Contribution of Electron Microscopy to the Study of Parasitic Protozoa

W. de Souza Instituto de Biofisica Carlos Chagas Filho, UFRJj Rio de Janeiro, Brazil

Tropical diseases represent a serious constraint on human development, imposing on 500 million people their heavy burden of sickness and incapacity, and killing some two millon people each year (World Health Organization,1995). Some of these tropical diseases are caused by parasitic protozoa which comprise a large number of species some of which are responsible for diseases with a high prevalence, such as (a) malaria, caused by members of the. genus Plasmodium, affecting about 500 millon people per year, (b) leishmaniasis, caused by members of the genus Leishmania, affecting about 16 millon people, (c) Chagas’ disease, caused by Trypanosoma cruzi, affecting about 14 millon people in Latin America, (d) sleepness disease, caused by members of the Trypanosoma brucei group, affecting about 3 millon people in Africa, (e) amebiais, caused by the anerobic protozoa Entamoeba histolytica, affecting about 16 millon people throughout the world. Some of these protozoa have a complex life cycle with developmental stages found in vertebrate and invertebrate hosts, as occur with Tcruzi and Leishmania. Others developed the ability to penetrate into mammalian cells, developing either in direct contact with cytoplasmic structures (T. cruzi) or within modified endocytic vacuoles of the host cell, known as parasitophorous vacuole ( Toxoplasma, Leishmania) . Still others only live in oxygen-poor media, as occur with Trichomonas, Entamoeba and Giardia. The analysis of the fine strucuture of these protozoa using several techniques associated with transmission electron microscopy revealed that some of them present organelles found in all eucaryotic cells, like mitochondria, peroxisomes, Golgi complex, etc. Others, do no present some of these structures. Still others show the presence of specialized organeles such as the kinetoplast and glycosome in protozoa of the Kinetoplastida order, and peripheral vesicle in Giardia. In addition they also present special cytoskeletal structures, such as the sub-pellicular microtubules and the paraflagellar rod in trypanosomatids, the axostyle and the costae in trichomonads. In this communication we will review some of the basic aspects of these structures. The kinetoplast is a compact array of extranuclear DNA associated with proteins, concentrated in the portion of the mitochondrion located close to the flagellar basal body. Is is formed by concatenated minicircles and peripheral maxicircles. Its morphology varies according to the parasite developmental stage. The glycosome is found in all members of the Kinetoplastida order. Usually it appears as a membrane bounded and rounded organelle with a homogenous matrix. In some species it may present an elongated form, as observed after three- dimensional reconstruction of the whole cell. Most of the time they are randomly distributed throughout the cell. Occasionally, however, they are closely associated to each other. In trypanosomatids which harbour an endosymbiont a close association between glycosomes and the ensosymbiont is observed. Biochemical studies have shown that most of the enzymes of the glycolytic pathway, which are usually localized in the cytosol of mammal cells, concentrate in the glycosome of trypanosomatids. These enzymes present a high pHi and can be localized in cells fixed in glutaraldehyde and incubated for a few hours in the presence of ethanolic phosphotungstic acid before embedding, a procedure usually employed to localize basic proteins. The glycosome is a specialized peroxisome. Cytochemical studies have shown the presence of catalase in the glycosome of some trypanosomatids. The peripheral vacuoles are found at the dorsal region of Giardia. Using different electrondense tracers we could show that macromolecules ingested via an endocytic process are localized in a few minutes in these vacuoles, remaining in these structures for several hours. In addition acid phosphatase, a well known marker of lysosomes, is also found in the vacuole, thus suggesting its lysosomal nature.Using three-dimensional reconstruction technique and electron tomography of cells incubated in cytochemical media for detection of acid phosphatase and glucose-6- phosphatase results were obtained suggesting that Giardia presents a primitive endo- lysosomal system in a single compartment. The cytoskeleton of trypanosomatids is mainly composed by sub-pellicular microtubulles arranged in a parallel array, running throughout the cell. They are localized below the plasma membrane and are connected to each other, as well as to profiles of the endoplasmic reticulum and the inner face of the plasma membrane, via small filamentous structures. The use of the quick freeze, freeze-tiacture, deep- etching and rotary replication technique revealed details of the cytoskeleton of trypanosomatids. No other cytoskeletal structures were found in the cytoplasm, except some filaments which connect the kinetoplast to the basal body. One characteristic feature of the flagellum of trypanosomatids is the presence of a complex array of filamentous structures, known as the paraflagellar or paraxial rod, located at the side of a typical axoneme. Thick and thin filaments were seen organized in a complex array. Special Y-shaped bridges connect the filaments to axonemal microtubules. This structure is not observed in trypanosomatids which harbour a cytoplasmic endosymbiont. The axostyle found in trichomonads is formed by a layer of microtubules associated to each other by filamentous bridges. The costae is a complex array of globular and filamentous proteins, which can be visualized in replicas of quick f&en, freeze- fractured and deep etched cells. Sunnorted bv PRONEX, FINEP. CNPa and FAPERJ