Introduction to Protozoan Infections
2.4 Harosa
2.4.1 Aveolata
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.
Historically named theSporozoa, this phylum is comprised of a diverse group of mitochondria bearing parasitic protozoa with life cycle stages that include an infective motile ‘zoite’ form. The apical end of the zoite is comprised of a specialised secretory organelle complex – the ‘apical complex’ – and this is the basis for the name of the phylum and a requisite for invasion and colonisation of host cells.
Generally, the life cycles of theApicomplexaare comprised of three stages. The first is a growth stage after infection of a host (or host cell) by the zoite. In many species, this is accompanied by mitotic reproduction. The second is a sexual cycle via the production of gametes, followed by fertilisation and the forma- tion of zygotes within a thick-walled structure called an oocyst. Finally, sporo- genesis occurs, during which there are successive divisions of the sporoplasm within the oocysts to form new infective zoites (now termed ‘sporozoites’). In thoseApicomplexathat infect new hosts via transit through the external envi- ronment, highly resistant spore structures (sporocysts), which shelter the de- veloping sporozoites, form in the oocyst.
TheApicomplexais a large phylum made of over 2,500 described species. It can be divided up into three major groups:
1. Gregarina, which infect invertebrates such as arthropods and prochordates.
2. Coccidia, which infect a wide variety of animals.
3. Aconoidasida, which is composed of the Piroplasmids and Haemosporidi- ans, whose heteroxenous life cycles alternate between a vertebrate and an arthropod vector.
Parasites within all three of these classes can either be generalists, which in- fect a wide range of hosts or related species, or can have extremely narrow host specificities.
Ultra-structural analysis of the apical complex of the invasive zoites has shown they are composed of a cap formed by two conoidal rings (in some species), a conoid which is composed of spirally arranged microtubules, and a polar ring which acts as an organising centre connecting the conoid to microtubules that extend backwards underneath the plasma membrane (Figure 2.7A and 2.7B).
This unique microtubule structure gives the complex its characteristic shape.
Within the complex, there are generally two pedunculate secretory organelles called rhoptries, and a variety of other organelles such as the micronemes and dense bodies that are believed to traffic material such as secreted proteins from the Golgi to the apical complex.
In theApicomplexa, the plastid organelle (or apicoplast) is surrounded by four membranes but has lost any photosynthetic capacity. Despite not being pho- tosynthetic, the inner membrane of the apicoplast in some species such as Plasmodiumstill retains tubular ‘whorls’ which resembles thylakoids found in photosynthetic plastids. The function of these membrane structures remains unknown. The apicoplast still contains its own organellular DNA and is be- lieved to perform a variety of important biosynthetic functions in lipid, heme
Nucleus
Golgi Rhoptry
Mitochondria Micronemes
Apicoplast
Conoid Conoid and
Polar rings
Posterior ring Dense Granule
(A) (B)
Figure 2.7 Apicomplexa basic anatomy.(A) A transmission electron microscopy (TEM) image of aToxoplasma gondii tachyzoite. (B) The illustration shows an idealised image of aToxoplasma gondiitachyzoite. The major organelles and other subcellular features are labelled.
and amino acid metabolism. The unique metabolic pathways used in the api- coplast are being developed as novel anti-apicomplexan drug targets.
Understanding the evolution of this important group of parasites, and theApi- complexain general, has offered a number of challenges. Traditional morphol- ogy or ultra-structure-based taxonomies are often conflicting and have not offered robust resolution within the genusPlasmodiumor the phylumApicom- plexa. Phylogenies based on molecular data have also offered conflicting re- sults, as genes which have served as workhorses for these types of analyses in other groups, such as the small subunit (SSU) ribosomal DNA (rDNA) gene, ap- pear to have heterogeneous evolutionary rates or atypical composition biases in someApicomplexa.
Figure 2.8A depicts a simplified phylogenetic tree, with only the major api- complexan groups and representative species listed. Surprisingly, in these and other analyses, theCryptosporidiaare consistently placed outside of the clade containing other Coccidea, such asEimeriaandToxoplasma. If these are truly unrelated groups, this may serve as a starting-point for elucidating why tra- ditional anti-coccidial drugs are not effective in treatingCryptosporidiainfec- tions. Recent phylogenetic analyses using mitochondrial genes has also helped resolve relationships with the Haemosporidians (Figure 2.8B) and has lent sup- port to the choice of some rodent and primatePlasmodiumspecies as mod- els for human malarial biology and vaccinology. MammalianPlasmodium sp.
P. malariae P. ovale P. reichenowi P. falciparium
P. vivax P.knowlesi
and other primate infecting species
Hepatocystis sp.
P. berghei P. yoelii P. chabaudi
and other rodent infecting species
P. gallinaceum
Bird infecting Plasmodium sp.
Reptile infecting Plasmodium sp.
Bird infecting Haemproteus sp.
Gregatines and Haemosporidians
(Plasmodium sp.)
Piroplasmids
(Theileria sp. and Babesia sp.)
Coccidians
(Eimeria sp., Neospora sp., Sarcocystis sp. and Toxoplasma sp.)
Cryptosporidians Outgroup
(A) (B)
Figure 2.8 The phylogeny of theApicomplexa.(A) The cladogram shows the phylogenetic relationship of the major Apicomplexagroups. This illustration, based the analysis of the SSU, has been adapted from Morrison, DA (2009).
Evolution of the Apicomplexa: where are we now?Trends in Parasitology25(8), 375–382. (B) This illustration is based the analysis of the mitochondrial cytochrome b gene and shows the phylogenetic relationships of the major Haemosporidian groups andPlasmodiumspecies of medical importance (adapted from Perkins, SL and Schall, JJ (2002). A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences.Journal of Parasitology88(5), 972–978)
are now believed to have evolved from an ancestral species infecting reptiles or birds, and species capable of infecting humans appear to have evolved sev- eral times potentially as species jumped back and forth between primate and human hosts.
Coccidia, Cryptosporidium sp.
The Cryptosporidia are parasites of the brush boarders of a variety of mam- mals, birds, reptiles and fish, causing diarrhoea in human infections. Cryp- tosporidium parvumandC. hominis are the species that cause most human infections (see Chapter 5), although other species have been documented to cause disease. Infection begins after ingestion of a sporulated oocyst that contains four infectious sporozoites. Sporozoites parasitise epithelial cells in the digestive tract (or occasionally the respiratory epithelium). In these cells, parasites undergo alternating cycles of asexual division as type I meronts (schizogony) and sexual reproduction as type II meronts (gametogony). Dur- ing gametogony, micro- and macrogamonts (gametocytes) develop. Microga- monts undergo asexual replication and then invade the adjacent tissue until they find and fertilise a macrogamont. The fertilised zygote then forms a thick- or thin-walled oocyte. The thick-walled oocytes exit the host in the faeces and
sporulate into infectious oocytes. The thin-walled oocytes sporulate within the host and sporozoites released initiate an autoinfectious cycle.Cryptosporidium infections are usually self-limiting, with only young and immunocompromised hosts developing serious complications.
Coccidia, Toxoplasma gondii
This cosmopolitan species has been found in almost every part of the world and in just about every warm-blooded mammal. In human populations, in- fection is widespread but is often asymptomatic outside of the very young or immunocompromised individuals. As an intracellular parasite, T. gondiican infect a wide variety of cells, including epithelia, muscle and neuronal cells.
Sexual reproduction (the enteroepithelial phase) is confined to the definitive feline host, while asexual reproduction (the extra-intestinal phase) and the for- mation of long-lived cyst structures occurs in humans and other intermediate hosts.
Extra-intestinal stages begin upon ingestion of environmental oocysts or cyst- like structures contained in the tissue of an infected intermediate host called bradyzoites. Once ingested, sporozoites released from the oocysts or brady- zoites released from cysts rapidly penetrate the gut and invade a host cell, where they live within a PV. The parasites develop into rapidly replicating tachy- zoite stages. These disseminate and invade muscle and neural tissue (includ- ing the brain). In most cases, after the acute phase, the onset of chronic in- fection is characterised by slower replication of the parasite and formation of bradyzoites. Cyst formation coincides with the onset of protective immune re- sponses, but these long-lived structures occasionally rupture, releasing para- sites that can re-initiate an acute phase of infection if the host becomes im- munosuppressed.
If a feline definitive host ingests an oocyst or tissue-encysted bradyzoite in ad- dition to the asexual systemic infection, a short phase of sexual reproduction occurs. Sporozoites or bradyzoites invade the intestinal epithelial, transform into replicating merozoites and produce microgametocytes and macrogame- tocytes. Microgametocytes divide and break out of the infected cell, and invade adjacent cells until they find and fuse with a macrogametocyte, where fertilisa- tion occurs. The fertilised zygote forms an immature oocyte, which passes out of the feline in it’s faeces.
Haemosporida, Plasmodium sp.
Composed of three major genera,Haemoproteus,Leucocytozoon, andPlasmod- ium, this group of organisms is one of the most successful parasitic assem- blages known. In humans, all major pathogens belong to the genusPlasmod- ium(see Chapter 3), with the five important species currently recognised being P. vivax,P. falciparum,P. ovale,P. malariae and P. knowlesei. All thePlasmod- iumspecies have very similar life cycles, requiring both an invertebrate host (mosquito) and a vertebrate host.
The mosquito acts as the relatively brief definitive host, where sexual repro- duction occurs, in contrast to several phases of asexual reproduction that oc- cur within the vertebrate intermediate host. Infection in the vertebrate host takes place after deposition of saliva-containing sporozoites during mosquito feeding on the host blood. These sporozoites are highly motile and they rapidly migrate to the liver, where they specifically invade hepatocytes. Specific recog- nition of hepatocytes by receptors on the surface of the sporozoite confers the cell tropism observed by this stage of the parasite. Invasion of hepatocytes is facilitated by secretion of proteins from the secretory organelles of the apical complex. Entry into the hepatocyte initiates a stage of asexual reproduction called the pre-erythrocytic cycle, where the parasite transforms into a tropho- zoite, feeds on intracellular material and begins a process of asexual reproduc- tion called schizogony. However, in some Plasmodium species (P. vivax and P. ovale in humans), some of the parasites develop into a state of dormancy and become hypnozoites, which can reactivate many years after the initial infection.
The schizont (also known as a cryptozoite) initially undergoes a series of nuclear divisions without cytokinesis, forming a large polynucleate cell. Once nuclear division is finished, other organelles also undergo division and, even- tually, individual merozoites are formed. When the merozoites leave the hep- atocytes, they initiate the erythrocytic cycle by invading a blood erythro- cyte. Within the erythrocyte, they again transform into a trophozoite and feed, forming a large food vacuole (ring stage), where haemoglobin digestion occurs. They undergo schizogony, and finally rupture the host erythrocyte, releasing a new generation of merozoites. After an indeterminate number of asexual generations, a proportion of merozoites enter erythrocytes and de- velop into macro- or micro-gametocytes. These are the sexual stages of the parasite and the only transmissible stages of Plasmodium that are infective to mosquitoes.
After ingestion by a suitable mosquito host, gamonts are released from the gametocytes. The microgametocytes undergo a series of cell divisions, and daughter cells seek a suitably mature macrogamete to penetrate and fertilise.
The resulting diploid zygote transforms into a motile ookinete, which pene- trates the gut wall of the mosquito to form an oocyst on the hemocoel side of the gut. The oocyst undergoes a complex cycle of DNA replication and segregation that culminates in the formation of thousands of new individual sporozoites, which break out of the oocyst. The free sporozoites then migrate and enter the salivary glands, where they are in a prime position to be deposited in a new host when the insect feeds again.
One particularly interesting molecule produced by the parasite is a heme poly- mer called hemozoin. Free heme released during the digestion of erythrocyte haemoglobin would build to toxic levels if left uncompartmentalised. Plas- modium’s solution to this problem is to sequester the heme in the form of a polymer, which is then released when the merozoites escape the erythro- cyte. Hemozoin’s activity on the immune cells is complicated and has been shown both to activate (via Toll-like receptor 9) and to suppress innate immune cell function. Formation of hemozoin is an important metabolic process for the parasite, and several drugs currently used to treat malaria infection target
various aspects of heme polymerisation or food vacuole function (chloroquine and artemisinin).