Antimicrobial actions of the human epididymis 2 (HE2) protein isoforms, HE2alpha, HE2beta1 and HE2beta2
© Yenugu et al; licensee BioMed Central Ltd. 2004
Received: 16 June 2004
Accepted: 24 August 2004
Published: 24 August 2004
The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis.
E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N)]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean ± S.D.
E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis.
The morphological changes observed in E. coli treated with epididymal HE2 peptides provide further evidence for their membrane dependent mechanism of antibacterial action. HE2 C-terminal peptides can inhibit E. coli macromolecular synthesis, suggesting an additional mechanism of bacterial killing supplementary to membrane permeabilization.
Antimicrobial proteins and peptides are widely expressed in both plants and animals. A variety of natural antibiotics belonging to different classes such as defensins, cathelicidins, cercopins and protease inhibitors  are found in epithelial tissues of organs that are most likely exposed to pathogens. Among them, the most studied in humans are the defensins, which are broadly classified into three types viz alpha, beta and theta defensins depending on their disulfide bonding, tissue distribution and genomic organization. They exhibit broad spectrum antimicrobial activity [2–5], thus may form an important component of the innate immune system. Antimicrobial proteins and peptides including defensins are generally cationic in nature  and are believed to exert their bactericidal effect by permeabilizing the bacterial membranes by forming pores , thinning the membrane , or by destabilizing the membrane bilayer . In addition to membrane permeabilization, antimicrobial proteins and peptides kill bacteria by inhibition of macromolecular biosynthesis [10–12] and/or interacting with specific vital components inside the bacteria [13, 14].
Recombinant peptide preparation and synthesis
HE2α and HE2β2 C-terminal peptides were synthesized at the Peptide Synthesis Facility, University of North Carolina, Chapel Hill by standard f-moc solid phase procedures using Rainin symphony multiple peptide synthesizer (Rainin Instrument, Woburn, MA). The purified peptides eluted as single peaks upon reverse phase high performance liquid chromatography (HPLC) and were further demonstrated to have their corresponding molecular weight by MALDI-TOF mass spectrometry. HE2β1 C-terminal peptide was expressed in E. coli and purified as described previously . Briefly, E. coli strain M15 (pREP4) was transformed with pQE30 vector (Qiagen, Valencia, CA, U.S.A) containing cDNA that codes for HE2β1 C-terminal peptide. Protein expression was induced with 1 mM isopropyl-β-D-thiogalactoside for 1 h at 37°C and the His-tagged recombinant peptide was purified using nickel-nitrilotriacetate agarose column (Qiagen, Valencia, CA, U.S.A). To avoid baseline expression of the protein prior to induction, 1% glucose was maintained in the bacterial medium and the induction time was kept to a minimum (1 h) to minimize the toxic effects of the peptide on E. coli. The peptide was dialyzed extensively against 10 mM sodium phosphate (pH 7.4) to remove urea.
Transmission electron microscopy
E. coli resuspended in 10 mM sodium phosphate buffer (pH 7.4) were treated with 50 μg/ml HE2α, HE2β1 or HE2β2 for 30 and 60 min. After incubation, bacterial cells were washed with 10 mM sodium phosphate buffer (pH 7.4) and fixed with an equal volume of 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, followed by centrifugation at 1000 rpm for 10 minutes to concentrate the cells in a pellet. The fixed samples were stored overnight to several days at 4°C in the fixative solution. The pellet was rinsed in 0.1 M sodium cacodylate buffer several times, and post-fixed with a combination of 1.25% potassium ferrocyanide and 1% buffered osmium tetroxide for one hour at room temperature. Following dehydration with a graded series of ethanols (30–100%) and two changes of propylene oxide, the cell pellet was infiltrated and embedded in PolyBed 812 epoxy resin (Polysciences, Inc., Warrington, PA). Ultra thin sections (70 nm) were cut and mounted on copper grids followed by post staining with 4% uranyl acetate and 0.4% lead citrate. The sections were examined and photographed at an accelerating voltage of 80 kV using a LEO EM 910 transmission electron microscope (LEO Electron Microscopy, Inc., Thornwood, NY) equipped with a Gatan BioScan digital camera (Gatan, Inc., Pleasanton, CA).
Scanning electron microscopy
The structural changes induced by HE2 peptides on E. coli were studied using scanning electron microscopy as described earlier . Bacterial cells suspended in 10 mM sodium phosphate buffer (pH 7.4) after treating with 50 μg/ml of HE2 peptide were fixed with an equal volume of 4% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4. Immediately following the addition of the fixative solution, the sample tube was mixed by gently inverting the tube up and down for several minutes to prevent clumping of the cells. 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 ethanols (30–100%). 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 with a Cambridge Stereoscan 200 scanning electron microscope (LEO Electron Microscopy, Inc., Thornwood, NY) using an accelerating voltage of 20 kV.
The effects of HE2 peptides on E. coli DNA, RNA and protein synthesis were studied as functions of incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N)]leucine respectively as described . 1 × 106 mid-log phase E. coli resuspended in 10 mM sodium phosphate buffer (pH 7.4) were treated with varying concentrations of HE2 peptides 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 periods. After incubation, bacterial suspensions were added to 10% ice-cold trichloroacetic acid and allowed to stand in ice for 40 min. Samples were then collected on 2.4 cm GF/C glass microfiber filters (Fisher Scientific, Pittsburgh, PA) using vacuum filtration and washed thoroughly with 5% TCA and 70% ethanol. The filters were then dried and placed in scintillation vials containing 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 ± S.D.
Transmission electron microscopy
Scanning electron microscopy
Earlier we demonstrated that HE2 proteins and their C-terminal peptides exhibit salt tolerant and structural dependent antimicrobial activities and their mechanism involved permeabilization of both outer and inner bacterial membranes . In this study, structural changes induced in E. coli by epididymal HE2α, HE2β1 and HE2β2 peptides as visualized by transmission and scanning electron microscopy provide further evidence of the membrane dependent mechanism of bacterial killing. Such structural changes induced in E. col i by other antimicrobial proteins and peptides were reported previously. Membrane thickening as shown in Fig 3A,3B,3C,3D,3E,3F was reported in E. coli treated with human neutrophil peptides 1 and 2 (defensins) . Similarly, retraction of cytoplasm and the appearance of vacuoles as shown in Fig 4A,4B,4C,4D,4E,4F were reported for E. coli treated with synthetic peptides of the antimicrobial protein apolipoprotein A-II . Highly granular cytoplasm with discontinuous membrane was reported for E. coli treated with the antimicrobial peptide tigerinin-1  similar to the changes shown in Fig. 5A,5B,5C,5D,5E,5F. Scanning electron micrographs of E. coli treated with HE2 peptides also revealed striking structural changes in their morphology. HE2 peptides caused membrane wrinkling, blebbing and leakage of fibrous material primarily at the dividing septa in E. coli. Such structural changes shown in Fig. 7,8,9 were earlier reported for other antimicrobial proteins viz the cathelicidin-derived peptide SMAP-29 , temporin-L , salmon antimicrobial protein  and the epididymal proteins ESC42 (DEFB118)  and EPPIN . An interesting observation in this study is the leakage of fibrous material primarily at the dividing septa. It is known that cell division in E. coli involves annular constriction of all layers of the cell envelope and synthesis and assembly of new septal materials . It is possible that during this dynamic remodeling process, the region of division septum formation to be particularly vulnerable to attack by antibacterial proteins.
The mechanism of action of antimicrobial proteins is primarily thought to be membrane dependent involving membrane permeabilization and disruption. Structural characteristics of antimicrobial peptides tend to play an important role in their mechanism of action. For example, β-defensins are cationic in nature and with β-sheet rich amphipathic structures stabilized by the three disulfide motif . The cationic nature of β-defensins favors them to bind to and disrupt target membranes that are rich in anionic phospholipids. Similarly, HE2α, HE2β1 and HE2β2 peptides are cationic in nature with basic pIs. Our three dimensional structural analysis of HE2β1 peptide revealed that it is rich in β-sheet structure and its tertiary structure presents regional concentrations of basic and hydrophobic amino acids similar to β-defensins . Such structural characteristics of HE2 peptides which resemble to those of β-defensins suggest that they bind to and disrupt the anionic target membranes and mediate bacterial killing similar to β-defensins. However, alternate mechanisms of antimicrobial action such as inhibition of macromolecular synthesis [10–12] and interaction with specific targets inside the bacterial cells [13, 14] are proposed. HE2 peptides at 10 and 25 μg/ml concentrations inhibited DNA, RNA and protein synthesis suggesting that their antimicrobial action may include interference with metabolic functions of E. coli. Inhibition of macromolecular synthesis was reported for bactenectins , human neutrophil peptide-1 , pleurocidin derived peptides  and the epididymal defensin DEFB118 . In this study, it appears that HE2 peptides were more effective in inhibiting the incorporation of [methyl-3H]thymidine than [5-3H]uridine and L-[4,5-3H(N)]leucine, suggesting DNA synthesis is more sensitive to their antimicrobial action. It is possible that in bacteria that are extensively damaged by HE2 peptides, inhibition of macromolecular synthesis may result simply from the total breakdown of the cells. However the electron micrographs show that only some bacteria appear to be exuding cell contents after the 30 minute treatment. Thus during the first 10–20 minutes exposure to HE2 peptides, some peptides may be entering through pores too small for major cytoplasmic release. The early inhibition of DNA and RNA synthesis in bacteria where little loss of cell contents has occurred, may result from specific interaction of the synthetic machinery with HE2 peptides. Further studies are required to identify specific molecular targets within the bacteria and to establish whether HE2 interactions with these targets can be beneficial to the host by slowing bacterial proliferation.
Increasing recognition of the ability of a number of proteins on the sperm surface to kill bacteria has led to the proposal that they may defend against microbial attack in both the male and female reproductive tracts. The cathelicidin hCAP18 on sperm is processed by the prostate-derived protease, gastricsin to release the active peptide ALL-38 and is found in the female reproductive tract after intercourse . A member of the β-defensin family, DEFB126 also appears to have a role in fertility as a capacitation factor on sperm . Similarly, the rat epididymis specific β-defensin Bin1b, appears to play an important role in sperm maturation . Thus, these defense proteins may enhance the probability of successful fertilization in addition to helping prevent the spread of sexually transmitted diseases.
In conclusion, we report that the epididymal antimicrobial peptides HE2α, HE2β1 and HE2β2 induce striking morphological changes in E. coli consistent with their membrane dependent mechanism of action . In addition to membrane permeabilization, their antimicrobial mechanism involves inhibition of E. coli DNA, RNA and protein synthesis.
We thank Victoria Madden, Microscopy Services Laboratory (MSL), University of North Carolina for her assistance with the electron microscopy. 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. The views expressed by the authors do not necessarily reflect the views of Contraceptive Research and Development or Consortium for Industrial Collaboration in Contraceptive Research. This work is also supported by NIH Grants R37-HD04466, by 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.
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