Introduction to Protozoan Infections
2.3 Excavata
2.3.3 Discoba, Euglenozoa
This diverse group of mitochondriate flagellates includes the euglenids, kine- toplastids (trypanosomes and bodonids), diplonemids and symbiontids. The Euglenozoaare united by the presence of two flagella, which are inserted paral- lel to one another in an apical or subapical pocket. These are usually associated with a unique feeding apparatus called a cytostome, which is used to ingest smaller prey or other material. Most members of this group are heterotrophic.
One lineage (the euglenids) has acquired a plastid derived from an endosymbi- otic association with a photosynthetic alga. Taxa from this group occupy almost every trophic niche, with species identified having free-living, commensal,
Nucleus
Mitochondria Kinetoplast
Flagellar Pockets
Flagella
Secretory vesicles and reticulum
Golgi
Pellicular microtubules Kinetosome/ Basal Body
Nucleus
(B) (A)
(C)
Figure 2.5 Kinetoplastida basic anatomy.(A) A TEM image of aLeishmaniapromastigote kinetoplast and kDNA complex at the base of the kinetosome and flagella. (B) The illustration shows an idealised image of aT. bruceistumpy
trypomastigotes. The major organelles and other subcellular features are labelled. (C) A light microscopy image of Wright Giemsa-stained thin blood smear containingT. bruceitrypomastigotes.
endosymbiotic, and parasitic lifestyles. TheKinetoplastidacontain the species that are of medical importance to humans.
2.3.3.1 Kinetoplastida
The kinetoplastids are an extremely diverse order, containing both free-living and parasitic members. As well as having the defining characteristics of the Eu- glenozoa, this order possess a single large mitochondrion containing a massed collection of mitochondrial DNA that forms a complex structure called the kinetoplast (kDNA). The kinetoplast is associated with the two kinetosomes at the base of the flagella (Figure 2.5A and 2.5B).
The Kinetoplastida contains two suborders: the predominately free-living Bodoninaand the parasiticTrypanosomatidae. TheTrypanosomatidaeare one of the most diverse groups of parasites known; they are unusual because they have members which not only infect animals and plants (Phytomonas sp.), but can do so in both terrestrial and aquatic environments using different groups of intermediate hosts.
Their life cycles can range from simple to complex and, while it is believed that the group may have evolved from parasites of insect digestive tracts, many
Prokinetoplastina (parasitic) Diplonemids (free-living) Neobondonida (free living)
Parabodonida (free-living and parasitic) Eubodonida (free-living)
T. brucei clade
T. cruzi clade
Rodent Trypanosomids
Avian Trypanosomids Aquatic Trypanosomids
Leishmania Phytomonas
Monoxenous insect trypanosomids
Trypanosomatidae
(parasitic)
Trypanosoma
Euglenids
Figure 2.6 The phylogeny of the Kinetoplastida.The cladogram shows the phylogenetic relationship of the different free living or parasitic kinetoplastid groups. This illustration is based the analysis of the small subunit (SSU) ribosomal RNA and several protein encoding gene families. Adapted from Simpson, AGet al.(2006). The evolution and diversity of kinetoplastid flagellates.Trends in Parasitology22(4), 168–174.
vertebrate-infecting species are transmitted via an invertebrate host. Under- standing how parasitism evolved in this group and their placement with in the Kinetoplastids, has been an area of great interest. Figure 2.6 shows a phyloge- netic tree that summarises our current knowledge of the evolutionary relation- ships of the members of this order.
The data suggests several important points. Importantly, the previous belief that the closest free-living ancestors of the Kinetoplastida are photosynthetic Euglenids is not supported by these analyses. Instead, they indicate the Kine- toplastida may have evolved from an obscure group of surface-associated het- erotrophes called the Diplonemids. Within the kinetoplastids, parasitism has evolved at least three different times. However, while there is support for the monophyly of the Trypanosomatids and the major groupings within this clade, the details of how many of these organisms are related to each other remain unresolved.
The two genera containing important humans pathogens areTrypanosomaand Leishmania. These genera undergo replicative cycles in both their insect and
vertebrate hosts and, while it is poorly described, there is evidence that these parasites also undergo periodic sexual cycles in their insect hosts. Besides the kinetoplast a second unusual organelle called the glycosome is found in the TrypanosomaandLeishmania. A peroxisome derivative, this specialised vesicle is bound by a single membrane and is the primary site of glycolysis and gly- colytic regulation in the parasite.
Trypanosoma brucei
This is a collection of three subspecies: T. brucei brucei,T. brucei gambiense, andT. brucei rhodesiense(see Chapter 8).T. brucei bruceiis a pathogen of ru- minants causing a disease called nagana. As well as infecting native game,T.
b. gambiense, andT. b. rhodesienseare the etiological agents of African sleep- ing sickness, causing chronic and acute forms of the disease respectively. In- fection is initiated by the bite of an infected tsetse flies (Glossina sp.), which can inoculate a host with thousands of metacyclic trypomastigotes. Once in the vertebrate host, the protozoa transform into slender trypomastigotes and begin replication in the blood and lymph. The parasites remain extracellu- lar, although there are reports of recovery of amastigote forms from certain tissues during experimental infection of animals. In chronic infections, the par- asites eventually invade the CNS and brain of the host. While in the vertebrate, the parasites are pleomorphic, with slender (containing sparse short tubu- lar cristae in their mitochondria), stumpy (containing many tubular cristae in their mitochondria) and intermediate trypomastigotes, forms easily detected in circulation.
Studies of isolated varieties of these pleomorphic forms have shown that mor- phological differences are linked to the forms of metabolism that the parasites are currently using. Slender forms are highly replicative and have repressed mi- tochondrial function, metabolising glucose as far as pyruvate. They do not use the TCA cycle or oxidative phosphorylation for further ATP generation. Plen- tiful glucose and oxygen supplies in the blood apparently favour this form of respiration, and the glycosomes serve as the centre of this respiratory process.
In contrast, the stumpy forms that are infectious to the tsetse fly do not divide and have active mitochondria, fully oxidising glucose. Changes in mitochon- drial respiration are linked with changes in kDNA localisation, but it is unclear whether the localisation of the kDNA regulates this process. Once the stumpy forms are taken up in a blood meal by a tsetse fly, the parasites migrate to the posterior midgut, where they replicate. After about ten days, slender form try- pomastigotes migrate to the foregut and then the salivary glands, via the oe- sophagus and pharynx. In the salivary glands, they develop into epimastigotes, undergo further replication and transform into infectious metacyclic trypo- mastigotes.
The major mechanism of immune evasion, variation of surface antigen reper- toires, is one of the best-studied aspects ofT. bruceibiology. One of the most interesting features of its genome is the organisation of more than 1,000 differ- ent variant-specific surface glycoprotein genes (VSG) in sub-telomeric arrays, and the mechanisms employed to restrict expression to a single VSG.
Trypanosoma cruzi
Restricted to the new world, the causative agent of Chagas disease,T. cruzi(see Chapter 9), has a radically different life cycle to its cousinT. brucei, opting in- stead for an intracellular lifestyle within the vertebrate host. A number of genet- ically distinctT. cruzipopulations have been identified in South America, and phylogenetic analyses estimate that some of these groups may have diverged at least ten million years ago.
Infection is initiated by the feeding of a reduviid bug and its subsequent defecation on the vertebrate host. Infectious metacyclic stages are passed onto the host in the faeces of the insect. These either invade the host directly, via the feeding wound, or by mechanical transfer to mucosal surfaces. Once in the blood, trypomastigotes of T. cruzido not replicate, but instead invade host cells. While a wide variety of cell types can be invaded, the most common are reticuloendothelial and muscle cells. The mechanisms underlying the invasion process are still being elucidated, but they involve the release of Ca+by the host cell Golgi and recruitment of lysosomes to the contact point with the parasite.T. cruzithen enters the cell via modification of these lysosomes into a parasitophorous vacuole (PV). As the lysosome re-acidifies, a porin-like toxin secreted by the parasite ruptures the PV membrane, releasing the parasite into the cell cytoplasm where they lose their flagella and develop into amastigotes.
The amastigotes replicate quickly, transform back to trypomastigotes and eventually rupture the infected cell. In certain larger cells, such as muscle cells, cyst-like pockets of parasites can form pseudocysts.
Released trypomastigotes can invade new cells or enter the blood, where they can continue their life cycle if they are ingested by feeding reduviid bugs. Once in the midgut of the insect, they transform into short epimastigotes, which replicate by longitudinal fission into a longer, slender form. Short, infectious metacyclic trypomastigotes appear in the rectum of the insect eight to ten days after it is infected.
Leishmania sp.
The genusLeishmania(see Chapter 7) contains over a dozen recognised species and several species complexes which have been defined by biochemical or molecular criteria. Distributed throughout both the old and the new world, two sub-genuses are recognised,LeishmaniaandViannia, both of which contain pathogens relevant to human health. The five most important species from a medical perspective areL. tropica,L. infantum, L. major,L. donovani,L. mex- icana, andL. brasiliensis, and these can cause visceral or cutaneous disease.
Infection in a vertebrate is initiated when promastigotes are transmitted into the skin by the bite of female phlebotomine sandfly. Promastigotes are quickly phagocytosed by macrophages that reside in or underneath the dermis. Instead of being dispatched by the macrophages, the parasites reside in a modified lysosome, which matures into a PV. Once in the PV, the parasites transform into replicative amastigotes, which eventually rupture out of the infected cell and can infect new macrophages or other phagocytic cells.
Morphologically, the different amastigote species look fairly similar and are among the smallest known eukaryotic cells (2.5–5 m wide). In stained
microscopic preparations, the nucleus and kinetoplast are the most prominent features, and the small cytoplasmic space appears vacuolated. Successive cy- cles of infection and intracellular replication ensue with different species dis- playing tropisms for specific organ sites. These tropisms eventually lead to the distinct pathologies that eachLeishmaniaspecies causes.
A new sandfly is infected whenLeishmania-containing cells in skin and blood are ingested during a meal. When infected cells are digested, amastigotes are released and migrate to the mid or hindgut, where they transform into pro- mastigotes, attach to the fly’s gut wall and multiply by binary fission. By the fourth or fifth day after infection, the promastigotes migrate to the oesoph- agus and pharynx, which they eventually clog. The fly clears the obstruct- ing parasites by pumping the contents of the oesophagus in and out, and this action inadvertently inoculates the promastigotes onto the skin of a new vertebrate host.