Aspects of the Biology of Small Free-Living and Facultative Parasitic Amoebae

Zoology, Parasitology

Mat Amin, Nakisah

University:University of Glasgow

关键词: Zoology, Parasitology;   

Others  :  http://theses.gla.ac.uk/75827/1/13818563.pdf

来源: University of Glasgow

【 摘 要 】

Aspects of the biology of small, naked, free-living amoebae which include some facultatively parasitic forms have been studied with emphasis on two selected genera, Naegleria and Hartmannella. In the former genus, non-pathogenic Naegleria gruheri (CCAP strain 1518/1 A), as well as the closely related pathogenic Naegleria fowleri, have been investigated. A combination of fluorescence and electron microscopy techniques have been employed to investigate mitosis in N. gruberi with special reference to the distribution of chromosomes/DNA and the possible existence of the microtubule organising centres. In transmission electron micrographs, the nucleolus appeared to persist during nuclear divison but its electron density changed during the course of nuclear division. During most of stages of nuclear division, chromosomal elements could not be distinguished with certainty from nucleolar material. Possible chromosomal structures could be observed as electron dense spherical profiles or as fluorescent dots only when the nucleus was at metaphase or anaphase stages.Intranuclear microtubules have been detected by transmission electron microscopy in most stages of nuclear division in Naegleria. Their profile length seemed to vary according to the stage of nuclear division and it is argued that this might reflect their function; the long microtubule profiles might represent continuous fibres responsible for the elongation of the nucleus in anaphase-telophase and the short microtubules may be important in transporting chromosomes to the poles i.e. the kinetochore microtubules. Both kinds of microtubule were observed in associations with the poles; the kinetochore microtubules were most evident when the nucleus was in metaphase. By both transmission microscopy and light microscopy, stages in both anaphase A (the moving apart of nuclear material to the daughter cells) and in anaphase B (the elongation of microtubules in order to move the poles apart) could be observed clearly. Colchicine-treatment was used to induce synchronous division in Naegleria cells. During colchicine treatment, Naegleria cells stopped dividing but resumed division almost synchronously ~1 to 2 h after the drug was removed from the medium. Staining of such cells by a-tubulin antibody makes detection of microtubule structures in cells possible. A centrosome with associated microtubules was found by immunofluorescence using anti-tubulin-conjugated antisera to be present in the cytoplasm of Naegleria during nuclear division and this finding contradicts the previous idea that this organelle does not exist in Naegleria. The centrosome appeared to divide during prophase but it could not be detected later at metaphase- telophase. Moreover, transmission electron microscopy did not detect this organelle, though crystalloid structures possibly induced by the colchicine treatment were found near the nuclear envelope in early stages of division. The presence of proteinases in Naegleria spp. and their relation to the life cycle was also investigated. These enzymes were found in all three stages of the amoeba's life cycle; trophozoite, cyst and flagellate. At pH 5.5 in the presence of DTT (dithiothreitol) at least 5 enzymes could be detected in gelatin SDS-PAGE gels for actively multiplying trophozoites; they were named bands A, B, C and DE enzymes . The apparent of band A was -200 kDa, band B was -148 kDa, band C was -116 kDa, band D was -98 kDa and band E was -92 kDa. Bands DE were the most prominent in gels. Flagellates appeared to posses enzymes A and DE, and cysts only band C. Amoebae grown on agar with living bacteria exhibited fewer proteinase bands than those grown in axenic medium; bacterised amoebae lacked bands B and C. The individual proteinases present in Naegleria were characterised on the basis of inhibitor studies, apparent molecular weight, substrate preferences and pH optimum. Band A which was inconsistently observed in gelatin SDS-PAGE gels, had a wide pH range of activity (pH 5.5 to 8.0) but its acitvity was higher at alkaline pH and inhibited by APMSF (4-(amidinophenyl)methanesulphonyl fluoride). Bands B and C were active only at slightly acidic pH (5.5 to 6.0), indicating they are lysosomal in origin; their activity was DTT-dependent, inhibited by E-64 (L-3-carboxy-2,3-trans-epoxypriopionyl-leucylamido-(4-guanidino) butane) and markedly inhibited by antipain, so they could be cysteine proteinases. Doublet DE enzymes, which were membrane-associated, exhibited bimodal activities; at lower pH, their activities were stimulated by DTT but at alkaline pH their activities were higher and not stimulated by DTT. Their activities were inhibited by APMSF, EDTA (ethylenediaminetetraacetic acid) , E-64 and antipain. Band B and C enzymes hydrolysed only the fluorogenic peptidyl-amido methylcoumarin substrate, H-Pro- Phe-Arg-NHMec, in contrast to doublet DE enzymes which hydrolysed substrates H- Pro-Phe-Arg-NHMec, Z-Pro-Arg-NHMec, Bz-Phe-Val-Arg-NHMec and Z-Arg- Arg-NHMec. A similar pattern of proteinases (in SDS-PAGE gelatin gels) was expressed by different stages of growth of amoebae in axenic culture but the intensity of each band appeared to be different and this may have been due to the condition of the trophozoites in cultures during growth.(Abstract shortened by ProQuest.).