Matthias Leippe

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 α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α 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 β-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.

Uncovering the evolutionary history of innate immunity: The simple metazoan Hydra uses epithelial cells for host defence

Although many properties of the innate immune system are shared among multicellular animals, the evolutionary origin remains poorly understood. Here we characterize the innate immune system in Hydra, one of the simplest multicellular animals known. In the complete absence of both protective mechanical barriers and mobile phagocytes, Hydras epithelium is remarkably well equipped with potent antimicrobial peptides to prevent pathogen infection. Induction of antimicrobial peptide production is mediated by the interaction of a leucine-rich repeats (LRRs) domain containing protein with a TIR-domain containing protein lacking LRRs. Conventional Toll-like receptors (TLRs) are absent in the Hydra genome. Our findings support the hypothesis that the epithelium represents the ancient system of host defence.

ENTAMOEBA HISTOLYTICA AND ENTAMOEBA DISPAR: DIFFERENCES IN NUMBERS AND EXPRESSION OF CYSTEINE PROTEINASE GENES

In order to identify molecules that might be responsible for the difference in pathogenicity between the two closely related protozoan parasites Entamoeba histolytica and Entamoeba dispar, we focussed on cysteine proteinases because this class of enzymes has been considered important for pathogenic tissue destruction. By screening a genomic library derived from an E. histolytica isolate, a total of six distinct genes (ehcp1–ehcp6) encoding typical prepro‐forms of cysteine proteinases were identified which differed from each other by 40% to 85% of their nucleotide sequences. Three of these genes, ehcp1, ehcp2, and ehcp5, which exhibited high levels of expression, were found to be responsible for approximately 90% of cysteine proteinase transcripts, whereas the remaining three were either not or only marginally expressed. Expression of the different genes directly correlated with the level of activity of the respective enzymes in trophozoite lysates. Purification of the enzymes and N‐terminal sequencing revealed that virtually all cysteine proteinase activity of E. histolytica can be attributed to three enzymes namely EhCP1, EhCP2 and EhCP5. Southern blot analysis indicated that just two of these abundantly expressed genes are missing in E. dispar. On the other hand, genes analogous to four of the six genes identified in E.histolytica were found to be present in E. dispar, but only two of these are expressed within the trophozoite stage.

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.

Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore bacterial cytoplasmic membranes

Three peptides with pore‐forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed‐phase high‐performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic α‐helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore‐forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram‐positive bacteria. The amoebapores represent a distinct family of membrane‐active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.

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.

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 ~ 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.

Hydramacin-1, Structure and Antibacterial Activity of a Protein from the Basal Metazoan Hydra

Hydramacin-1 is a novel antimicrobial protein recently discovered during investigations of the epithelial defense of the ancient metazoan Hydra. The amino acid sequence of hydramacin-1 shows no sequence homology to any known antimicrobial proteins. Determination of the solution structure revealed that hydramacin-1 possesses a disulfide bridge-stabilized αβ motif. This motif is the common scaffold of the knottin protein fold. The structurally closest relatives are the scorpion oxin-like superfamily. Within this superfamily hydramacin-1 establishes a new family of proteins that all share antimicrobial activity. Hydramacin-1 is potently active against Gram-positive and Gram-negative bacteria including multi-resistant human pathogenic strains. It leads to aggregation of bacteria as an initial step of its bactericidal mechanism. Aggregated cells are connected via electron-dense contacts and adopt a thorn apple-like morphology. Analysis of the hydramacin-1 structure revealed an unusual distribution of amino acid side chains on the surface. A belt of positively charged residues is sandwiched by two hydrophobic areas. Based on this characteristic surface feature and on biophysical analysis of protein-membrane interactions, we propose a model that describes the aggregation effect exhibited by hydramacin-1.

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.

ANCIENT WEAPONS : NK-LYSIN, IS A MAMMALIAN HOMOLOG TO PORE-FORMING PEPTIDES OF A PROTOZOAN PARASITE

Peptides from natural sources with biological activity toward bacteria and tumor cells are attracting increasing attention (Boman, 1991; Zasloff, 1992). In a time of ongoing search for such factors throughout the animal kingdom (Boman, 1995) it is not amazing that relatives of peptides once discovered as unique in one organism are subsequently found in other species. Nevertheless, I was surprised to find that NK-lysin, the novel effector molecule recently identified in porcine cytotoxic lymphocytes (An-dersson et al., 1995), structurally and functionally resembles amoebapore. The latter molecule is a polypeptide that forms ion channels in target cell membranes (for review see Leippe and Miiller-Eberhard, 1994). Amoebapore was isolated from Entamoeba histolytica (Leippe et al., 1991), a protozoan parasite that causes life-threatening disease in humans. Interestingly, this organism is considered a living relic of the earliest phase of eukaryotic evolution , as revealed by protein sequence data (Hasegawa et al., 1993). Andersson et al. (1995) describe the isolation from por-cine intestinal tissues and the molecular cloning of NK-lysin, a 78 residue peptide with antibacterial activity. They report a moderate sequence divergence (seven residues) between the purified NK-lysin and the translation products of the two identical cDNA clones isolated. This may reflect the existence of NK-lysin isoforms or may be due to allelic variations in pig populations, as suggested by the authors. Since the purified peptide also shows lytic activity toward tumor cells and is synthesized by cytotoxic T cells and natural killer cells, it has been assumed that NK-lysin is an additional component of the cytotoxic armament of these cells. Likewise, amoebapore has been considered the primary candidate for mediating the extraordinary cytolytic activity of the protozoan parasite. The 77 residue peptide displays cytotoxic and antibacterial activity (Leippe et al., 1992, 1994a). Recently, we discovered a family of amoebapores in amoebic cytoplasmic granules consisting of three iso-forms with considerable divergence in their primary structure but similar properties (Leippe et al., 1994b). Until recently, researchers have suggested, as the aforemen-tioned authors have with regard to NK-lysin, that structurally the amoebapores represent a unique class of membrane active peptides that functionally may have analogs in the well-known defensins, disulfide-bonded antibacte-rial and cytotoxic peptides from the cytoplasmic granules of neutrophils (Lehrer et al., 1993). It is therefore important to notice the obvious structural homology between the amoebic peptides and NK-lysin (Figure 1). An initial alignment was produced using the program BESTFIT (University of Wisconsin Genetics …