Motile unicellular eukaryotes (Protozoa)
“Phylum Sarcomastigophora” is an umbrella term for protozoans that move either by one or more flagella (“subphylum Mastigophora or Flagellata” – the flagellates) or by pseudopods (“subphylum Sarcodina” – the amoeboids). The quotations marks indicate that all listed groups are no longer recognized as taxons and, with the possible exclusion of amoeboids, are thought to be polyphyletic, i.e. to join together groups with fairly distant evolutionary relationship. However, this obsolete classification is convenient for our course and therefore we are still using it, at least for now.
Phylum Sarcomastigophora includes both free-living and parasite protozoans. The cell size is usually between 10 and 30 micrometers. Like other protozoans, they reproduce by closed mitosis, i.e. nuclear envelope remains intact throughout division and all movements of chromosomes happen within the nucleus.
The group Kinetoplastida, listed as order by some authors and as class by others, includes unicellular eukaryotes moving by one or two flagella (in all medically important species – one). The nucleus is diploid. The cell possesses a single mitochondrion located near the basal body of the flagellum. The mitochondrial DNA is unusually complex, organized in a set of interlocked circular molecules. They form a particle called kinetoplast, after which the group is named. The kinetoplastids reproduce by closed mitosis (i.e. the nuclear envelope never breaks down). The chromosomes, which do not condense, are segregated by an intranuclear spindle formed independently from the basal body. In most kinetoplastids, sexual process has not been identified.
The name Trypanosoma derives from trypanon (Greek) – borer and soma – body. In turn, the popular histological dye trypan blue is named so because of its ability to kill trypanosomes.
The cell of trypanosomes is elongated, spindle-shaped and easily bends during movement. The nucleus is located in its broadest central part. The flagellum protrudes from the anterior end (compare with animal spermatozoa). However, the basal body is located in a more posterior part of the cell. (“Anterior” and “posterior” are defined based on the direction of motion.)
Before reaching the anterior end of the cell, the axoneme (axial apparatus) of flagellum runs parallel to the main cellular mass and supports a fin-like extension of cytoplasm called undulating membrane. It is an efficient, although energy-consuming, motility organelle.
Schematic drawing of a trypanosome with trypomastigote morphology
In the drawing, the basal body is shown near the posterior end of the cell, and the undulating membrane is quite long. This life form is called trypomastigote and is typical for trypanosomes living in the extracellular fluids of a vertebrate host. Generally, flagellated forms are extracellular parasites – in intracellular environment, flagellum is useless. The trypomastigote has the typical size for sarcomastigophoran cell, 10 – 30 micrometers long.
Trypanosomes have a peculiar strategy to evade the host’s immune system: They change their dominant antigen. Their genome contains multiple genes (about 1000, or 10% of all genes) for surface glycoproteins. At any moment, however, only one of these genes is active, and the glycoprotein encoded by it is covering the parasite surface. The active gene is periodically changed, resulting in new antigenic properties of the cell and, hence, resistance to the antibodies developed against the previous surface glycoprotein.
Trypanosomes feed by absorbing nutrients from the body fluids of their host.
With one exception (T. equiperdum), trypanosomes have a life cycle alternating between two hosts – a vertebrate and a blood-sucking insect. In the insect, sexual reproduction is thought to occur by meiosis and fertilization, but data are still scarse.
In our course, we describe the trypanosome morphology on Trypanosoma equiperdum. This species actually has no medical importance because it does not affect humans. It is a horse parasite (“equiperdum” is from equus – horse) and is important in veterinary parasitology. The disease caused by it is called dourine or covering sickness. Horses are infected by sexual intercourse. T. equiperdum is the only trypanosome transmitted sexually. It lives as an extracellular parasite in blood plasma and has trypomastigote morphology.
Giemsa-stained blood smear from a mouse experimentally infected with T. equiperdum. The nucleus, flagellum and undulating membrane are well visible. The purple dot seen near the posterior end of some cells is the kinetoplast.
Trypanosoma equiperdum is included in our course because it is morphologically very similar to the medically important species Trypanosoma brucei, the pathogen causing sleeping sickness.
Trypanosoma brucei is found in sub-Saharan
Africa. It has three subspecies: T. brucei brucei, T. brucei gambiense and T. brucei rhodesiense. The first subspecies, T. brucei brucei, is destroyed by human innate immunity and therefore is not pathogenic for humans. It infects other mammals, causing a disease called nagana. T. brucei gambiense and T. brucei rhodesiense infect humans, causing African trypanosomiasis (sleeping sickness). T. brucei gambiense causes slow onset chronic trypanosomiasis and humans are its main vertebrate host. T. brucei rhodesiense causes fast onset acute trypanosomiasis and uses as hosts different ungulates and other non-human mammals. For this parasite, humans are accidental hosts.
Trypanosoma brucei has a life cycle typical for the genus. It includes a vertebrate host (often called reservoir, especially when non-human) and an invertebrate host (blood-sucking arthropod, often called vector). For T. brucei, the vertebrate host is a mammal and the vector is the tse-tse fly Glossina (order Diptera). The distribution of the disease in Sub-Saharan Africa matches that of the vector.
In the mammal, the trypanosome is in the trypomastigote form we already know. In the insect vector, it has another morphology called epimastigote form. The difference is that in the epimastigote, the basal body is located between the nucleus and the anterior end of the cell and, hence, the undulating membrane is much shorter than for trypomastigotes.
When an infected tse-tse fly bites a human, trypanosomes from the saliva are inoculated into the wound. They first multiply in the blood plasma. Then, several weeks after infection with T. brucei rhodesiense and several months or even years after infection with T. brucei gambiense, the parasite spreads to the cerebrospinal liquor, invading the central nervous system. Severe headache appears. Circadian regulation of sleep is disturbed, patients tend to sleep at daytime and stay awake at night. If untreated, the disease progresses, leading to personality changes, impaired mental functions, coma and death.
Sleeping sickness is a major public health problem in
Africa. Its prevalence is currently rising.
Trypanosoma cruzi is found in Central and
South Americaand causes Chagas disease (American trypanosomiasis). The disease is named after Carlos Chagas, a Brazilian doctor who first discovered the disease in 1909.
Vertebrate hosts of T. cruzi include a large range of mammals. Humans have joined them relatively recently, after migrating to the
New World. The invertebrate host (vector) is the “kissing bug” Triatoma (insect belonging to order Hemiptera, family Reduviidae).
In the insect, the trypanosome has epimastigote morphology. It multiplies in midgut. When ready to infect the vertebrate host, it moves to the hindgut and transforms into trypomastigote. Unlike T. brucei, it is not inoculated into the mammal by saliva but is deposited on the skin with vector excrements (the insect defecates while sucking blood) and then enters the body through the wound.
In the mammalian host, T. cruzi exists in two morphologies: extracellular trypomastigote similar to that of T. equiperdum and T. brucei; and a small form (2-4 micrometers) without a flagellum called amastigote and adapted for intracellular parasitism. The parasite can invade a wide range of host cells – macrophages, muscle fibers, fibroblasts, epithelial cells. It penetrates the host cell by “hijacking” a pathway of vesicular traffic normally used to repair lesions in cell membrane. Because cardiac muscle cells actively use this pathway, they are preferentially used as host cells by T. cruzi, leading to cardiomyopathy. Gastrointestinal tract (oesophagus and intestines) and central nervous systems are also affected.
Once inside its host cell, the trypomastigote transforms into amastigote and multiplies by mitosis. Daughter cells turn again into trypomastigotes and burst from the host cell back into the bloodstream.
Life cycle of T. cruzi. Image courtesy of CDC:
Clinically, the Chagas disease begins with an acute phase, then becomes chronic and lasts many years. If untreated, it can even be fatal. Its prevalence is falling due to vector control, but it still infects millions of people.
Leishmania, named after the Scottish pathologist William Leishman, is a parasite related to Trypanosoma and resembling it in many respects. The life cycle includes a vertebrate host (typically canid or rodent mammal, can also be human) and an invertebrate host (vector) – the sandfly Phlebotomus, a dipteran insect. Only the female sandfly sucks blood and can be host and vector of Leishmania.
In the insect vector, Leishmania exists in the so-called promastigote form. The basal body is at the anterior end of the cell and the flagellum comes out free; there is no undulating membrane. Otherwise, the morphology and size (14-20 micrometers) is similar to that of trypanosomes. The cell is spindle-shaped, the nucleus is in its broadest part, not far from the middle. The cytoskeleton is more rigid than in Trypanosoma, so we do not see the cell bending. This form, similarly to the trypomastigote of trypanosomes, is adapted for extracellular parasitism.
L. donovani promastigotes obtained by culture in a medium resembling the conditions in the insect host. The flagella are difficult to see in this photo
The infected Phlebotomus harbours the Leishmania promastigotes in its anterior gut, pharynx and proboscis. When sucking blood from a mammal, it inoculates the promastigotes into the wound. In its vertebrate host, Leishmania is an intracellular parasite. Unlike T. cruzi and the apicomplexan parasites that we shall discuss later, Leishmania has no mechanism to actively penetrate the host cell. Therefore, it uses as host cells those who themselves bring it to their cytoplasm, i.e. phagocytes. It lets itself to be engulfed by macrophages and their relations collectively known as mononuclear phagocytes. We could suppose that a parasite restricted to mononuclear phagocytes would not be very dangerous, but in reality the infection of these cells causes widespread damage to the surrounding tissue and has severe consequences for the organism (see below the three types of leishmaniasis).
Once inside the host cell, Leishmania dramatically transforms its morphology, reducing its size and losing its flagellum, and turns into amastigote forms adapted for intracellular parasitism. Leishmania amastigotes, similarly to those of T. cruzi, are 5 to 10 times smaller than the promastigotes and are among the smallest eukaryotic cells – 2-5 micrometers. Of course, an intracellular parasite must be small to fit inside the host cell.
L. donovani amastigotes in liver section of an experimentally infected mouse. The parasite cells, visible as small brown ovals as a result of a peculiar staining protocol, are clustered in two infected host cells (left of center and bottom right)
The phagocyte of course has the “intention” to kill Leishmania, but the parasite resists killing. Moreover, it takes control over the phagocyte, obtaining nutrients and dividing inside its cytoplasm until the infected cell ruptures. The released amastigotes are engulfed by other phagocytes and restart the cycle. Alternatively, they may be sucked by a female Phlebotomus, where they will turn into promastigotes. Unlike T. cruzi, Leishmania does not produce any flagellated forms in the vertebrate host.
Life cycle of Leishmania. Image courtesy of CDC:
Similarly to trypanosomes, Leishmania is thought to have sexual reproduction in its insect host Phlebotomus.
There are three species of Leishmania (to be precise, complexes of species) pathogenic for humans. They cause three distinct types of leishmaniasis.
Leishmania donovani complex
Leishmania donovani and related species are found in
Asia, Africaand the Mediterranean region. They cause visceral leishmaniasis, also known as kala-azar or dumdum fever. Unlike other leishmanias, members of L. donovani complex are quickly cleared from the site of infection and no local lesion is produced. However, the parasites spread to internal organs – spleen, liver, lymph nodes, bone marrow, intestinal mucosa. The patient suffers of fever and muscle wasting, the liver and spleen become progressively enlarged. Untreated visceral leishmaniasis is fatal.
Leishmania tropica complex
The distribution of Leishmania tropica complex overlaps with that of L. donovani complex. L. tropica and related species cause cutaneous leishmaniasis (Oriental sore). It is restricted in the vicinity of the infection site (i.e. the sandfly bite). First, a nodule (papule) forms. After growing, it forms an ulcer. Cutaneous leishmaniasis is the mildest form of leishmaniasis. Even if untreated, it heals spontaneously after several months to more than a year. However, it leaves a disfiguring scar, and in immunocompromised patients can disseminate.
Leishmania braziliensis complex
Parasites belonging to Leishmania braziliensis complex are found in Central and
South America. They cause cutaneous leishmaniasis in the New World. However, in a proportion of patients, they cause a much more severe disease – mucocutaneous leishmaniasis. It starts like the cutaneous leishmaniasis. However, the parasites settle in the facial mucosa, often years after healing of the initial cutaneous lesion. This occurs by spreading, if the bite has been on the face, or by “metastasis” via blood, if the bite has been somewhere else. If untreated, the mucocutaneous leishmaniasis slowly destroys the nasal septum, palate and other mucosal structures, leading to complete disfiguring of the face. Because of the visible deformity, the patient is ostracised from community. In some cases, the disease is fatal due to airway damage or secondary infection.
The group Metamonada, which is not yet quite official, includes protozoans which have anaerobic metabolism and have lost their mitochondria. Most of them are extracellular parasites or symbionts living in anaerobic environment inside animal bodies, in the digestive tract or other cavities. A few are free-living. The metamonads have flagella assembled in groups of four and often associated with the nucleus. Many metamonads have an axostyle – longitudinal supporting structure made of cross-linked microtubules. Reproduction is by closed mitosis.
The cell is 15-20 micrometers long. It is pear-shaped due to the presence of an axostyle, which protrudes from the posterior end of the cell and may facilitate attachment of the parasite to mucosal tissues of the host. The nucleus is located near the broader anterior end of the cell. It is haploid. The genome is among the largest genomes studied so far, with 26,000 genes (comparable to human). Trichomonas has five flagella: four protruding freely from the anterior end and one bending backward and supporting an undulating membrane. Close to the anterior end, there is an invagination called cytostome and used for phagocytosis. For the purpose of phagocytosis, Trichomonas can switch from pear-like to amoeboid shape. Data about sexual reproduction are still insufficient.
Trichomonas vaginalis inhabits the mucosal surface of vagina in women and urethra in both sexes. It feeds on bacteria, probably damaging the symbiotic normal vaginal microflora. It also damages the mucosa by adhering to epithelial cells, releasing hydrolases on them, phagocytosing erythrocytes and maybe also leukocytes and epithelial cells.
T. vaginalis is transmitted by sexual intercourse. In women, it often causes vaginitis (colpitis) often associated with discharge and unpleasant odour. In men, the infection is usually asymptomatic but nevertheless it is important to treat both sexual partners, because asymptomatic individuals still can transmit the parasite. Trichomoniasis is widespread – the most common non-viral sexually transmitted disease, and T. vaginalis is the most common pathogenic protozoan in industrialized countries.
In vitro cultured Trichomonas vaginalis. The nucleus is visible inside the cell, and the axostyle can be seen protruding from its posterior end.
Trichomonas tenax is closely related and morphologically similar to T. vaginalis. It is often found in the oral cavity, especially in individuals with periodontitis and/or poor dental hygiene. It feeds on bacteria and is considered a harmless commensal rather than a parasite. However, in some immunocompromised patients, T. tenax is found in the lungs (pulmonary trichomoniasis), apparently settled after being inhaled from the mouth or throat. For that reason, some researchers consider it an opportunistic pathogen.
Genus Giardia. Giardia lamblia (Lamblia intestinalis, Giardia intestinalis)
Giardia lamblia was named after two scientists, Alfred Giard and Vilem Lambl. The metabolically active form is called trophozoite. It is 10-20 micrometers long, pear-shaped, had a dorsal and a ventral side and is dorsoventrally flattened. In the anterior part of the trophozoite, on the ventral side, there is a sucking (adhesive) disk serving for attachment to the intestinal mucosa. The cell has bilateral symmetry and its organelles are paired. This way, Giardia has two diploid nuclei and a total of eight flagella (two sets of four flagella each). Two of the flagella have a median position and span the cell body before coming out free from the posterior end. The intracellular portion of their axonemes is called by some authors axostyle(s); others describe Giardia as having no axostyle. Data about sexual reproduction are still insufficient.
In vitro cultured Giardia trophozoites. Image contributed by the Oregon State Public Health Laboratory, downloaded from the CDC site: http://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/G-L/Giardiasis/body_Giardiasis_il9.htm
Giardia lives in the lumen of the small intestine and absorbs nutrients by its surface. Giardiasis, when symptomatic, manifests as foul-smelling diarrhoea and malabsorbtion. It is not quite clear how the parasite harms its host but it may be due to the action of suction disks. After detachment, they leave imprints on mucosal cells.
Infection with Giardia happens by the so-called alimentary or fecal-oral route typical for intestinal parasites: the infective stage comes out of the current host’s organism with stool and relies on being eventually ingested by a new host. All parasites using this way of transmission have a metabolically quiescent, highly resistant stage to endure weeks or months in external environment (soil or water) waiting to be ingested. In unicellular parasites, this dissemination stage is the cyst form (as opposed to the trophozoite form) which is oval and supplied by a thick resistant outer sheath.
Giardia cysts are generally smaller than trophozoites (about 10 micrometers) and, when mature, have four nuclei as a result of mitosis. The transition from trophozoite to cyst happens when the trophozoite is carried downstream by intestinal content.
Trichrome-stained Giardia cyst. Sometimes, as in this photo, the cytoplasm may retract from the cyst wall. Image courtesy of CDC: http://www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/G-L/Giardiasis/body_Giardiasis_il4.htm
Cysts can survive weeks to months in cold water. It is also important that chlorination does not guarantee full destruction of Giardia cysts at low temperature. Humans are usually infected by drinking contaminated water.
G. lamblia is primarily a human parasite but can infect also other mammals, both domestic (cats, dogs, pigs etc.) and wild (beavers, bears). It has cosmopolitan (worldwide) distribution.
These are the amoeboid protozoans moving by pseudopods (pseudopodia) and feeding primarily by phagocytosis.
Entamoeba includes several species living as parasites or commensals inside the digestive tract of humans and other animals. Similarly to metamonads, Entamoeba has lost its mitochondria. There are yet no data about sexual reproduction.
The most important representative of Entamoeba is E. histolytica which causes amoebiasis. It inhabits the large intestine. Human is the only natural host.
The trophozoites are 15 – 30 micrometers long. In most cases, they live as commensals. They adhere to the mucous layer covering the intestinal epithelium and feed by phagocytosis of bacteria and debris.
Some of the trophozoites detach, are carried to the distal parts of the large intestine and turn into oval cysts measuring 12 – 15 micrometers. Immature cysts have 1 or 2 nuclei while mature ones have 4 nuclei. The cysts can survive in the environment for weeks. Humans are infected when they ingest cysts from contaminated food or water. In the intestine, cysts excyst. By one round of mitosis and three rounds of cytokinesis, each of them gives rise to eight uninuclear trophozoites.
Trichome-stained cyst of E. histolytica or E. dispar. Two nuclei are visible in this focal plane (black arrows). The red arrow indicates the so-called chromatoid body often found in cysts and consisting of ribosomes. Image courtesy of CDC: http://dpd.cdc.gov/dpdx/HTML/ImageLibrary/Amebiasis_il.htm
Finding cysts and (in some cases) trophozoites in faeces is necessary but not enough for diagnosis because of the morphological similarity between E. histolytica and related non-pathogenic amoebae, notably E. dispar. For that reason, identification by molecular biology methods (PCR for species-specific DNA sequences) is required today to diagnose amoebiasis.
The pathogenicity of E. histolytica depends on the strain and on the host’s condition. Larger strains tend to be more invasive, and the hosts affected are mostly children under 5, pregnant women, elderly and immunocompromised patients. Invasive trophozoites not only adhere to the intestinal mucosa but also secrete cytotoxic enzymes (proteases, phospholipases) and a pore-forming protein. These molecular “weapons” lyse mucosal cells and erode the extracellular matrix, leading to ulceration of the interstinal wall and disentery.
The patient suffers of abdominal pain and diarrhoea with mucus and/or blood. In the lesions, E. histolytica trophozoites can phagocytose erythrocytes. Perforation of the intestinal mucosa and peritonitis may result. If trophozoites reach a blood vessel, they can be carried with the bloodstream to extra-intestinal organs – liver, brain or lungs, where they produce life-threatening abscesses. Most of the deaths resulting from aboebiasis are due to liver abscess.
Trophozoites of E. histolytica with ingested erythrocytes stained with trichrome. The erythrocytes appear as dark inclusions easily distinguishable from the nucleus. Image courtesy of CDC: http://dpd.cdc.gov/dpdx/HTML/ImageLibrary/Amebiasis_il.htm
A non-pathogenic species related to and morphologically indistinguishable from E. histolytica, lives in the large intestine as a commensal. The confusion of E. dispar with pathogenic E. histolytica has been a major obstacle in the diagnosis and research of amoebiasis.
This species lives in the oral cavity, most often of people with gum disease. It is still discussed whether it is a parasite or just a commensal taking advantage of the rich bacterial flora associated with periodontitis. Unlike other Entamoeba species, E. gingivalis has no cyst stage and cannot endure for long in the environment. Trophozoites are transferred directly from one host to another, e.g. by kissing or sharing utensils.
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Published in 2013
Copyright © Maya Markova