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Infection and Immunity, October 2007, p. 4780-4791, Vol. 75, No. 10
0019-9567/07/$08.00+0     doi:10.1128/IAI.00557-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Human Defensin -1 Causes Trypanosoma cruzi Membrane Pore Formation and Induces DNA Fragmentation, Which Leads to Trypanosome Destruction

M. Nia Madison, Yuliya Y. Kleshchenko, Pius N. Nde, Kaneatra J. Simmons, Maria F. Lima, and Fernando Villalta^*

Department of Microbial Pathogenesis and Immune Response, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208

Received 17 April 2007/ Returned for modification 20 May 2007/ Accepted 9 July 2007

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Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that human defensin -1 displays a trypanocidal role against Trypanosoma cruzi, the causative agent of Chagas' disease. The toxicity of human defensin -1 against T. cruzi is mediated by membrane pore formation and the induction of nuclear and mitochondrial DNA fragmentation, leading to trypanosome destruction. Exposure of trypomastigote and amastigote forms of T. cruzi to defensin -1 significantly reduced parasite viability in a peptide concentration-dependent and saturable manner. The toxicity of defensin -1 against T. cruzi is blocked by anti-defensin -1 immunoglobulin G. Electron microscopic analysis of trypomastigotes exposed to defensin -1 revealed pore formation in the cellular and flagellar membranes, membrane disorganization, and blebbing as well as cytoplasmic vacuolization. Furthermore, human defensin -1 enters the trypanosome when membrane pores are present and is associated with later intracellular damage. Trypanosome membrane depolarization abolished the toxicity of defensin -1 against the parasite. Preincubation of trypomastigotes with defensin -1 followed by exposure to human epithelial cells significantly reduced T. cruzi infection in these cells. Thus, human defensin -1 is an innate immune molecule that causes severe toxicity to T. cruzi and plays an important role in reducing cellular infection. This is the first report showing that human defensin -1 causes membrane pore formation in a human parasite, leading to trypanosome destruction.

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Trypanosoma cruzi is a blood and tissue protozoan parasite that causes the debilitating inflammatory disease Chagas' disease (38). The World Health Organization estimates that almost one-quarter of the Latin American population is permanently at risk, with over 15 million people infected with T. cruzi (http://www.who.int/tdr/diseases/chagas/default.htm). When Chagas' disease is associated with human immunodeficiency virus type 1 coinfection, it causes severe brain damage (27). Another serious health problem is the incidence of human-to-human transmission through exposure to blood contaminated with trypomastigotes during pregnancy or blood transfusion (25). The latter has been an issue of great concern in the United States due to the millions of Latin American immigrants in the United States (15). The American Red Cross has only recently begun to screen for T. cruzi in the U.S. donor blood supply (21, 39). Currently, there is no cure for chronic Chagas' disease. The drugs available for the acute stage of infection are highly toxic to humans (5). The development of an efficacious prophylactic vaccine faces many challenges, and progress is slow despite several years of effort (11).

The infective trypomastigote form of T. cruzi must rapidly evade the barrage of innate immune mechanisms mounted against it in the host circulatory system by entering and establishing infection in mammalian host cells. During this journey, invasive trypomastigotes manage to evade some innate immune molecules, such as complement (8); however, it is unknown how T. cruzi responds to innate immune molecules such as defensins.

Defensins are small peptides that are produced by leukocytes and epithelial cells and have important effector roles in innate immunity against some microbes (9). Defensins are cysteine-rich cationic peptides that lack enzymatic activity, with ß-pleated sheet structures that are stabilized by three intramolecular disulfide bonds between the cysteine residues (9, 31, 46). Mammalian defensins are classified into three subfamilies, the -, ß-, and -defensins, which differ in their distribution of disulfide links between the six conserved cysteine residues. The disulfide linkages of cysteine residues in -defensins are between the first and sixth cysteine residues (Cys¹-Cys⁶), Cys²-Cys⁴, and Cys³-Cys⁵, whereas in ß-defensins, the linkages are Cys¹-Cys⁵, Cys²-Cys⁴, and Cys³-Cys⁶. In contrast, -defensins have a circular structure, with the cysteine residues linked as Cys¹-Cys⁶, Cys²-Cys⁵, and Cys³-Cys⁴ (37). Six human -defensins have been identified (17), including -1, -2, -3, -4, HD5, and HD6. Human defensins -1, -2, and -3 differ only in the first amino acid (30), whereas the amino acid sequence of human defensin -4 varies greatly in comparison with those of human defensins -1, -2, and -3. Finally, the crystal structures of HD5 and HD6 show significant conformational variability compared to the other -defensins (35).

Human defensin -1 is a 3.5-kDa, 30-amino-acid peptide that has shown effector functions in host innate immunity against some microorganisms (18, 19), yet the role of human defensin -1 against T. cruzi has never been investigated. It was initially reported that this innate immune molecule was produced by neutrophils (10). It was recently shown, however, that other cells of the immune system, such as natural killer cells, B cells, and T cells, can also produce human defensin -1 (1).

Because human epithelial cells rapidly respond to early T. cruzi infection by up-regulating the expression of defensin -1 (M. N. Madison et al., unpublished data), in the present study we investigate the possible role of human defensin -1 against T. cruzi. Here we show that human defensin -1 displays a trypanocidal role against the trypomastigote and amastigote forms of Trypanosoma cruzi and that the toxicity against the parasite is mediated by membrane pore formation, the induction of DNA fragmentation, and consequently, a significant reduction in trypanosome infection of human cells. Thus, human defensin -1 is an innate immune molecule that plays an important role in reducing cellular infection by T. cruzi. This is the first report showing that human defensin -1 causes membrane pore formation in a human parasite, leading to trypanosome destruction.

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Organisms. The highly infective trypomastigote clone MMC 20A, derived from the Tulahuen strain of T. cruzi (20), was used. Pure-culture trypomastigotes were obtained from the supernatants of heart myoblast monolayers as described previously (20). Amastigotes were obtained as described previously (41). Trypomastigotes in blood collected from infected C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were also used (42).

Peptides. Mature human defensin -1 (ACYCRIPACIAGERRYGTCIYQGRLWAFCC) (6, 40) was synthesized and highly purified by reverse-phase high-performance liquid chromatography (HPLC), resulting in a single sharp chromatographic peak. Its analysis by mass spectrometry showed a single peak at mass 3,445.07 (m/z). A scrambled defensin -1 control peptide (CACRPGCRIQYECRARLTAICIGYFAWYCG) with no discernible similarity to existing human proteins was designed using RJE-SEQ software (http://bioinformatics.ucd.ie/shields/redwards/). This control peptide was synthesized, highly purified to a single chromatographic peak by reverse-phase HPLC, and analyzed by mass spectrometry, which showed a single peak at mass 3,445.03 (m/z). The peptides were synthesized, HPLC purified, and analyzed by mass spectroscopy by Genemed Synthesis (San Francisco, CA).

Viability assays. To investigate the ability of human defensin -1 to kill T. cruzi parasites, trypomastigotes or amastigotes (2 x 10⁶/ml) were incubated in Dulbecco's modified Eagle medium (DMEM) with several concentrations of human defensin -1 in DMEM, ranging from 3.7 to 35 µM, alone or in the presence of goat anti-defensin -1 immunoglobulin G (IgG) or goat preimmune IgG (MBL International Corp., Woburn, MA), for 1 h at 37°C. Controls were performed by exposing the same number of parasites to the same concentrations of a scrambled defensin -1 control peptide or medium alone for the same incubation period. The number of killed trypomastigotes or amastigotes was determined microscopically (44, 45), using the following formula: % parasite killing = [1 – (number of live parasites after treatment/number of live parasites in DMEM)] x 100. Three independent experiments were performed, with each done in triplicate.

To investigate if defensin targets the membranes of trypomastigotes, we preexposed parasites to carbonyl cyanide m-chlorophenylhydrazone (CCCP) (32), a membrane depolarization agent. The same concentration of trypomastigotes was preincubated with 10 µM of CCCP (Sigma, St. Louis, MO) or mock treated for 15 min, washed in DMEM, and then exposed to 20 µM of defensin -1 for 1 h at 37°C. Additional controls included mock-treated trypomastigotes exposed to 20 µM of scrambled defensin -1 control peptide or defensin -1 preincubated with anti-defensin -1 IgG or preimmune IgG and trypomastigotes incubated with 10 µM CCCP alone or medium alone for 1 h at 37°C. The number of killed trypomastigotes was determined microscopically as described previously (45). Three independent experiments were performed in triplicate.

To study the ability of human defensin -1 to kill trypomastigotes in blood, trypomastigotes collected from the blood of T. cruzi-infected mice (3 x 10⁶ trypomastigotes/ml in the presence of 1.5% sodium citrate) were exposed to 25 µM defensin -1 or 25 µM scrambled defensin -1 peptide control or were mock treated in triplicate for 24 h at 4°C. The number of killed blood trypomastigotes was determined microscopically as described previously (42). Three independent experiments were performed, with each done in triplicate.

Electron microscopy. To investigate the ability of human defensin -1 to cause membrane pore formation and ultrastructural alterations in T. cruzi trypomastigotes, samples containing 10⁷ parasites/ml under different conditions, as indicated below, were subjected to scanning electron microscopy (SEM), negative stain electron microscopy (nsEM), negative stain immunogold electron microscopy (ns-immunogold EM), and transmission electron microscopy (TEM). Additionally, immunogold TEM was used to explore whether human defensin -1 enters trypomastigotes and whether internalized defensin -1 is associated with intracellular damage in the trypanosome.

For SEM, trypomastigotes (10⁷ parasites/ml) were exposed to various concentrations of human defensin -1 (3.7, 5, and 20 µM), 20 µM scrambled defensin -1 control peptide, or 20 µM defensin -1 preincubated with anti-defensin -1 IgG or preimmune IgG for 1 h as described above. Cells were processed as described previously (44), followed by postfixation with OsO₄, and were sputter coated with gold-palladium and imaged using a Hitachi 2700 scanning electron microscope.

For nsEM, trypomastigotes (10⁷ parasites/ml) were exposed to 3.7 µM defensin or 3.7 µM scrambled defensin -1 control peptide for 1 h. Samples were stained with phosphotungstic acid (13) and observed with a Phillips CM-12 electron microscope.

For ns-immunogold EM, trypomastigotes (10⁷ parasites/ml) were treated with 3.7 µM defensin -1 and processed as described for nsEM. Samples were incubated with anti-defensin -1 IgG or preimmune IgG diluted in saline solution supplemented with 1% bovine serum albumin and probed with donkey anti-goat IgG conjugated with 12-nm gold particles (Amersham Bioscience, Piscataway, NJ) diluted in saline solution supplemented with 1% bovine serum albumin. Samples were stained with phosphotungstic acid and observed with a Phillips CM-12 electron microscope.

To determine the percent frequencies of different pore diameters caused by human defensin -1 on the membranes of T. cruzi trypomastigotes, the sizes of 320 pores were measured in three independent experiments.

For TEM, trypomastigotes (10⁷ parasites/ml) were exposed to 15 µM defensin -1, 15 µM scrambled defensin -1 control peptide, 15 µM defensin -1 preincubated with anti-defensin -1 IgG, 15 µM of defensin -1 preincubated with preimmune IgG, or medium alone at 37°C for different periods, from 15 min to 1 h. Samples were fixed in 2.5% glutaraldehyde, postfixed with OsO₄, and embedded in Spurrs resin. Ultrathin sections were observed with a Phillips CM-12 electron microscope as described previously (43).

For immunogold TEM, trypomastigotes (10⁷ organisms) were incubated with 10 µM defensin -1 for 15 to 60 min and embedded in low-acryl resin (43). Ultrathin sections of trypomastigotes previously exposed to defensin -1 were probed with anti-defensin -1 IgG or preimmune IgG, followed by incubation with IgG complexed with gold particles as described previously (43), stained with 0.25% phosphotungstic acid, and analyzed by TEM as described above.

TUNEL assay. For monitoring T. cruzi DNA cleavage, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (in situ cell death detection kit; Roche Diagnostics Inc., Indianapolis, IN) was used. T. cruzi trypomastigotes (10⁶/ml) were exposed to 10 µM human defensin -1 for 5 min at 37°C in DMEM free of phenol red (Invitrogen Corporation, Carlsbad, CA). Controls were performed by exposing 10⁶ parasites/ml to human defensin -1 preincubated with anti-defensin -1 IgG, medium alone, or exogenous DNase (Promega, Madison, WI) as a positive control. TUNEL assay was performed as described by the manufacturer. Fluorescence was visualized with a Nikon fluorescence microscope (16) and by fluorescence-activated cell sorting (FACS) analysis (12).

Infection assays. The ability of human defensin -1 to block T. cruzi infection of HeLa cells was evaluated. HeLa cells were used because they are a good model for studying cellular infection by T. cruzi in vitro. Trypomastigotes were preincubated in triplicate for 1 h with a sublethal dose of 3.7 µM human defensin -1 (a human basal blood concentration of defensin -1), 3.7 µM scrambled defensin -1 control peptide, medium alone, or 3.7 µM defensin -1 preincubated with either anti-defensin -1 IgG or preimmune IgG. Parasites were washed with DMEM and exposed to HeLa cell monolayers at a ratio of 10 parasites per cell for 2 h (24). After the 2-h incubation period, unbound parasites were removed, and trypomastigote binding to HeLa cells was evaluated by fluorescence microscopy, using fluorescein isothiocyanate-labeled antibodies specific for a trypomastigote surface protein and DAPI (4',6'-diamidino-2-phenylindole) staining (33). The number of internalized parasites at 2 h was obtained by subtracting the number of bound fluorescent parasites from the total number of DAPI-stained parasites per 200 host cells. Parasite multiplication within cell monolayers at 24 h, 48 h, and 72 h was evaluated microscopically in Giemsa-stained monolayers by determining the number of parasites per 200 cells (24, 33).

Presentation of results and statistical analysis. The results shown in this work are representative of three independent experiments performed in triplicate by identical methods. The results are expressed as means ± 1 standard deviation. Differences were considered statistically significant if the P value was <0.05 by Student's t test.

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Human defensin -1 kills T. cruzi, and trypanosome membrane depolarization inhibits defensin -1-mediated killing. To investigate whether human defensin -1 kills invasive T. cruzi trypomastigotes or amastigotes, we exposed these mammalian forms of T. cruzi to increasing physiological concentrations of human defensin -1 (3.7 to 35 µM) for 1 h. These concentrations were used to clearly observe the effects of defensin -1 on T. cruzi. Our results shown in Fig. 1A indicate that human defensin -1 kills T. cruzi trypomastigotes in a peptide concentration-dependent and saturable manner. Minimal trypomastigote killing was seen with 3.7 to 5 µM human defensin -1, whereas maximum killing and saturation were observed with 30 µM human defensin -1 (Fig. 1A). Preincubation of defensin -1 with anti-human defensin -1 IgG abolished the trypomastigote killing effect of human defensin -1, whereas preincubation of defensin -1 with preimmune IgG alone did not (Fig. 1A). Furthermore, preincubation of trypomastigotes with the same concentrations of a scrambled defensin -1 control peptide did not have any consequences for the trypomastigotes (Fig. 1A). Human defensin -1 also killed amastigotes in a peptide concentration-dependent manner, and this killing was abolished by anti-human defensin -1 IgG (Fig. 1B). Amastigotes were more susceptible than trypomastigotes at concentrations of defensin -1 ranging from 3.7 to 10 µM. Scrambled defensin -1 did not have any consequence on amastigotes under the same conditions (Fig. 1B). Human defensin -1 was also able to kill trypomastigotes present in blood collected from infected mice when these trypomastigotes were exposed to 25 µM defensin -1 and incubated at 4°C, a storage condition used in blood banks. We observed that defensin -1 killed 35% ± 1.8% of blood trypomastigotes in a period of 24 h with respect to those in mock-treated infected blood or infected blood exposed to 25 µM scrambled defensin -1 control peptide (P < 0.05). Defensin -1 needs to be present at all times for the effects to be seen in the parasite, since when it was neutralized with antibodies no effects were seen (Fig. 1). The minimal time of exposure for trypomastigote killing with 20 µM defensin -1 was 15 min; we observed that 20 µM defensin -1 killed 81% ± 2% of trypomastigotes at 15 min with respect to mock-treated parasites (P < 0.05). Washing did not reverse parasite killing.

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FIG. 1. Defensin -1 kills T. cruzi trypomastigotes and amastigotes in a concentration-dependent and saturable manner. The same number of parasites were incubated in triplicate with several concentrations of defensin -1, ranging from 3.7 to 35 µM, alone or in the presence of defensin -1 preincubated with anti-defensin -1 IgG or preimmune IgG, for 1 h. Controls included the same number of parasites incubated with the same concentrations of a scrambled defensin -1 control peptide or medium alone for the same incubation time. The number of killed trypomastigotes or amastigotes was determined microscopically as described in Materials and Methods. Data are means ± 1 standard deviation for one representative experiment of three independent experiments performed in triplicate with similar results. Differences between parasites exposed to defensin -1 or to defensin -1 in the presence of preimmune IgG and parasites exposed to the scrambled defensin -1 peptide control, defensin -1 in the presence of anti-defensin -1 IgG, or medium alone were statistically significant (P < 0.05).

Our results show that CCCP, an agent that depolarizes the cytoplasmic membrane, reverses defensin -1-mediated killing of T. cruzi trypomastigotes (Fig. 2). This is an indication that trypanosome membrane depolarization protects the parasite from human defensin -1-mediated trypanocidal activity and provides the first evidence that defensin -1 activity starts at the membrane of the trypanosome. Accordingly, preexposure of trypomastigotes to 10 µM CCCP followed by exposure to human defensin -1 abolished human defensin -1-mediated killing, whereas preexposure of trypomastigotes to 10 µM CCCP followed by incubation in medium alone resulted in 100% trypanosome survival (Fig. 2). Again, anti-human defensin -1 IgG abolished the killing effect induced by defensin -1, whereas preimmune IgG did not, supporting the specificity of defensin -1 effects on the trypanosome. This first observation encouraged us to investigate the effects of defensin -1 at the membrane of the trypanosome, using several ultrastructural approaches.

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FIG. 2. Susceptibility of T. cruzi trypomastigotes to a lethal concentration of defensin -1 in the presence or absence of CCCP. Trypomastigotes at a final concentration of 2 x 106 parasites/ml were preincubated with 10 µM of CCCP or mock treated for 15 min and then exposed to 20 µM of defensin -1 for 1 h. Additional controls included trypomastigotes treated with 10 µM CCCP alone and mock-treated trypomastigotes exposed to 20 µM of scrambled defensin -1 control peptide or 20 µM of defensin -1 preincubated with anti-defensin -1 IgG or preimmune IgG for 1 h. The number of killed trypomastigotes was determined microscopically as described in Materials and Methods. Bars represent means plus 1 standard deviation for triplicate experiments with similar results. *, significant differences between the lethal effect of defensin -1 or defensin -1 in the presence of preimmune IgG and the effect on trypomastigotes preexposed to CCCP, CCCP followed by exposure to defensin -1, medium alone, scrambled defensin -1 control peptide, or defensin -1 preincubated with anti-defensin -1 IgG (P < 0.01).

Human defensin -1 causes membrane pore formation and membrane disruption, followed by cellular destruction, in T. cruzi trypomastigotes. The facts that human defensin -1 is trypanocidal against T. cruzi trypomastigotes and that this process starts at the trypanosome cell membrane prompted us to determine whether defensin -1 causes ultrastructural alterations in the parasite membrane as part of our investigation into the initial action of defensin -1-mediated trypanocidal activity. Accordingly, we performed SEM, nsEM, ns-immunogold EM, and TEM on T. cruzi trypomastigotes exposed to the indicated concentrations of human defensin -1 for the incubation periods described in the figure legends. Control SEM shows the normal membrane structure of the parasite and its membrane integrity when trypomastigotes were incubated with a scrambled defensin -1 control peptide (Fig. 3A). Figure 3B shows that exposure of trypomastigotes to 3.7 µM human defensin -1 for 1 h resulted in membrane disorganization in the trypanosome membrane, as indicated by the arrows, and plasma membrane blebbing, as indicated by the arrowheads. Exposure to 3.7 µM defensin -1 resulted in membrane alterations in all trypomastigotes exposed to the molecule. These effects induced by human defensin -1 in the trypanosome membrane were abolished when trypomastigotes were exposed to human defensin -1 that was previously preincubated with anti-human defensin -1 IgG (Fig. 3C). SEM analysis of trypomastigotes exposed to 5 µM human defensin -1 for 1 h, shown in Fig. 3D, revealed that the parasites developed plasma membrane blebbing, as indicated by the arrowhead, and plasma membrane disruption with cytoplasmic eruption, as indicated by arrows, indicating that an increase of defensin -1 results in severe membrane disorganization. Exposure of trypomastigotes to a higher concentration of human defensin -1 (20 µM) for 1 h resulted in total destruction of the parasite, with detachment of the flagellum from the trypanosome body, as seen in Fig. 3E. We observed that the detached flagellum had part of the flagellar membrane with flagellar membrane blebbing, as indicated by the arrowhead in Fig. 3E.

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FIG. 3. Defensin -1 kills T. cruzi trypomastigotes, resulting in structural alterations in the trypomastigote membrane, as evidenced by SEM. (A) Trypomastigotes incubated with 20 µM scrambled defensin -1 control peptide. (B) Trypomastigotes incubated with 3.7 µM defensin -1. Arrows point to membrane disorganization, and arrowheads point to membrane blebbing. (C) Trypomastigotes exposed to 20 µM defensin -1 preincubated with anti-defensin -1 IgG. (D) Trypomastigotes exposed to 5 µM defensin -1. Arrows point to cytoplasmic eruption, and the arrowhead points to membrane blebbing. (E) Exposure to 20 µM defensin -1 causes trypomastigote destruction with detachment of the flagellum. The arrowhead points to membrane blebbing associated with the detached flagellum. All incubations were done for 1 h, and samples were processed for SEM as described in Materials and Methods. Bars, 1 µm. The results are representative of three independent experiments performed with similar results.

The fact that exposure of trypomastigotes to 3.7 µM human defensin -1 resulted in parasite membrane disruption (Fig. 3B) prompted us to explore whether human defensin -1 causes pore formation in the membranes of trypomastigotes by using nsEM, which is a standardized procedure to demonstrate membrane pore formation (4). nsEM analysis of trypomastigotes exposed to human defensin -1 indicates that human defensin -1 causes pore formation in the trypanosome membrane, as can be seen in Fig. 4. Human defensin -1 causes the formation of numerous pores in the membrane that covers the flagellum and in the flagellar membrane that attaches the flagellum to the body of the trypanosome, as evidenced by nsEM (Fig. 4A) and shown in the clear area, where pores caused by human defensin -1 are most noticeable. Figure 4B shows two pores from Fig. 4A in proximity to each other at a high magnification, and Fig. 4C shows two amplified pores being fused to each other in the flagellar membrane of a trypomastigote. Figure 4D shows a larger pore in the flagellar membrane of the trypanosome with the combined diameter of the pores seen in panels B and C. Much larger pores can also be seen in the flagellar membrane of the trypanosome (Fig. 4A).

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FIG. 4. Defensin -1 causes membrane pore formation in T. cruzi trypomastigotes, as evidenced by nsEM. (A) Exposure of trypomastigotes to defensin -1 causes many pores along the parasite flagellar membrane. FM, flagellar membrane; PB, parasite body. Bar, 100 nm. (B) High magnification of two individual pores, each with a diameter of 46 nm, coming into fusion proximity on the flagellar membrane of a trypomastigote exposed to defensin -1. (C) Two individual pores undergoing fusion, forming a transitional pore complex with a diameter of 92 nm. (D) Large, pronounced pore with a diameter of 92 nm. Bars in panels B, C, and D, 25 nm. Samples were processed for nsEM as described in Materials and Methods. The results are representative of three independent experiments performed in triplicate with similar results.

We also investigated the insertion of human defensin -1 molecules into the membranes of trypomastigotes by using ns-immunogold EM. This standardized approach has been used to demonstrate the insertion of molecules that cause membrane pore formation (e.g., aquaporin) into the membranes of mammalian cells (4). Our results, presented in Fig. 5, clearly indicate that defensin -1 integrates into the membranes of trypomastigotes in concentric binding patterns leading to pore formation, as observed by ns-immunogold EM. Negative staining of trypomastigotes probed with goat anti-defensin -1 IgG followed by gold-conjugated anti-goat IgG shows that defensin -1 integrates into the membranes of trypomastigotes in circular patterns, whereas probing negatively stained trypanosome samples with preimmune IgG followed by conjugated anti-goat IgG does not show any reactivity (Fig. 5A). Figure 5B shows that human defensin -1 integrates into the trypomastigote membrane in a concentric manner with different diameters (panels B', B''', and B''') as early as 15 min after exposure. Accordingly, the pore shown in panel B' has a diameter of 52.6 nm, the pore in panel B''' has a diameter of 142 nm, and the pore in panel B''' has a diameter of 126 nm. Figure 5C shows an intermediate pore at 30 min, whereas Fig. 5D shows late pore formation caused by defensin -1 in T. cruzi at 1 h. Figure 5E shows the distribution of pore diameters caused by a 1-h exposure of the trypanosome membrane to defensin -1. The highest percent frequency of pore diameters (75%) is for pores ranging in diameter from 10 to 39 nm, whereas the lowest percent frequency of pore diameters (25%) is for pores ranging in diameter from 3 to 6 nm and 40 to 190 nm.

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FIG. 5. Defensin -1 integrates into the membranes of trypomastigotes, forming concentric binding patterns leading to pore formation, as evidenced by ns-immunogold EM. (A) Defensin -1 binds to the membranes of T. cruzi parasites in circular patterns. (A') Early circular integration of defensin -1 molecules into the trypomastigote membrane at 15 min, as revealed by anti-defensin -1 IgG and developed by ns-immunogold EM. (A''') Control trypomastigotes incubated with preimmune IgG at the same time and developed by ns-immunogold EM do not show reactivity. (B) Pattern of early integration of defensin -1 molecules into the membranes of trypomastigotes in a concentric manner. (B', B''', and B''') Circular integration of defensin -1 into the trypanosome membrane, with different diameters, at 15 min. (C) Intermediate pore formation caused by defensin -1 at 30 min. (D) Late pore formation caused by defensin -1 at 1 h. Bars, 0.1 µm. (E) Frequencies of pore diameters caused by 1-h exposure of the trypanosome membrane to defensin -1. Trypomastigotes were exposed to 3.7 µM defensin -1 and processed for ns-immunogold EM as described in Materials and Methods. The results are representative of three independent experiments performed with similar results. Histogram bars represent means ± 1 standard deviation for triplicate samples in one representative experiment selected from three experiments with similar results. Significant differences were observed between groups marked with single and double asterisks and among groups marked with double asterisks (P < 0.05).

To investigate the ultrastructural alterations caused by defensin -1 in the whole trypanosome, TEM studies were performed. First, we studied the kinetics of ultrastructural alterations caused by 15 µM of human defensin -1 in the trypanosome, looking first at ultrastructural membrane alterations, followed by cytoplasmic and organelle alterations in the trypanosome. TEM revealed that incubation of trypomastigotes with 15 µM scrambled defensin -1 control peptide does not cause toxicity to the trypanosome (Fig. 6A). Anti-human defensin -1 completely protected the integrity of the trypanosome structures when parasites were incubated with 15 µM of human defensin -1 (Fig. 6B). Exposure of trypomastigotes to 15 µM defensin -1 caused ultrastructural changes that worsened with increasing incubation periods. As shown in Fig. 6C, we observed that a 15-min incubation with 15 µM human defensin -1 resulted in trypanosome plasma membrane disorganization with a loss of plasma membrane regions (indicated by arrows), a reduction in membrane microtubules (indicated by arrowheads), and swelling of the mitochondria and alterations in the mitochondrial membranes (indicated by the asterisk) compared to trypanosomes exposed to medium alone (Fig. 6C'). We observed that at 15 min, there is flagellar membrane disorganization with patch formation (indicated by arrowheads), as shown in Fig. 6D, compared to trypanosomes exposed to medium alone (Fig. 6D'). Figure 6E shows that a 30-minute incubation with 15 µM defensin -1 caused rapid vacuolization of the trypanosome cytoplasm compared to that of trypanosomes in medium alone (Fig. 6E'). At this time, multiple vacuoles were induced by human defensin -1 and some vacuoles started to fuse to each other, as indicated by asterisks in Fig. 6E. At 45 min, all vacuoles fused to form a giant vacuole in the trypanosome cytoplasm, as shown in Fig. 6F, in contrast to the case for trypanosomes exposed to medium alone (Fig. 6F'). Figure 6G shows that at 1 h of incubation with defensin -1, trypanosomes are destroyed, the body is severely swollen, the trypanosome membrane cannot be seen, no clear structures and organelles are seen in the cytoplasm, and the giant cytoplasm vacuole persists. Control trypanosomes in medium alone are intact (Fig. 6G').

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FIG. 6. Exposure of T. cruzi trypomastigotes to defensin -1 results in ultrastructural alterations, as evidenced by TEM. (A) Trypomastigotes exposed to 15 µM scrambled defensin -1 control peptide for 1 h. (B) Trypomastigotes incubated with 15 µM of defensin -1 preincubated with anti-defensin -1 IgG for 1 h. (C) Defensin -1 at 15 µM causes membrane disorganization and disappearance, reduction of membrane microtubules, and mitochondrial swelling, including alteration of mitochondrial membranes, at 15 min. Arrows point to trypanosome membrane disappearance, arrowheads show reductions of membrane microtubules, and the asterisk points to mitochondrial membrane alteration. (C') The arrowhead shows normal membrane microtubular structures in control trypanosomes incubated in DMEM. The asterisk points to a normal mitochondrial membrane. (D) Defensin -1 causes flagellar membrane disorganization at 15 min. Arrowheads point to flagellar membrane disorganization. (D') Control trypomastigotes incubated in DMEM. (E) Defensin -1 causes rapid cytoplasmic vacuolization at 30 min. Asterisks point to vacuoles undergoing fusion. (E') Control for panel E. (F) Defensin -1 causes large cytoplasmic vacuole with loss of intracellular compartmentalization at 45 min. (F') Control for panel F. (G) Defensin -1 destroys trypomastigotes at 1 h. F, flagellum. (G') Control trypomastigotes in DMEM. Bars in panels A, B, F, F', G, and G', 0.5 µm; bars in panels C, C', D, D', E, and E', 0.1 µm. The results are representative of three independent experiments performed with similar results.

We then explored whether human defensin -1 gains entry into trypomastigotes and whether intracellular defensin -1 is associated with intracellular damage by using immunogold TEM. The results shown in Fig. 7 indicate that, indeed, human defensin -1 gains early entry into the cytoplasm and organelles of T. cruzi trypomastigotes. Figure 7A shows early entry of defensin -1 into trypomastigotes and indicates that intracellular defensin -1 is associated with minimal organelle damage upon 15 min of trypomastigote exposure to defensin -1, when minimal trypanosome damage was observed, and under conditions where membrane pores are present. This indicates that defensin -1 gains entry into the parasite cytoplasm through membrane pores. Internalized defensin -1 was observed within the nucleus, kinetoplast, and cytoplasm of trypomastigotes (Fig. 7A). At 30 min of trypomastigote exposure to defensin -1, we observed pronounced trypomastigote cytoplasmic damage associated with internalized defensin -1 (Fig. 7B) and saw that entry of defensin -1 into the kinetoplasts of trypomastigotes was associated with mitochondrial damage (Fig. 7C). Severe trypomastigote cellular damage associated with internalized defensin -1 was seen at 1 h of trypomastigote exposure to defensin -1 (Fig. 7D).

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FIG. 7. Human defensin -1 gains early entry into T. cruzi trypomastigotes when minimal cellular damage is observed and later is associated with cellular damage, as seen by immunogold TEM. (A) Early entry of defensin -1 into trypomastigotes at 15 min is associated with minimal damage of organelles, as denoted by immunogold particles. The arrow points to kDNA. N, nucleus. (B) Entry of defensin -1 into the cytoplasm of trypomastigotes at 30 min reveals intracellular human defensin -1 associated with cytoplasmic damage. (C) Human defensin -1 gains entry into the kinetoplast of trypomastigotes at 30 min and is associated with mitochondrial damage. K, kinetoplast. (D) Entry of human defensin -1 into trypomastigotes showing intracellular defensin -1 associated with severe cellular damage following 1 h of exposure to defensin -1. The arrow points to kDNA. N, nucleus. EM grids containing ultrathin sections of trypomastigotes previously exposed to 10 µM defensin -1 for the times indicated above were probed with anti-defensin -1 IgG followed by incubation with anti-IgG complexed with gold particles, stained with phosphotungstic acid, and observed by TEM. Bars in panels A and D, 500 nm; bar in panel B, 100 nm; bar in panel C, 250 nm. The results are representative of three independent experiments performed with similar results.

Human defensin -1 induces DNA fragmentation in T. cruzi trypomastigotes. We performed TUNEL assays to investigate whether defensin -1 induces DNA fragmentation (29) in trypomastigotes. TUNEL analysis indicated that the trypomastigote DNA became fragmented within 5 min of exposure to 10 µM human defensin -1, as evidenced by FACScan analysis (Fig. 8A). The positive control included trypomastigotes incubated with exogenous DNase, which underwent DNA fragmentation to a similar degree as trypomastigotes incubated with human defensin -1 (Fig. 8A). Furthermore, parasites incubated with human defensin -1 that was preincubated with anti-human defensin -1 IgG did not exhibit DNA fragmentation, nor did parasites incubated in medium alone, as shown by FACS analysis (Fig. 8A). Nuclear DNA and mitochondrial DNA (kDNA) fragmentation induced by human defensin -1 was observed by fluorescence microscopy, as shown in Fig. 8B. Trypomastigotes exposed to 10 µM of human defensin -1 showed nuclear and kinetoplastid DNA fragmentation, as determined by the fluorescence seen in both the nuclei and kDNAs of trypanosomes (Fig. 8B, panel b). The positive control, T. cruzi trypomastigotes exposed to exogenous DNase, showed similar fluorescence in the nuclei and kDNAs of trypanosomes, as shown in Fig. 8B, panel a. Negative controls of trypomastigotes incubated in medium alone (Fig. 8B, panel c) and trypomastigotes incubated with human defensin -1 previously preincubated with anti-human defensin -1 IgG (Fig. 8B, panel d) did not show fluorescence.

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FIG. 8. Detection of DNA fragmentation in T. cruzi trypomastigotes exposed to human defensin -1 by TUNEL assay. (A) Flow cytometric TUNEL analysis of trypomastigotes exposed to human defensin -1. (a) Control permeabilized trypomastigotes incubated with exogenous DNase. (b) Trypomastigotes incubated with defensin -1. (c) Trypomastigotes incubated with medium alone. (d) Trypomastigotes exposed to defensin -1 preincubated with anti-defensin -1 IgG. (B) Fluorescence microscopic TUNEL analysis of trypomastigotes exposed to human defensin -1. (a) Control permeabilized trypomastigotes incubated with exogenous DNase. (b) Trypomastigotes incubated with defensin -1. (c) Trypomastigotes incubated with medium alone. (d) Trypomastigotes exposed to defensin -1 preincubated with anti-defensin -1 IgG. The results are representative of three independent experiments performed in triplicate with similar results.

Our results indicate that human defensin -1 induces DNA fragmentation in 94% ± 2% of trypanosomes with respect to trypanosomes exposed to exogenous DNase, as seen by triplicate FACScan TUNEL analysis (P < 0.05). These results clearly demonstrate that human defensin -1 induces DNA fragmentation of the nuclear DNA and kDNA of the trypanosome.

Exposure of T. cruzi trypomastigotes to human defensin -1 inhibits T. cruzi infection of human cells. To investigate whether exposure of T. cruzi trypomastigotes to defensin -1 inhibits the ability of these parasites to infect mammalian cells, we preincubated T. cruzi parasites with a sublethal concentration (3.7 µM) of defensin -1, followed by their exposure to human epithelial cells, and examined the kinetics of cellular infection. We had previously observed that this selected sublethal concentration of defensin -1 affected the membrane of the trypanosome (Fig. 3B). We observed that T. cruzi invasive trypomastigotes exposed to human defensin -1 showed a reduced ability to bind and enter host cells at 2 h, to enter host cells at 24 h, and to multiply as amastigotes within host cells at 48 to 72 h compared to trypomastigotes exposed to medium alone, a scrambled defensin -1 control peptide, or defensin -1 preincubated with anti-defensin -1 IgG (Fig. 9A). Analysis of the kinetics of cellular infection of human epithelial cells exposed to trypomastigotes incubated with human defensin -1 that was preincubated with preimmune IgG showed a significant reduction in the kinetics of cellular infection, as evaluated by determining the number of T. cruzi organisms per 200 cells (Fig. 9A). This reduction in T. cruzi cell infectivity was similar to the reduction seen when trypomastigotes were incubated with human defensin -1 (Fig. 9A). We also observed that trypomastigotes exposed to human defensin -1 under the same conditions were less able to infect human epithelial cells than were trypomastigotes exposed to medium alone, scrambled defensin -1 control peptide, or defensin -1 preincubated with anti-defensin -1 IgG (Fig. 9B). Exposure of trypomastigotes to human defensin -1 or to human defensin -1 previously incubated with preimmune IgG under the same conditions resulted in a 50% reduction in the percentage of infected cells compared to the percentage of infection caused by either mock-treated trypanosomes or trypanosomes incubated with defensin -1 previously preincubated with anti-human defensin -1 (Fig. 9B).

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FIG. 9. Preexposure of trypomastigotes to a sublethal concentration of defensin -1 significantly reduces T. cruzi infection of human cells. (A) Pretreatment of trypomastigotes with defensin -1 followed by exposure to HeLa cells reduces the number of T. cruzi organisms per 200 cells during the course of infection. (B) Pretreatment of trypomastigotes with defensin -1 followed by exposure to HeLa cells reduces the percentage of infected cells over time. Trypomastigotes were preexposed to 3.7 µM defensin -1, followed by incubation with HeLa cell monolayers at a ratio of 10 parasites per cell. Parasite binding, entry, and multiplication were analyzed as described in Materials and Methods. Each point represents the mean ± 1 standard deviation for triplicate samples of one representative experiment selected from three experiments with similar results. Significant differences were seen between trypomastigotes incubated with medium alone, scrambled defensin -1 control peptide, or defensin -1 preincubated with anti-defensin -1 IgG and trypomastigotes incubated with defensin -1 or defensin -1 preincubated with preimmune IgG (P < 0.05).

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In this study, we describe for the first time that human defensin -1 displays a trypanocidal role against T. cruzi trypomastigotes and amastigotes. The toxicity of human defensin -1 against T. cruzi is mediated by membrane pore formation and the induction of DNA fragmentation leading to trypanosome destruction. We also describe for the first time the early internalization of human defensin -1 into the trypanosome when minimal cellular damage is observed and under conditions where membrane pores are present and show that, later, intracellular defensin -1 is associated with damage to the cytoplasm and organelles of trypomastigotes. Adding a sublethal dose of human defensin -1 to trypomastigotes resulted in a significant reduction in the infection of human cells. Thus, human defensin -1 is an important innate immune molecule that causes severe toxicity to T. cruzi and plays an important role in reducing cellular infection.

This is the first report showing that human defensin -1 integrates into the membrane of a human parasite to cause pore formation leading to parasite death, expanding the biological role of human defensin -1 against microbes. The concentrations of human defensin -1 that kill T. cruzi trypomastigotes or amastigotes in vitro (Fig. 1) are comparable to the basal concentrations of human defensin -1 present in the sera of healthy individuals (up to 3.7 µM) and to the serum concentrations that are present during inflammation, which are amplified up to 13-fold (14, 26). Since Chagas' disease is a severe inflammatory disease, we suggest that this parasite may be exposed to these concentrations of defensin -1 in the bloodstream. In fact, our observations also indicate that exposure of contaminated blood to human defensin -1 kills blood trypomastigotes. This also suggests that human defensin -1 has an important role in innate immunity against T. cruzi. The fact that defensin -1 kills T. cruzi in blood in vitro at 4°C, a storage condition in blood banks, suggests that defensin -1 could be used in blood banks in blood infected with T. cruzi or suspected of T. cruzi infection. Since our results indicate that defensin -1 is an innate immune molecule that plays a role in killing both mammalian forms of T. cruzi and consequently reducing infection, we suggest that human defensin -1 may play a beneficial role in the host in reducing early infection or during the chronic phase of the disease. A potential way to bolster defensin -1 to ameliorate infection may be by activating neutrophils in the host with colony-stimulating factor, interleukin-8, and tumor necrosis factor alpha to increase the production of defensin -1 in the body (3, 23, 36). It may also be possible to engineer defensin -1 molecules to avoid potential undesirable interactions with the host and to retain trypanocidal activity, which might prevent Chagas' disease transfusion.

Here we have documented the toxicity of defensin -1 against T. cruzi. The early event in this process involves the integration of defensin -1 molecules into the membrane of the trypanosome in a concentric manner. This integration causes pore formation in the trypanosome membrane (Fig. 4 and 5), leading to DNA fragmentation (Fig. 8) and ultimately to trypanosome destruction (Fig. 6G and 7D). The facts that defensin -1 causes early pore formation after exposure of trypanosomes to defensin -1 for 15 min and that we also observed early entry of defensin -1 into the cytoplasm of trypomastigotes when minimal cellular damage was observed under conditions where membrane pores were present, which later was associated with intracellular damage (Fig. 7), indicate that defensin -1 can gain entry into the cell through the pores and possibly disrupt internal membranes to gain entry into organelles. This was evidenced by the loss of intracellular compartmentalization and by the contents causing eventual lysis, as observed in EM analysis. We suggest that cellular membrane damage, the intracellular damage caused in the membrane or organelles by internalized defensin -1, and possible downstream effects all contribute to cellular damage.

The action of human defensin -1 on T. cruzi trypomastigotes has not been investigated before. A previous study has shown that murine enteric defensin (cryptin-4), which differs from human defensin -1, does not show any trypanocidal effect on bloodstream forms of Trypanosoma brucei (22). Exposure of Giardia lamblia to defensin -1 kills this organism (2), but the mechanism that mediates the toxicity is unknown.

The fact that a sublethal concentration of defensin -1 can block T. cruzi trypomastigote infection of human cells by directly acting on the surface of the trypanosome reinforces the concept that this immune molecule plays an important direct role in interfering with T. cruzi infection.

The hallmarks of apoptosis include membrane blebbing, DNA fragmentation (7), and as recently reported, pore formation (34, 48). The fact that we show that human defensin -1 causes pore formation (Fig. 5), membrane blebbing (Fig. 3B, E, and D), and nuclear DNA and kDNA fragmentation (Fig. 8) in the trypanosome might suggest that human defensin -1 induces apoptosis in T. cruzi trypomastigotes, leading to trypanosome death (Fig. 1, 6G, and 7D). Our observations that human defensin -1 gains entry into the kinetoplast (Fig. 7A and C) and is associated with nuclear DNA (Fig. 7A) support the fact that human defensin -1 induces nuclear DNA and kDNA fragmentation (Fig. 8B).

The current model of defensin antimicrobial activity was derived from experiments using synthetic membranes and suggests that the cationic, amphipathic nature of defensins enables them to bind to anionic moieties on the membranes of select microbes and to insert into the membrane in a membrane voltage potential-dependent manner (47). Our findings showing that the trypanocidal action of human defensin -1 in trypomastigotes is completely inhibited by CCCP favor this hypothesis. The effect of depolarizing the membrane of the trypanosome with CCCP on the trypanocidal activity of defensin -1 was examined to determine if defensin-mediated trypanocidal activity is initiated on the parasite membrane. CCCP is an ionophore that dissipates the proton motive force across membranes, resulting in neutralization of the membrane potential and proton electrical potential. Since defensin -1 was not effective against T. cruzi in the absence of a membrane voltage potential, these data indicate that the trypanocidal action of defensin -1 is initiated at the membrane of the parasite.

It is thought that defensin molecules aggregate in microbial membranes to form pores through which more defensin can enter the organism to mediate intracellular damage (18, 19, 28). Our findings show that, indeed, human defensin -1 molecules insert into the trypanosome membrane to form pores with typical ring structures, as shown in Fig. 5, which facilitate entry of defensin -1 through the pores to cause further cellular damage (Fig. 7).

The fact that we found that the most sensitive areas in trypomastigotes where defensin -1 causes pore formation are the flagellar membrane of the trypanosome and the membrane that attaches the flagellum to the parasite body (Fig. 4A) suggests that these topological areas in the trypanosome could be prime targets for trypanocidal defensin peptides.

In summary, these results indicate that human defensin -1 plays an important beneficial role in reducing T. cruzi infection of human cells. Understanding the complex function of human defensins in innate immunity against human protozoan parasites has implications for the prevention and treatment of parasitic diseases and suggests that defensin -1 derivatives might be exploited for use as trypanocidal agents against T. cruzi.

 
This work was supported in part by NIH grants 1SC1 GM081168, GM 08037, GM 059994, AI 07281, AI 056667, HL 007737, MD 000104, RR 003032, and DK 20539.

We thank Michael Linde for his help with flow cytometry and Richard Edwards for his help with the design of scrambled peptides.

 
* Corresponding author. Mailing address: Department of Microbial Pathogenesis and Immune Response, School of Medicine, Meharry Medical College, 1005 Dr. D. B. Todd Jr. Blvd., Nashville, TN 37208. Phone: (615) 327-6667. Fax: (615) 327-6072. E-mail: fvillalta{at}mmc.edu

Published ahead of print on 16 July 2007.

Editor: W. A. Petri, Jr.

M.N.M. and Y.Y.K. contributed equally to this work.

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Infection and Immunity, October 2007, p. 4780-4791, Vol. 75, No. 10
0019-9567/07/$08.00+0     doi:10.1128/IAI.00557-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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