ReviewLipid synthesis in protozoan parasites: A comparison between kinetoplastids and apicomplexans
Introduction
Diseases caused by protozoan parasites are among the most pressing concerns on the global health agenda. Within their ranks are not only some of the most widespread and important infectious diseases, but also many of the most sorely neglected [1]. In this review we will focus on two protozoan phyla in particular, Apicomplexa and Kinetoplastida, which stand out in their overall public health impact with respect to incidence and severity of the diseases they cause [2]. Not only are these the most important groups of parasites, they also are the best studied. Facile experimental model systems have been established for several members of both phyla. Driven by powerful genetics these models have yielded considerable insight into parasite metabolism. There are numerous significant metabolic differences between these parasites and their human host, and we will highlight these differences, as they afford opportunities for drug development. We will provide a brief introduction to the parasite models that are the focus of this article for readers unfamiliar with the organisms. We will describe the particular importance of lipid metabolism for pathogens to introduce the subsequent systematic and comprehensive discussion of the biosynthesis pathways of major lipid classes for both phyla.
Apicomplexans are eukaryotic pathogens named after the complex of secretory and cytoskeletal organelles located at the apical end of the parasite cell. Apicomplexa are intracellular parasites and the organelles of the apical complex are required for host cell invasion. Three genera represent a particular threat for human health and therefore will be the main focus of this review: Plasmodium, Toxoplasma and Cryptosporidium. Note, however, that there are numerous additional apicomplexans that are important veterinary pathogens.
Five species of Plasmodium collectively are the causative agents of malaria, a disease that claims the lives of about a million individuals every year. Most of these deaths are due to Plasmodium falciparum and occur in small children in sub-Saharan Africa. Plasmodium has a complex life cycle, and as we will see, profound metabolic and biochemical changes are associated with each life cycle transition. Infection of a human occurs through the bite of a mosquito, whereby a small number of sporozoites are injected with the saliva. The sporozoites travel to the liver and invade hepatocytes, where they massively proliferate. Merozoites are then released into the bloodstream, where they invade and replicate in red blood cells. It is this intraerythrocytic phase that is responsible for the disease. Control of malaria rests on the prevention of transmission (e.g. through treated bed nets) and drug treatment. The genetic malleability of the parasite and its propensity to evolve drug resistance [3] has long haunted malaria control. This famously includes the loss of chloroquine as an effective therapeutic. There is grave concern about the future of the current artemisinin-based treatment regimen with first signs of treatment failure emerging in South East Asia [4], [5]. A constantly evolving portfolio of new anti-malarials is required to keep up with the ever-changing parasites. Understanding parasite metabolism is an important cornerstone of this agenda.
Toxoplasma and Cryptosporidium were initially recognized as opportunistic infections in immunocompromised individuals and received particular attention as late stage manifestations of AIDS [6], [7]. The sexual phase of the Toxoplasma life cycle occurs in the intestinal tract of cats and results in the shedding of spore-like oocysts, which then become highly infectious to other animals and humans upon consumption of contaminated food or water. Within intermediate hosts, tachyzoites cause rapid systemic infection. The onset of immune control eliminates tachyzoites, but bradyzoites persist within tissue cysts for the life of the host. In immunocompromised adults, chronic infection is reactivated leading to Toxoplasma encephalitis. In addition, congenital toxoplasmosis is a significant concern during pregnancy. While anti-folate treatment is effective in controlling tachyzoites and acute disease, there are no drugs available to eliminate chronic infection, which is problematic in a variety of clinical settings [8].
Cryptosporidiosis is an acute enteric disease that typically is self-limiting. However, recent studies show that in particular in malnourished children the disease can be severe, protracted, and life threatening [9]. Cryptosporidium has a single host life cycle restricted to the intestinal epithelium, and oocysts are the only mode of transmission. Nitazoxanide was approved by the FDA for treatment but only shows moderate efficiency in immunocompetent children and produces no benefit in immunocompromised patients. Finding a reliable drug remains an important goal [10].
Kinetoplastids are flagellated protists that share a mitochondrial genome of unique organization and localization called the kinetoplast. There are numerous human pathogens among these early-branching eukaryotes, including African and American trypanosomes and many different species of Leishmania. All of these pathogens have insect and mammalian hosts. The different life cycle forms can be morphologically distinguished based on the position and length of the flagellum. More importantly, they also show remarkable metabolic differences. Stages from insect and mammalian hosts can be cultured axenically, which has greatly facilitated biochemical and genetic studies.
Trypanosoma cruzi is the causative agent of Chagas disease, which is endemic in large parts of South America (note that although both the parasite and the vector are present in North America, transmission is rare). T. cruzi is transmitted by reduvid, or kissing bugs. Promastigote stages replicate within the bug’s midgut and infective metacyclic trypomastigotes are deposited with the feces onto the skin of the mammalian host. In the mammalian host, the parasite cycles between replicative intracellular amastigotes and trypomastigotes. Importantly, replicating amastigotes are free in the host cell cytoplasm providing intimate access to host metabolites. Chagas disease is characterized by chronic and progressive inflammatory tissue damage, in particular of the heart muscle. Treatment is available but is not consistently effective and suffers from significant adverse effects [11].
Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei brucei cause Nagana, an important cattle disease that severely limits ranching and dairy production in many parts of Africa. T. brucei gambiense and rhodesiense are morphologically indistinguishable from the bovine subspecies and are the causative agents of Human African Trypanosomiasis, also known as sleeping sickness. All African trypanosomes are vectored by tsetse flies, where they are present as procyclic trypomastigotes, epimastigotes and metacyclic trypomastigotes. In the mammalian host, trypomastigotes replicate extracellularly in the bloodstream causing anemia and cachexia. In humans, the blood phase is ultimately followed by invasion of the central nervous system. Untreated infection is invariably fatal. Several drugs are available for treatment of Human African Trypanosomiasis and Nagana, but there are severe limitations. Some of the drugs have grave and even life threatening adverse effects, are limited in their potency once the parasites have entered the brain, or are only active against certain species or subspecies. Lastly, there is treatment failure and overt resistance to several drugs, in particular in veterinary practice [12], [13], [14].
Parasites of the genus Leishmania are transmitted by a variety of sand flies, where promastigotes replicate in the midgut and are mechanically introduced into the bite site. In the mammalian host, Leishmania parasites proliferate as intracellular amastigote in macrophages, and they do so within a fully matured and acidified phagolysosome [15].
A large number of different Leishmania species are responsible for a broad spectrum of disease manifestations, with currently 12 million people being infected worldwide. In its most benign form, leishmaniasis presents as a self-limiting skin ulcer at the bite site (cutaneous leishmaniasis). In contrast, progressive non-healing erosion of mucosal tissue in the vicinity of the bite site is characteristic for mucocutaneous forms. In the most severe visceral disease, parasites spread systemically causing hepatosplenomegaly, cachexia and immunosuppression. The etiology of these various forms appears to be closely linked to an unbalanced immune response to the chronic infection resulting in too much or too little inflammation and parasite control. Visceral leishmaniasis is fatal if not treated. Several drugs are available [16], but treatment of leishmaniasis remains challenging due to adverse effects, the requirement of lengthy regimens, limited drug availability and expenditure [17].
Lipids are critical to the biology of all cells and organisms. They are the main structural elements of all biological membranes, they anchor glycoconjugates and many proteins to membranes, they serve as signaling molecules within and between cells, and they represent a highly efficient store and source of energy and reduction power. Lipids are of particular importance for pathogens, and some pathogens deliberately seek out lipid-rich host niches [18], or enhance the availability of lipids by manipulating the host [19], [20]. Intracellular pathogens have evolved sophisticated mechanisms to manipulate and tap into the lipid metabolism of their host cells. These include interference with vesicular and non-vesicular cellular lipid trafficking in viral [21], bacterial [22] and protozoal [23] pathogens. Within host cells, intracellular pathogens often develop in specialized vacuoles and the flow of lipids between host and pathogen-controlled membranous compartments is key to the pathogen’s ultimate success [15], [24], [25], [26].
Lipids are not only used by pathogens as food or structural building blocks, but are also important pathogenesis factors that allow the pathogen to evade immune responses, manipulate host processes, and cause disease. In many cases these are specialized lipids synthesized by the pathogen [27]. The best-characterized example in protozoan parasites is a class of specialized glycosylphosphatidylinositol (GPI) lipids. They include GPI-anchored lipophosphoglycans of Leishmania species, which are crucial for host specificity and survival of the parasite in the sand fly vector [28], [29], and are also thought to modulate the initial interaction with the mammalian host [30]. In African trypanosomes, GPI-anchored variant surface glycoproteins are at the heart of the antigenic variation mechanism used to enable chronic infection. In Plasmodium and Toxoplasma, precursor GPI lipids are believed to play important roles as toxins and immune modulators [31], [32], [33].
Due to the importance of specialized lipids in mycobacteria, lipid synthesis has been a major target of drug development for tuberculosis [34]. Such examples highlighting the important roles of lipids are also present in protozoan parasites. As we will describe in detail, kinetoplastids and apicomplexans rely on a number of mechanisms for lipid synthesis that are not found, or different from those used in the mammalian host. These include fatty acid synthesis in the mitochondrion and the plastid, specialized elongation and desaturation pathways, and differences in downstream pathways of phospholipid synthesis. The success of miltefosine as an orally available Leishmania drug is one important validation of lipid metabolism as a drug target. Lipid turnover in pathogens is complex and involves numerous mechanisms of uptake and synthesis. In the following we will systematically review fatty acid and phospholipid synthesis and uptake pathways in both apicomplexans and kinetoplastids.
Section snippets
Fatty acid synthesis in protozoan parasites
The genomes of protozoan parasites encode the enzymes for three distinct biochemical pathways involved in fatty acid synthesis. While there are significant differences between these pathways, the underlying chemistry and sequence of enzymatic reactions is highly conserved (Fig. 1). All pathways synthesize fatty acids by successive addition of two carbon units to a growing carboxylic acid chain that is held via the pantothenyl group of acyl carrier protein (ACP; see Table 1 for names of enzymes
Phospholipids of Kinetoplastida and Apicomplexa
Growth and proliferation of parasites depends on their ability to recruit the building blocks for membrane assembly. According to the paradigm of parasitism, it has long been thought that Kinetoplastida and Apicomplexa scavenge lipids for membrane formation from their hosts. However, recent genetic, biochemical, and pharmacological data have made it clear that de novo lipid synthesis plays a crucial role for parasite viability at different life cycle stages.
Phospholipids are the major lipid
Fatty acid and phospholipid synthesis pathways as drug targets
Because lipid biosynthetic pathways have long been considered of little importance for parasite survival, their potential as drug targets has not been systematically addressed. After a brief discussion of selected compounds known to affect lipid metabolism in Kinetoplastida and Apicomplexa, we highlight potentially interesting lipid pathways unique to certain members of these groups of parasites, or alternatively, are common to several parasite species but differ from the corresponding pathways
Acknowledgments
Work on T. brucei lipid metabolism in the lab of P.B. was supported by Swiss National Science Foundation Grant 31003A-130815. P.B. thanks B.J. Armstrong and A. Niederer for valuable input. Work on apicomplexan lipid metabolism was supported by grants from the National Institutes of Health (AI 64671 and AI 84415) to B.S. B.S. is a Georgia Research Alliance Distinguished Investigator and S.R. received a pre-doctoral fellowship from the American Heart Association.
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- 1
These authors contributed equally.
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Present address: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.