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M Leippe

Publications and source records attributed to M Leippe.

At least 19 recordsLinked to original sources
PubMed2002

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.

Animals↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Journal Article↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

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.

Amino Acid Motifs↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1997

Amoebapores.

The enormous cytolytic potential of Entamoeba histolytica appeals to parasitologists and immunologists because it kills target cells in a contact-dependent reaction resembling that of cytotoxic lymphocytes. In this review, Matthias Leippe summarizes what is currently known about a family of pore-forming peptides termed 'amoebapores', to which the cytolytic effect has been attributed, and describes the structural and functional properties of these potent factors, as well as their structure-activity relationships. Finally, a comparison is made with effector molecules of the mammalian defensive system.

Journal Article↗
PubMed1997

Molecular modeling of amoebapore and NK-lysin: a four-alpha-helix bundle motif of cytolytic peptides from distantly related organisms.

BACKGROUND: Amoebapore of the protozoan Entamoeba histolytica and NK-lysin of porcine cytotoxic lymphocytes are effector peptides from organisms separated extremely early in their evolutionary paths. The peptides intrigued us, however, with indications of some functional similarity. We thus wanted to derive and compare predictions for their as yet unknown three-dimensional structures as a guide for and to be tested by further experiments. RESULTS: Molecular models were generated by use of a genetic algorithm that selects according to basic protein structure principles exploiting available information such as the primary structures, secondary structure predictions and positions of disulfide bonds. Topological differences aside, the structural motif of an antiparallel four-alpha-helix bundle with adjacent connections and intramolecular crosslinks is predicted for both types of peptides. It combines the feature of amphipathic alpha-helices with a disulfide-bonded compact structure known from the beta-sheeted defensins and small toxins. CONCLUSIONS: The models presented here strengthen the notion that amoebapore and NK-lysin are particular among cytolytic and antibacterial polypeptides and share a similar function and structural motif. They also allow experimental testing and a better comparison of the two proteins in view of the predicted similarities and differences of their respective folds.

Amino Acid Sequence↗
PubMed1997

Necrosis versus apoptosis as the mechanism of target cell death induced by Entamoeba histolytica.

The human pathogen Entamoeba histolytica is known to kill a variety of host cells, including leukocytes. Using human myeloid cells as targets, we studied whether cytotoxicity of amoebic trophozoites in vitro is equivalent to the induction of apoptosis or whether these target cells die via necrosis. Based upon morphological criteria, incubation of target cells with amoebae resulted in necrosis, with cell swelling, rupture of plasma membrane, and release of cell contents including nucleic acids being detected by light and transmission electron microscopy. On the other hand, the characteristic features of apoptosis such as cell shrinking, surface blebbing, and chromatin condensation were not observed. Moreover, internucleosomal fragmentation of genomic DNA within target cells as a characteristic feature of apoptotic cell death did not occur as judged by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique in combination with flow cytometry. Consistently, cleavage of DNA was detectable upon agarose gel electrophoresis only after a substantial part of the target cell population had already been lysed. We also analyzed the mechanism of cell death induced by amoebapores, pore-forming peptides and primary candidate molecules for mediating the cytolytic activity of E. histolytica. At a time point at which the majority of target cells showed membrane injury upon incubation with purified amoebapores, no DNA degradation was detectable in the victim cells. The data suggest that the target cells used in our study undergo necrosis rather than apoptosis when they are killed by viable trophozoites as well as by isolated amoebapores.

Animals↗