Killing of Trypanozoon Parasites by the Equine Cathelicidin eCATH1.

Trypanozoon parasites infect both humans, causing sleeping sickness, and animals, causing nagana, surra, and dourine. Control of nagana and surra depends to a great extent on chemotherapy. However, drug resistance to several of the front-line drugs is rising. Furthermore, there is no official treatment for dourine. Therefore, there is an urgent need to develop antiparasitic agents with novel modes of action. Host defense peptides have recently gained attention as promising candidates. We have previously reported that one such peptide, the equine antimicrobial peptide eCATH1, is highly active against equine Gram-positive and Gram-negative bacteria, without cytotoxicity against mammalian cells at bacteriolytic concentrations. In the present study, we show that eCATH1 exhibits an in vitro 50% inhibitory concentration (IC50) of 9.5 µM against Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum Its trypanocidal mechanism involves plasma membrane permeabilization and mitochondrial alteration based on the following data: (i) eCATH1 induces the rapid influx of the vital dye SYTOX Green; (ii) it rapidly disrupts mitochondrial membrane potential, as revealed by immunofluorescence microscopy using the fluorescent dye rhodamine 123; (iii) it severely damages the membrane and intracellular structures of the parasites as early as 15 min after exposure at 9.5 µM and 5 min after exposure at higher concentrations (19 µM), as evidenced by scanning and transmission electron microscopy. We also demonstrate that administration of eCATH1 at a dose of 10 mg/kg to T. equiperdum-infected mice delays mortality. Taken together, our findings suggest that eCATH1 is an interesting template for the development of novel therapeutic agents in the treatment of trypanosome infections.

Localization of antimicrobial peptides in the tunic of Ciona intestinalis (Ascidiacea, Tunicata) and their involvement in local inflammatory-like reactions.

Tunicates comprising a wide variety of different species synthesize antimicrobial peptides as important effector molecules of the innate immune system. Recently, two putative gene families coding for antimicrobial peptides were identified in the expressed sequence tag database of the tunicate Ciona intestinalis. Two synthetic peptides representing the cationic core region of one member of each of the families displayed potent antibacterial and antifungal activities. Moreover, the natural peptides were demonstrated to be synthesized and stored in distinct hemocyte types. Here, we investigated the presence of these natural peptides, namely Ci-MAM-A and Ci-PAP-A, in the tunic of C. intestinalis considering that the ascidian tunic is a body surface barrier exposed to constant microbial assault. Furthermore, as the tunic may represent a major route of entry for pathogen invasion after its damage we monitored the location of these peptides upon a local inflammatory-like reaction induced by injection of foreign cells. Using immunocytochemistry and electron microscopy both peptides were localized to the tunic and were massively present in granulocytes of inflamed tissue. Conclusively, antimicrobial peptides may constitute a chemical barrier within the tunic of urochordates.

Antimicrobial properties of the equine alpha-defensin DEFA1 against bacterial horse pathogens.

Defensins are small effector molecules of the innate immune system, synthesised by various organisms including plants and animals. The peptides act as endogenous antibiotics with an antimicrobial activity against a broad spectrum of microbes including bacteria, fungi and viruses. alpha-Defensins are a subgroup of the defensin family, their synthesis is limited to some tissues and furthermore to some mammalian species including the horse. Equine DEFA1 is an enteric alpha-defensin exclusively produced in Paneth cells. The peptide showed an activity against a broad spectrum of microbes, but typical pathogens of the horse were not included in the previous antimicrobial studies. Here, we report the antibacterial properties of DEFA1 against clinical isolates of typical horse pathogens including Rhodococcus equi, various streptococci strains, Salmonella choleraesuis, and Pasteurella multocida. The recombinantly expressed DEFA1 peptide exerted potent activity against these pathogenic bacteria. The highest susceptibility showed R. equi. Three genetically different strains of R. equi were killed at low micromolar concentrations, comparable with conventionally used antibiotics.

Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

Recombinant expression and purification of an enzymatically active cysteine proteinase of the protozoan parasite Entamoeba histolytica.

Cysteine proteinases and in particular cysteine proteinase 5 (EhCP5) of Entamoeba histolytica are considered important for ameba pathogenicity. To study EhCP5 in more detail a protocol was elaborated to produce considerable amounts of the enzyme in its active form. The protein was expressed in Escherichia coli as a histidine-tagged pro-enzyme and purified to homogeneity under denaturing conditions in the presence of guanidine-HCl using nickel affinity chromatography. Renaturation was performed by 100-fold dilution in a buffer containing reduced and oxidized thiols, which led to soluble but enzymatically inactive pro-enzyme. Further processing and activation was achieved in the presence of 10 mM DTT and 0.04% SDS at 37 degrees C. Recombinant enzyme (rEhCP5) was indistinguishable from native EhCP5 purified from E. histolytica lysates. Both runs in SDS-PAGE under reducing and nonreducing conditions at positions corresponding to 27 and 29 kDa, respectively, had the same pH optima and displayed similar specific activity against azocasein. Moreover, both enzymes were active against a broad spectrum of biological and synthetic substrates such as mucin, fibrinogen, collagen, human hemoglobin, bovine serum albumin, gelatin, human IgG, Z-Arg-Arg-pNA, and Z-Ala-Arg-Arg-pNA, but not against Z-Phe-Arg-pNA. The identity of rEhCP5 as a cysteine proteinase was confirmed by inhibition with specific cysteine proteinase inhibitors. In contrast, various compounds known to specifically inhibit aspartic, metallo, or serine proteinases had no effect on rEhCP5 activity.

Novel putative saposin-like proteins of Entamoeba histolytica different from amoebapores.

Amoebapores, the pore-forming proteins of Entamoeba histolytica, have been shown to play a pivotal role in the pathogenicity of the protozoan parasite. They belong to the functionally diverse family of saposin-like proteins (SAPLIPs) characterized by a conserved pattern of cysteine residues and the ability to interact with lipids. Here, we report the identification of genomic sequences encoding presumably novel SAPLIPs in E. histolytica and classify them in the structural and functional context provided by known family members. The genes of altogether 15 SAPLIPs are transcribed in the axenically cultured trophozoites as evidenced by reverse transcriptase-polymerase chain reaction. Interestingly, a remarkable sequence variety with a strong resemblance to that of known, functionally diverse SAPLIPs is present in this archaic, unicellular organism.

Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans.

Natural products are the major source of lead compounds for drugs against human pathogens. Among the first natural peptides from animals for which a potent antibacterial activity has been recognized were the cecropins. The 30- to 40-residue alpha-helical peptides display their activity by permeabilizing the membranes of bacteria. Although originally isolated from insect hemolymph, a structural and functional correlate was also found in a mammal. Here, we report on the finding that cecropin A and B from the silk moth Cecropia as well as the porcine cecropin P1 are capable of inhibiting the growth of and to kill yeast-phase Candida albicans. The peptides were tested in radial diffusion and microbroth dilution assays. They displayed potent activity against a clinical isolate as well as against defined culture strains of the pathogenic yeast but are of exceedingly low cytotoxicity towards the human cell line Jurkat. The candidacidal properties of the intensely studied molecules known to be highly active against Gram-positive and Gram-negative bacteria may renew the interest in these natural broad-spectrum peptide antibiotics and their limited cytotoxicity to human cells may be exploited for the development of topical therapeutics against pathogens resistant to classical antibiotics.

Membrane-permeabilizing polypeptides of amoebae - constituents of an archaic antimicrobial system.

Amoebae may be viewed as primitive, actively phagocytosing eukaryotic cells, many of which use bacteria as a major nutrient source. At a very archaic level, amoebae exert mechanisms which kill bacteria comparable to those found in phagocytic cells of higher organisms. Accordingly, it is tempting to suggest that the ancestors of effector cells of the innate immune system were bacteria-feeding amoebae and that their molecular armament is ancient. Here, we summarize the characteristics of antimicrobial and cytolytic 77-residue polypeptides from the protozoon Entamoeba histolytica for which correlates were found in effector cells of the mammalian immune systems. Based on the current knowledge about these small membrane-destabilizing proteins of phylogenetically extremely diverse origin, we discuss similarities and differences in their structure and activities.

Two novel calcium-binding proteins from cytoplasmic granules of the protozoan parasite Entamoeba histolytica.

We report on the molecular characterisation of two novel granule proteins of the protozoon and human pathogen Entamoeba histolytica. The proteins, which were named grainin 1 and 2, show a considerable structural similarity to calcium-binding proteins, particularly within EF-hand motifs. Each grainin possesses three of these putative calcium-binding sites. Based on careful inspection of known structures of protein families containing EF-hands, a domain of grainin 1 covering two EF-hand motifs was modeled by homology. Calcium-binding activity of grainins was demonstrated by two independent methods. These granule proteins may be implicated in functions vital for the primitive phagocyte and destructive parasite such as control of endocytotic pathways and granule discharge.

Granulysin, a T cell product, kills bacteria by altering membrane permeability.

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Evidence that hyaluronidase is not involved in tissue invasion of the protozoan parasite Entamoeba histolytica.

As previous reports suggested that a hyaluronidase is involved in tissue invasion of Entamoeba histolytica, we searched for such an activity in trophozoite extracts. A hyaluronidase activity was not detectable in long-term cultures or in amoebae freshly passaged through a gerbil liver, as evidenced by four different techniques.

Antisense inhibition of amoebapore expression in Entamoeba histolytica causes a decrease in amoebic virulence.

Amoebapores have been proposed to be a major pathogenicity factor of the protozoan parasite Entamoeba histolytica, which is responsible for the killing of target cells. These 77-residue peptides are structural and functional analogues of NK-lysin and granulysin of porcine and human cytotoxic lymphocytes. Inhibition of amoebapore gene expression in amoebae was obtained following transfection with a hybrid plasmid construct (pAP-R2) containing the Neo resistance gene and the gene coding for amoebapore A, including its 5' and 3' untranslated region (UTR) sequences, in reverse orientation under a promoter (g34) taken from one of the E. histolytica ribosomal protein (RP-L21) gene copies. Transfectants of virulent E. histolytica strain HM-1:IMSS, in which the expression of amoebapore was inhibited by approximately 60%, were significantly less pathogenic. Cytopathic and cytolytic activities of viable trophozoites against mammalian nucleated cells, as well as lysis of red blood cells, were markedly inhibited. Moreover, trophozoite extracts of pAP-R2 transfectant displayed lower pore-forming activity and were less potent in inhibiting bacterial growth compared with controls. Notably, liver abscess formation in hamsters by the pAP-R2 transfectant was substantially impaired. These results demonstrate for the first time that amoebapore is one of the pathogenicity factors by which trophozoites of E. histolytica exert their remarkable cytolytic and tissue destructive activity.

Pore-forming peptides of Entamoeba dispar. Similarity and divergence to amoebapores in structure, expression and activity.

Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.

Comparative modeling of amoebapores and granulysin based on the NK-lysin structure-structural and functional implications.

Amoebapores, the pore-forming polypeptides of the protozoan parasite Entamoeba histolytica, and effector proteins of porcine and human lymphocytes, namely NK-lysin and granulysin, reveal a substantial sequence similarity despite their enormous evolutionary distance. Moreover, all these polypeptides display antibacterial activity and are in higher concentrations cytolytic to eukaryotic cells. The recently solved NMR structure of NK-lysin enabled us to build the three dimensional structures of amoebapores and granulysin by comparative modeling. The generated models revealed the expected similarities, but also fundamental differences with respect to charge distribution, hydrophobicity and core packing. The combination of these structural properties and known biochemical data provides insight in the different membrane-interacting mechanisms of the proteins. For amoebapores, exposed hydrophobic grooves and a locally loosely packed protein core may allow a rearrangement of the protein and therefore may account for its ability to penetrate the target membrane and to form defined ion channels in planar lipid bilayers. In contrast, the structural features of NK-lysin and granulysin appear to be suitable for a membrane-perturbing mode of action rather than for channel formation.

Antimicrobial and cytolytic polypeptides of amoeboid protozoa--effector molecules of primitive phagocytes.

Amoebae are primitive, actively phagocytosing eukaryotic cells, many of which use bacteria as a major nutrient source. One may suppose that amoebae possess an array of potent antimicrobial molecules acting in synergy to combat bacterial growth inside their phagosomes. Lysosome-like granular vesicles of Entamoeba histolytica contain a family of 77-residue peptides with a compact alpha-helical, disulfide-bonded fold. These polypeptides, named amoebapores, exhibit antibacterial and cytolytic activity by forming pores in membranes of various origin. It is of particular interest that amoebapores are structurally and functionally most similar to polypeptides of mammalian cytotoxic lymphocytes. In addition, amoebic granules contain bacteriolytic proteins with lysozyme-like properties. Some amoebic polypeptides may represent archaic analogs of effector molecules from invertebrates and vertebrates.

Candidacidal activity of shortened synthetic analogs of amoebapores and NK-lysin.

Natural antimicrobial peptides and synthetic analogs thereof have emerged as compounds with potentially significant therapeutical application against human pathogens. Amoebapores are 77-residue peptides with cytolytic and antibacterial activity considered to act by forming ion channels in cytoplasmic membranes of the victim cells. A functionally and structurally similar peptide named NK-lysin exists in mammalian lymphocytes. Several synthetic analogs of amoebapores and NK-lysin, which are substantially reduced in size compared to the parent molecules, were tested for their ability to inhibit the growth of and to kill Candida albicans. Some of the peptides displayed potent activity against a clinical isolate as well as against defined culture strains. Among the most active peptides found are some shortened substitution analogs of amoebapore C and a cationic core region of NK-lysin. As these peptides are also highly active against Gram-positive and Gram-negative bacteria but are of low cytotoxicity towards a human keratinocyte cell line they may provide promising templates for the design of broad-spectrum peptide antibiotics.

Molecular characterization of an exceptionally acidic lysozyme-like protein from the protozoon Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica contains a second antibacterial protein with lysozyme-like properties. The newly recognized bacteriolytic protein was purified from extracts of amoebic trophozoites to allow amino-terminal sequencing. Subsequent molecular cloning revealed that it is an isoform of the amoeba lysozyme described previously but also demonstrated a substantial sequence divergence of the two forms. As lysozymes typically are basic proteins, the novel amoebic protein differs markedly in having a pI of 4.5. There is no significant similarity of both amoeba lysozymes with any bacteriolytic protein of other organisms reported so far; however, striking sequence identity is found with predicted gene products of unknown function derived from the bacteria-feeding nematode Caenorhabditis elegans.

Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica.

Major pathogenic functions of Entamoeba histolytica involved in destruction of host tissues are the degradation of extracellular matrix proteins mediated by secreted cysteine proteinases and contact-dependent killing of host cells via membrane-active factors. A soluble protein with an affinity for membranes was purified from amoebic extracts to apparent homogeneity. N-terminal sequencing and subsequent molecular cloning of the factor revealed that it is a member of the cysteine proteinase family of E. histolytica, which we termed CP5. Further experiments with the purified protein showed that it has potent proteolytic activity that is abrogated in the presence of inhibitors specific for cysteine proteinases. The enzyme firmly associates with membranes retaining its proteolytic activity and it produces cytopathic effects on cultured monolayers. A model of the three-dimensional structure of CP5 revealed the presence of a hydrophobic patch that may account for the potential of the protein to associate with membranes. Immunocytochemical localization of the enzyme to the surface of the amoeba in combination with the recent finding that the gene encoding CP5 is missing in the closely related but non-pathogenic Entamoeba dispar suggests a potential role of the protein in host tissue destruction of E. histolytica.