated for 1 h (Fig. 5Go). The inability of DEFB118 to hemolyze erythrocytes suggests that these eukaryotic membranes are not sensitive to its membrane-destructive effects.



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  		FIG. 5. Hemolytic activity of DEFB118. Hemolysis caused by Triton X-100 and saline are considered 100 and 0%, respectively. Values shown are mean ± SD. *, P ≤ 0.001 compared with Triton X-100.

 
Electron microscopy
The morphological changes induced by DEFB118 on E. coli were examined by scanning electron microscopy. Untreated E. coli had smooth, normal surface morphology without any noticeable membrane abnormalities (Fig. 6Go, A–C). In contrast, DEFB118-treated E. coli showed time-dependent changes in morphology. After 30-min exposure to DEFB118, membrane wrinkling was noticed (Fig. 6Go, D–F). Exposure for 60 and 120 min resulted in surface blebbing and roughening of the membrane. Accumulation of fibrous material, particularly at the dividing septa, and cellular debris, likely arising from cell lysis, were observed (Fig. 6Go, G–L). These structural changes brought about by DEFB118 provide further evidence of potent bacterial membrane disruption and permeabilizing activity.



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  		FIG. 6. Scanning electron micrographs of untreated and DEFB118-treated E. coli. E. coli were incubated without DEFB118 (A–C) or with DEFB118 (50 µg/ml) for 30 min (D–F), 60 min (G–I), and 120 min (J–L).

 
Macromolecular synthesis
To assess the effect of DEFB118 on the macromolecular synthesis in E. coli, the incorporation of radioactive precursors viz. [methyl-3H]thymidine, [5-3H]uridine, and L-[4,5-3H(N)]leucine into DNA, RNA, and proteins was monitored. Incorporation studies were carried out over a range of concentrations, starting with a near-sublethal 2 µg/ml, as determined by the CFU assay. Eighty percent of bacteria survived when incubated for 60 min in the presence of 2 µg/ml DEFB118, and survival was close to 92% after a 20-min incubation (Fig. 7AGo). Higher concentrations of 4, 6, and 8 µg/ml DEFB118 further decreased bacterial survival (Fig. 7AGo). DEFB118, when added at 2 µg/ml, had little effect on DNA, RNA, and protein synthesis (Fig. 8Go, A–C). Similarly, 4 µg/ml DEFB118 had little effect on DNA and RNA synthesis but inhibited protein synthesis after a 40-min incubation (Fig. 8Go, A–C). Concentrations of 10 and 25 µg/ml caused significant decreases in RNA synthesis within 10 min, in DNA synthesis within 20 min, and in protein synthesis after a 40-min incubation. It appeared that DEFB118 first inhibits RNA synthesis, followed by DNA and protein synthesis (Fig. 8Go, A–C). The positive controls, ciprofloxacin, rifampicin, and tetracycline, caused inhibition of DNA, RNA, and protein synthesis when incubated for 60 min, thus validating the incorporation assays (data not shown).



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  		FIG. 7. Bacterial survival and inner membrane permeabilization with low concentrations of DEFB118. A, Bacterial survival: 2 µg/ml ({square}); 4 µg/ml ({triangleup}); 6 µg/ml ({triangledown}); and 8 µg/ml ({diamond}).Values shown are mean ± SD. B, Inner membrane permeabilization: 0 µg/ml ({bullet}); 2 µg/ml ({blacksquare}); 4 µg/ml ({blacktriangleup}); 10 µg/ml ({blacktriangledown}); and 25 µg/ml ({diamondsuit}).

 


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  		FIG. 8. Effect of DEFB118 on macromolecular synthesis in E. coli. A, [Methyl-3H]thymidine incorporation into DNA. B, [5-3H]uridine incorporation into RNA. C, L-[4,5-3H(N)]leucine incorporation into proteins. {bullet}, 0 µg/ml; {blacksquare}, 2 µg/ml; {blacktriangleup}, 4 µg/ml; {blacktriangledown}, 10 µg/ml; and {diamondsuit}, 25 µg/ml. Values shown are mean ± SD. *, P < 0.05–0.01; **, P < 0.01–0.001; and ***, P < 0.001 compared with 0 µg/ml at the corresponding time point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that the sperm binding protein, characterized earlier as ESC42, is a product of the ß-defensin gene cluster on human chromosome 20q11.1. ESC42/DEFB118 is an antibacterial protein that acts by a bacterial membrane-disrupting mechanism. The six-cysteine motif of DEFB118 is assumed to form three structure-stabilizing disulfide bonds characteristic of other ß-defensins. Reduction and alkylation of these cysteines causes complete loss of the membrane-permeabilizing activity of DEFB118 and indicates the importance of the structural integrity of this domain. Similar loss of antimicrobial activity after disulfide-bond reduction was reported for protegrins (31); sepacins (32); tachylepsins (33); HE2{alpha}, ß1, and ß2 (4); and other antimicrobial peptides (34, 35). Similarly, reduction of human ß-defensin 2 caused loss of its ability to permeabilize artificial lipid vesicles (36).

DEFB118 contains a long anionic C-terminal domain, which is unusual among ß-defensins. Such an anionic long C-terminal extension following the ß-defensin-specific cysteine core is reported for another ß-defensin, DEFB125 (37). However, other antimicrobial peptides, such as the HE2 family, have N-terminal extensions (4). The physiological significance of the long anionic C-terminal extensions is still not clear, although for some antimicrobial peptides, the N-terminal propieces have been shown to regulate their activity. For example, the antimicrobial and membrane permeabilization activities of human neutrophil peptide-1 are known to be inhibited by its N-terminal anionic propiece (38). The N-terminal anionic propieces of {alpha}-defensins (cryptidins) are reported to be regulatory domains (39). Such a regulatory effect on the activity of DEFB118 by its C-terminal propiece remains to be investigated.

Disulfide linkages within defensins are thought to preserve the presentation of surface positive charges that interact with negatively charged phospholipids on bacterial surfaces. Increasing salt concentrations tend to reduce antibacterial activity of defensins by weakening these electrostatic charge interactions required for initial contact. The effect of increasing NaCl concentrations on the bactericidal activity of DEFB118 differed markedly from the effect on human ß-defensin-1, which exhibited complete loss of activity in 300 mM NaCl (4). By contrast, DEFB118 had potent antibacterial activity in the presence of 300 mM NaCl and maintained strong activity over the normal physiological sodium concentration range of 75–222 mM reported for seminal plasma of normal humans (40, 41, 42, 43, 44). The effect of salt concentration on DEFB118 antimicrobial activity may result from changes in conformation of the protein. The cationic cysteine-rich domain may interact with bacterial membranes best under lower salt conditions, as reported for human ß-defensin-1 (4) and other defensins (29, 45, 46). However, the effect of increasing salt concentrations on the anionic C-terminal domain and conformation of the protein is not known. Reported effects of NaCl on anionic antibacterial peptides suggest slightly decreased killing potency as salt concentration increases (47), although these are aspartate-rich peptides, unlike DEFB118. Other types of antimicrobial peptides (48, 49, 50, 51), including cathelicidin-derived peptides (10) and HE2 (4), maintain activity in the presence of high salt concentrations. Sustained activity in high salt has been attributed to {alpha}-helical content in some peptides, but the predicted {alpha}-helix in DEFB118 is only 16% of the protein, ß-strand is 23%, and 61% is predicted to form a random coil (Protein Structure Prediction Server, http://bioinf.cs.ucl.ac.uk/psipred/) (52).

DEFB118 caused rapid outer and inner E. coli membrane permeabilization similar to that previously shown for the HE2 family of epididymal proteins (4). Membrane permeabilization and disruption mechanisms are involved in the bactericidal action of many peptides, including human defensins (21), indolicidins (53, 54), bactenectins (55), plant defensins (56), and other classes of antimicrobial peptides (57). Changes in E. coli after DEFB118 treatment observed by scanning electron microscopy revealed membrane wrinkling, blebbing, and cellular debris leaking out through damaged membranes. Similar effects of other antibacterial proteins have been reported. Surface roughening and blebbing that were more frequent at the division septa, similar to those in Fig. 6Go, G and J, were previously reported for Staphylococcus aureus treated with the cathelicidin-derived peptide SMAP-29 (58). E. coli exposed to salmon antimicrobial protein showed pronounced wrinkling as well as bent and elongated morphologies (59) similar to what is shown in Fig. 8EGo. The antimicrobial peptide Tigrenin-1 caused E. coli membrane thickening followed by formation of blebs and leakage of cytoplasmic contents (60) similar to what is shown in Fig. 6Go, G–L. Structural degradation that was associated with accumulation of electron-dense material in the periplasmic space and on the external face of the membranes was observed by transmission electron microscopy in E. coli treated with defensins (21). Defensin-treated S. aureus showed lamellar mesosome-like structures on the cell membrane, and a portion of the cell membrane peeled off from the cell walls (61).

Antimicrobial peptides that disrupt membranes of pathogenic organisms are sometimes toxic to eukaryotic cells (54, 62), in which case they are not suitable to be used as a systemic drug. In this study, DEFB118 did not exhibit any hemolytic activity on rat erythrocytes. This observation suggests that DEFB118 preferentially disrupts bacterial membranes. The lack of damaging effects of DEFB118 on eukaryotic membranes may be due to the presence of cholesterol in the membrane, which generally reduces the activity of antimicrobial proteins and peptides due to either stabilization of the lipid bilayer or to the interaction between the antimicrobial agent and cholesterol (63). Binding to sperm surface represents a nondamaging interaction of DEFB118 with an eukaryotic cell surface, where it may protect the sperm against pathogens and additionally have a role in fertility (22). Sperm carry other epididymal defensins and defensin-like proteins, including macaque ESP13.2/DEFB126, a capacitation factor (64); the rat E-3 form of defb22 (65); and HE2 proteins (66, 67). These reports suggest multiple functions for defensins in the male reproductive tract that may include promotion of fertility as well as host defense.

Bactenectins (68), human neutrophil peptide-1 (69), and pleurocidin-derived antimicrobial peptides (70) were shown to inhibit macromolecular synthesis. However, because of the different sensitivities of the assays, it has been difficult to determine whether inhibition of macromolecular synthesis is the primary bactericidal event or is secondary to membrane disruption. DEFB118 at the relatively low concentration of 2 µg/ml caused a small increase in membrane permeability (Fig. 7BGo) associated with measurable inhibition of bacterial survival (Fig. 7AGo), but there was little effect on inhibition of macromolecular synthesis at this concentration. These results are consistent with a primary effect of DEB118 on membrane permeability, but assays of specific metabolic functions might yet reveal that biochemical changes within the cell occur before or coincidentally with the increase in membrane permeability. Further studies on the nature of the interaction of DEFB118 with the bacterial cell will likely provide new insights into the mechanism of its antimicrobial activity. The antimicrobial effects of DEFB118 on reproductive pathogens are currently being investigated.


   Acknowledgments
 
We thank Dr. R. E. W. Hancock (Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada) for his valuable suggestions regarding the permeabilization assays. We also thank Dr. Ashutosh Tripathy (Macromolecular Interaction Facility, University of North Carolina) for permitting us to use the fluorescence spectrophotometer. We thank Victoria Madden (Microscopy Services Laboratory, University of North Carolina) for her help with the scanning electron microscopy.


   Footnotes
 
This work was supported by the Consortium for Industrial Collaboration in Contraceptive Research Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School, Norfolk, Virginia. (The views expressed by the authors do not necessarily reflect the views of the Contraceptive Research and Development Program or the Consortium for Industrial Collaboration in Contraceptive Research.) This work was also supported by the National Institutes of Health (NIH) Grant R37-HD04466; by the National Institute of Child Health and Human Development/NIH through Cooperative Agreement U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research; and by the Fogarty International Center Training and Research in Population and Health Grant D43TW/HD00627.

Abbreviations: CFU, Colony-forming unit; DEFB118, human ß-defensin 118; diSC3–5, 3,5-dipropylthiadicarbocyanine iodide; LCN6, epididymis-specific lipocalin; NPN, N-phenyl-1-napthylamine; pI, calculated theoretical isoelectric point.

Received December 15, 2003.

Accepted for publication March 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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