Review
Determinants of disease phenotype in trypanosomatid parasites Laura-Isobel McCall and James H. McKerrow Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, California, USA
Trypanosomatid parasites infect over 21 million people worldwide, with a range of disease phenotypes. Trypanosoma cruzi causes American trypanosomiasis, wherein 30–40% of infected individuals develop disease manifestations, most commonly cardiomyopathy but also digestive megasyndromes. In the case of Trypanosoma brucei, the etiological agent of African trypanosomiasis, disease progression can be rapid or slow, with early or late central nervous system involvement. Finally, Leishmania species cause leishmaniasis, a disease that ranges from self-healing but scarring cutaneous lesions to fatal visceral leishmaniasis in which parasites disseminate to the liver, spleen, and bone marrow. This review highlights parasite factors involved in disease phenotype in all three trypanosomatid diseases, with a particular focus on recent advances using large-scale ‘omics’ techniques. Trypanosomatid parasites: spectrum of disease Leishmania spp, Trypanosoma cruzi, and Trypanosoma brucei are protozoan parasites transmitted to mammalian hosts via their insect vectors, sandflies, triatomines, and tsetse flies, respectively. During their lifecycles Leishmania and T. cruzi have an intracellular mammalian stage, whereas T. brucei remains extracellular. T. cruzi causes American trypanosomiasis, commonly referred to as Chagas disease, in Central and South America (see Glossary) [1]. T. brucei causes African trypanosomiasis, also known as sleeping sickness [2]. Leishmania are the causative agents of leishmaniasis and are found worldwide in tropical and subtropical regions [3]. Jointly, these diseases cause over 4 million disabilityadjusted life years (DALYs). Twenty-one million people are currently infected, with over 2 million new cases per year [4]. Chagas disease is divided into an acute, usually asymptomatic phase, followed by the chronic stage of the disease. 60– 70% of infected individuals remain asymptomatic throughout their lifetime. Patients who progress to chronic symptomatic Chagas disease usually present with cardiomyopathy, but can also present with megacolon and/or megaesophagus, with or without cardiomyopathy [1]. Sleeping sickness manifests initially with fever and headache, and progresses to sleep disturbances and neurological disorders. It is typically fatal. Corresponding author: McKerrow, J.H. ([email protected]). Keywords: trypanosomes; leishmaniasis; Chagas disease; sleeping sickness; virulence factors; omics techniques. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.05.001
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Disease can be acute when caused by Trypanosoma brucei rhodesiense or chronic in the case of Trypanosoma brucei gambiense infection [2]. Leishmaniasis is found in three main forms: (i) self-healing but scarring cutaneous lesions; (ii) mucocutaneous disease with parasite dissemination to the nose, mouth and throat; and (iii) potentially fatal visceral
Glossary Amastin: small surface glycoproteins in trypanosomatids, implicated in disease progression. Cardiomyopathy: deterioration of heart muscle function, usually leading to heart failure. Chagas disease: American trypanosomiasis; disease caused by T. cruzi parasites. Chagas disease progresses from an acute phase immediately following parasite infection to a chronic, lifelong phase. Both phases can be asymptomatic; only 30–40% of infected individuals develop clinically manifest chronic Chagas disease with cardiac, digestive, or combined cardiodigestive symptoms. Cutaneous leishmaniasis (CL): skin disease caused by Leishmania parasites, characterized by parasite replication and lesion formation at the site of vector bite. Lesions are usually self-limiting and can include papules, nodules, or ulcers. Discrete typing units (DTUs): subdivision used to classify T. cruzi strains. Strains within a DTU are genetically more similar to each other than to strains from other DTUs (Box 1). gp90 and gp35/50 mucins: surface glycoproteins in T. cruzi. Their levels relative to other T. cruzi surface glycoproteins modulate the ability of the parasite to invade host cells. Hemolymphatic stage: initial stage of African trypanosomiasis (sleeping sickness) in which parasites are found in the blood and lymph. Symptoms include fever and headaches. Isolate: refers here to clinical isolates. Parasites isolated from a patient. Megacolon: enlargement of the colon. Megaesophagus: enlargement of the esophagus. Meningoencephalitic stage: second stage of African trypanosomiasis in which parasites cross the blood–brain barrier and are found in the central nervous system. Symptoms include sleep disturbances and neurological manifestations. Mucocutaneous leishmaniasis: disease caused by metastasis of some Leishmania spp to facial mucosal tissues, leading to ulceration and destruction of mucosal tissues in the nose, mouth, and throat. Serum resistance-associated (SRA) gene: gene responsible for T. brucei rhodesiense resistance to human serum. The encoded protein promotes TLF1 degradation. Strain: genetically distinct variant of a given species. Surface antigens: markers found on the parasite surface that elicit a host immune response. Tropism: preferential colonization of specific tissues by the parasite. Trypanosome lytic factors (TLF): human serum high-density lipoproteins that cause lysis and killing of T. brucei parasites that infect animals. The toxic components of TLF1 are apolipoprotein L-1 and haptoglobin-related protein. Human-pathogenic T. brucei subspecies have evolved mechanisms to resist killing by these factors. Trypomastigote small surface antigen (TSSA): a surface glycoprotein expressed in the trypomastigote stage of the T. cruzi lifecycle and which is involved in host cell invasion. Visceral leishmaniasis (VL): disease caused by some Leishmania spp in which parasites disseminate to the liver, spleen, and bone marrow. Symptoms include high fever and enlargement of the liver and spleen.
Review leishmaniasis with liver and spleen parasitemia [3]. Although host and environmental factors clearly influence disease, a common link between all three trypanosomatids is that parasite factors are a key determinant of disease phenotype. Underlying genetic differences between parasite strains and species lead to changes in the structure, activity, or levels of these factors. They are usually proteins, but can also include carbohydrates and nucleic acids. Parasite determinants of disease manifestation in Chagas disease Differences in tropism between discrete typing units (DTUs) T. cruzi is now classified into six DTUs, T. cruzi I (TcI), TcII, TcIII, TcIV, TcV, and TcVI, with multiple strains within each DTU (Box 1, Table 1). DTUs vary with regard to main geographic location and ecological niche, host, and vector preference [5]. Digestive syndromes are more prevalent in areas where TcII, TcV, and TcVI parasites predominate, including Argentina, Brazil, Bolivia, and Chile, but are rarer in areas where TcI and TcIV are found [1,5], suggesting that there may be an association between infecting DTU and disease manifestation, although this has yet to be confirmed. The strongest link between DTUs and disease phenotype was observed in Colombia: infection with TcI was associated with increased prevalence of cardiac alterations compared to infection with TcII, even after controlling for the lower overall prevalence of TcII [6]. Different strains were also observed between heart and esophagus biopsy samples in Brazil. However, the heart and esophagus samples came from separate patients in all but two cases, making it difficult to distinguish between variations in patient exposure to parasite strains and variations in parasite tropism [7]. Similarly, a possible link was suggested between TcII, TcV, and TcVI and megacolon [8]. By contrast, a later Colombian study using cardiac tissue samples rather than peripheral blood was unable to find a correlation between strain and disease manifestation [9]. A similar lack of association was observed in Brazil [10] and Bolivia [11,12]. Overall, a major limitation is that many of these studies were performed with samples only from peripheral blood [6,8,10,11]. There are reports of variations between circulating and organ-associated parasites [13], suggesting that Box 1. T. cruzi classification into DTUs There is considerable genetic diversity and significant variations in disease manifestations and disease severity within the T. cruzi species. To address this, T. cruzi has recently been reclassified into six discrete typing units (DTUs), TcI to TcVI, in which TcV and TcVI are hybrids derived from TcII and TcIII. DTUs differ in their geographical distribution, ecological niche, and vector and reservoir hosts. Their associations with specific disease phenotypes are an important topic currently under investigation. Each DTU can include multiple parasite strains which are more closely related to each other than to strains from other DTUs. This is manifested by a greater genetic similarity within a DTU than between DTUs, such that members of one DTU can be distinguished from other DTUs by the presence of common molecular markers. However, strains within a DTU are not necessarily identical and can also be distinguished from each other within the DTU using other molecular markers. The concept of DTUs has recently been reviewed in detail by Zingales et al. [5].
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Table 1. Representative T. cruzi strains by DTU DTU TcI
TcII
TcIII TcIV TcV TcVI a
Ref [87]
[87]
[87] [87] [87] [87]
Unless otherwise specified, isolates are from a human source.
b c
Strain a CA-I Colombian Dm28cb Gb Sylvio X10 12SF 21SF Peruvian Y 3869 Can III NRcl3 CL-Brenerc Tulahuen
Isolated from reservoir.
Isolated from vector.
peripheral blood samples may not be representative of the situation within affected organs. This is further complicated by the fact that coinfection with multiple parasite strains may be common [6]. Finally, these studies are limited to associations between local parasites and disease, making it difficult to determine whether the absence of a specific DTU in patients with a given disease phenotype is due to parasite factors or to lack of patient exposure to this DTU. Variations in pathology between T. cruzi strains from TcI and TcII were maintained in separate mouse strains, demonstrating that parasite factors are involved in determining disease severity in mice [14]. This forms the basis for the comparison of disease tropism between T. cruzi strains in mice. With regard to studies of cardiac pathology, some TcII strains caused more severe lesions than TcI or TcVI strains, whereas other TcII strains were less virulent [14–16]. Inflammation during chronic infection caused by TcVI was higher than during TcI infections, whereas TcV did not cause any inflammation [17]. In organs other than the heart, higher parasitism of spleen, liver, smooth muscle, and bone marrow was observed for TcII than for TcVI [18]. A large-scale study of 25 T. cruzi strains from TcI or TcIV found a broader organ tropism for TcI, whereas TcIV inflammation was restricted to skeletal muscle [19]. By contrast, CA-I strain (TcI) infected only cardiac and skeletal muscle of immunocompetent mice, leading to cardiac lesions and cardiac failure that recapitulate the disease seen in human infections [20]. Generalized conclusions are difficult to form from these studies. There is significant intra-DTU variation [21]; most of these studies used a single isolate from each DTU, and none compared all six DTUs. In addition, many of these studies used single reference strains, and often detailed patient information is lacking. This makes it difficult to determine whether disease severity and phenotype in mouse models accurately reflect disease phenotype in humans. Parasite factors influencing disease phenotype The variations in tropism and virulence described above in murine models are the basis for investigations into factors responsible for this variation. The diversity of surface 343
Review antigens between DTUs is most likely responsible for the variability in the cell invasion process between strains [22]. For instance, lower levels of gp90 and gp35/50 mucins have been suggested to promote higher invasiveness of CLBrener (TcVI) and Y (TcII) than G (TcI) [23]. The trypomastigote small surface antigen (TSSA) varied between DTUs [24], and TSSA from CL-Brener but not Sylvio X10 (TcI) was involved in adhesion to host cells [25]. Amastin sequence and expression levels also differed between DTUs [26,27]. Cells expressing higher amastin levels were detected earlier in the liver, suggesting a possible role in tropism [26]. Variations in other virulence factors have been observed; in particular, alterations in the glutathione binding site and expression levels of Tc52, a secreted virulence factor that alters host immune responses, were observed between DTUs [28]. Variation in expression of antioxidant tryparedoxin peroxidases and in glucose-6-phosphate and 6-phosphogluconate dehydrogenases were associated with increased resistance of TcVI to hydrogen peroxide and peroxynitrite [29,30]. The contribution of omics studies The CL-Brener (TcVI) strain was the first T. cruzi genome to be sequenced. TcVI are hybrids of TcII and TcIII; thus, the TcVI genome was divided into TcII-like and TcIII-like haplotypes [31]. More recently, the Sylvio X10/1 (TcI) [32] and the Dm28c (TcI) genomes were sequenced [33], providing representative genomes from four DTUs. Sequencing efforts are ongoing for representative strains from TcIV and TcV [34]. Comparison of the Sylvio X10/1 and CLBrener genomes highlighted many differences, including 17791 non-synonymous changes leading to chemically dissimilar amino acids. In addition, there were 1861 coding insertions or deletions in 1271 genes. Six very short open reading frames (ORFs) found in CL-Brener were absent in Sylvio X10/1. Finally, multigene families are significantly expanded in CL-Brener compared to Sylvio X10/1 [32]. An additional 16 strains were compared by genomic DNA hybridization to arrays designed from the CL-Brener genome. This highlighted significant copy-number variations, especially in large gene families of surface proteins such as mucins and trans-sialidases, as well as in enzymes involved in glycan synthesis, and in mitogen-activated protein kinases [35]. With regard to proteomics studies, comparison of 28 strains from all six DTUs identified nine DTU-specific proteins, two for TcIII and one each from the remaining DTUs. Most interesting are the proteins representative of TcV and TcVI, heat shock protein 85 and elongation factor 2, respectively. The remaining DTU-specific proteins are tubulins, making the link with virulence and disease manifestations unclear [36]. Parasite determinants of disease manifestation in African trypanosomiasis Trypanosoma brucei rhodesiense versus Trypanosoma brucei gambiense virulence Sleeping sickness progresses through two phases: the first hemolymphatic stage, where trypanosomes are found in the peripheral circulation, followed by the second meningoencephalitic stage, involving the central nervous system. 344
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Patients infected with T. b. rhodesiense, endemic to East Africa, progress to the second stage in a matter of weeks; conversely, patients infected with T. b. gambiense, found in West and Central Africa, may take over 1 year to progress to the first stage. T. b. rhodesiense is therefore generally considered to cause acute disease, and T. b. gambiense chronic disease. Fever for the first stage of disease, and second-stage neurological disorders and tremors are more common for T. b. rhodesiense versus T. b. gambiense infections. Insomnia, neurological disorders, and hepatosplenomegaly are more common in travelers infected with T. b. gambiense, whereas psychiatric disorders are more common in those infected with T. b. rhodesiense [37]. However, there is also variation within a subspecies; most human studies have focused on variations between T. b. rhodesiense foci (e.g., [38]). Similarly, T. b. gambiense has been subdivided into two groups, with group 1 being milder and more chronic than group 2 [39]. Similarly to human infections, most T. b. gambienseinfected BALB/c mice developed chronic infections whereas all tested T. b. rhodesiense isolates caused acute disease in this model, with accelerated mortality and elevated parasitemia. As in humans, variability in disease manifestations were also observed, with some T. b. gambiense isolates having high virulence and others causing chronic or sub-chronic infections [40]. These animal experiments, however, indicate that the observed disparities in human disease phenotype are due at least in part to parasite factors. Parasite factors influencing disease phenotype Comparative studies of T. b. gambiense and T. b. rhodesiense have focused mainly on resistance to trypanosome lytic factors (TLFs) found in human serum. T. b. gambiense group 1 express lower levels of a lower-affinity mutant of the T. brucei gambiense haptoglobin–hemoglobin receptor (TbgHpHbR) involved in the uptake of TLF1 [41–43]. T. b. gambiense also expresses T. b. gambiense-specific glycoprotein TgsGP, which promotes parasite membrane stiffening, thereby limiting membrane insertion and killing potential of lytic factors [44,45]. By contrast, serum resistance in T. b. rhodesiense is via the serum resistanceassociated (SRA) gene that is found only in this subspecies and is absent in T. b. gambiense. SRA interacts with TLF1 and promotes its degradation [46]. The contribution of omics studies Although T. b. rhodesiense usually causes acute disease, some strains have been associated with chronic disease. Genomes of representative T. b. gambiense and T. b. rhodesiense isolates have now been sequenced [47,48]. Sites at which classical acute disease-causing T. b. rhodesiense strains differ from the reference T. b. gambiense may be especially interesting because they could represent genes required for the establishment of chronic or acute disease. These are found on several chromosomes, in particular chromosomes 1 and 3. However, there are many more sites at which the T. b. rhodesiense isolate associated with chronic disease diverged from T. b. gambiense and where the acute disease-causing T. b. rhodesiense isolate resembled T. b. gambiense. These include large segments
Review of chromosomes 5, 8, and 10. This suggests that the mechanisms involved in the establishment of chronic versus acute disease may vary between T. b. gambiense and T. b. rhodesiense, but awaits confirmation through whole-genome sequencing of more strains. The 4000–6500 homozygous mutations shared by T. b. rhodesiense strains may be more relevant to identify factors influencing disease phenotype in East Africa than factors influencing disease phenotype in general [47]. Unfortunately, there have been few proteomics studies comparing T. brucei endemic to East versus West Africa. Within T. b. gambiense, the secretomes of strains from groups 1 and 2 were compared. A few strain-specific proteins were identified, including 36 specific to group 1 and 14 specific to group 2, the latter of which include proteins involved in folding and degradation [49].
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Table 2. Leishmania species and disease phenotype Old World
Cutaneous a
Visceral a New World
Cutaneous a Cutaneousa with the ability to cause mucocutaneous leishmaniasis
Visceral a a
L. L. L. L. L. L. L. L.
major tropica b aethiopica c donovani d infantum d mexicana c peruviana braziliensis e
L. L. L. L.
guyanensis e panamensis amazonensis f chagasi (= L. infantum) d
Most common disease manifestation.
b
Parasite determinants of leishmaniasis: self-contained or disseminated In contrast to studies of American and African trypanosomiasis, the link between the infecting parasite and disease phenotype is much clearer for leishmaniasis. Members of the Leishmania donovani species complex, including L. donovani, Leishmania chagasi, and Leishmania infantum, almost invariably cause visceral leishmaniasis (VL), usually in the absence of cutaneous manifestations in immunocompetent humans, whereas the remaining humaninfective Leishmania spp usually cause cutaneous leishmaniasis (CL), with varying degrees of severity: localized cutaneous, diffuse cutaneous, and disseminated or mucocutaneous leishmaniasis [3]. Cure of VL can be followed by post-kala-azar dermal leishmaniasis (PKDL), but this considered to be influenced mainly by host rather than parasite factors [50], and as such will not be discussed further here. Visceral versus cutaneous disease phenotype Most of the comparisons between CL- and VL-causing species have focused on the Leishmania major– L. donovani dichotomy. Importantly, disease phenotype can be replicated in mouse models; L. major causes progressive footpad swelling in susceptible mouse strains such as BALB/c following subcutaneous injection [51] and persisted at higher levels at intradermal injection sites [52]. Intradermal or visceral injection of L. donovani is associated with high visceral organ parasite levels [53,54]. Similarly, in hamsters, parasite load is much higher in the spleen and liver in L. donovani infection compared to L. major infection [55]. The ability to colonize internal organs and cause VL has been tied to thermotolerance [56,57]. The A2 family of proteins, expressed in L. donovani but not in L. major, promote resistance to the warmer temperatures found in these organs [58]. Originally identified as an amastigote (mammalian stage)-specific gene [59], A2 was subsequently shown to be stress-inducible and to protect against heat shock and oxidative stress [58,60], although the exact mechanism remains to be resolved. Introduction of the L. donovani A2 gene into L. major improves its ability to infect visceral organs [54] while decreasing its ability to cause footpad swelling [61].
Also causes visceral, viscerotropic leishmaniasis and lupoid leishmaniasis (leishmaniasis recidivans).
c
May be associated with diffuse cutaneous leishmaniasis.
d e f
Some reports of cutaneous leishmaniasis.
May cause disseminated leishmaniasis.
Some reports of visceral leishmaniasis.
Cutaneous versus mucocutaneous disease phenotype Mucocutaneous leishmaniasis is caused by parasites from the Viannia subgenus (Leishmania Viannia guyanensis, Leishmania Viannia panamensis, and Leishmania Viannia braziliensis) and from the Leishmania amazonensis species (Table 2) [62]. Much work has focused on comparing the host response in primary cutaneous lesions to mucocutaneous lesions (e.g., [63]) and on the role of host genetic background [64]. However, parasite factors are also involved. In L. guyanensis, metastatic ability has been tied to elevated peroxidase activity and increased resistance to oxidative stress [65]. Infection of L. guyanensis [66] or L. braziliensis [67] with the Leishmania RNA virus, promoting an exacerbated proinflammatory host response, may also promote mucosal disease [66]. Atypical strains: potential to identify additional determinants of disease There are several reports of L. donovani causing CL rather than VL. Sri Lanka is a major focus of CL caused by L. donovani [68], but atypical leishmaniasis cases caused by L. donovani or L. infantum have been described worldwide (e.g., [69]). Symptoms for these cases may be dissimilar from classical cutaneous leishmaniasis, for instance with regards to age of patients, parasite burden, and increased frequency of non-ulcerative lesions [70]. Characterization of L. donovani in Sri Lanka is currently ongoing. The reverse situation has also been identified. For example, Leishmania tropica was found to cause VL (e.g., [71]). L. tropica parasites isolated from patients with VL were associated with higher visceral parasite burden in hamsters infected by the intracardiac route than L. tropica isolated from CL patients. Increased visceralization was also observed for the former following subcutaneous footpad infection in hamsters [72]. However, these parasites have not been further characterized. L. amazonensis has also been reported to cause VL in the New World [73–75], 345
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but comparison of L. amazonensis isolates from CL and VL patients has so far led to conflicting results in animal models [73,76] and therefore awaits further investigation. The contribution of omics studies Genomic sequencing of L. infantum, L. major, L. braziliensis, and Leishmania mexicana highlighted 19 L. infantumspecific genes, two L. mexicana-specific genes, 14 L. majorspecific genes, and 67 L. braziliensis-specific genes [77,78]. Recently, the genome sequence of another member of the L. mexicana species complex, L. amazonensis, was also published, allowing the identification of factors characteristic of this species complex. These include a 30 -nucleotidase/ nuclease, expansion of the aminotransferase gene family, and the presence of amastin clades distinct from those found in other Leishmania species [79]. Genes present in L. infantum but absent in L. major were ectopically expressed in L. major. Three of these, the hypothetical LinJ.28.0340, the nucleotide sugar transporter LinJ.15.0900, and the cytosolic glyceraldehyde-3-phosphate dehydrogenase LinJ.36.2480, were able to increase L. major liver and spleen parasite burden, indicating a possible role in the ability to cause VL [80,81]. However,
LinJ.15.0900 and LinJ.36.2480 are present in L. mexicana (Table 3). Significant differences in chromosome ploidy have been identified between Leishmania strains and species [77,79,82], possibly leading to gene dosage effects. Similarly, multigene family loci were amplified in certain Leishmania spp [79]. However, the role of these in disease phenotype has not been examined; mutations in key genes may also be particularly important. Given the evolutionary distance between L. major and L. donovani, the identification of the key disease-determining mutations is difficult using L. major and L. donovani genome sequences. Sequencing of atypical clinical isolates, such as L. donovani isolates that cause CL rather than VL, may be able to identify such key mutations. Sequencing of multiple clinical isolates of metastatic and non-metastatic subgenus Viannia parasites may also help to identify additional factors that promote mucocutaneous disease. With regards to proteomic studies, comparison of metastatic and non-metastatic L. guyanensis identified variations in expression patterns for elongation factor 1b (EF-1b) and cytosolic tryparedoxin peroxidase (TXNPx). However, the pattern observed in the metastatic versus
Table 3. Leishmania species-specific genes L. infantum-specific genes L. infantum
L. major
L. mexicana
L. braziliensis
LinJ.02.0720 LinJ.08.0750 LinJ.09.1580
(LmjF.08.0795) c (LmjF.09.1500)
(LmxM.08_29.0795) (LmxM.09.1500)
(LbrM.09.1560)
LinJ.10.1170 LinJ.15.1660 LinJ.18.1030 LinJ.19.1120 LinJ.20.1200 LinJ.21.1810 LinJ.22.0300 LinJ.22.1570 LinJ.24.0450 LinJ.24.2130 LinJ.28.0340 LinJ.29.0320
(LmjF.10.1081) (LmjF.20.1175) (LmjF.24.2045) (LmjF.28.0420)
(LmxM.10.1085) (LmxM.20.1175) (LmxM.28.0420)
(LbrM.10.1190) (LbrM.28.0430)
LinJ.29.0650
LinJ.31.2120
LmxM.30.2465 (not pseudogene) (LmxM.31.1505) (LbrM.32.1680) LinJ.32.1580 Visceralizing genes absent in L. major but present in L. mexicana LinJ.15.0900 (LmjF.15.0840) LmxM.15.0840 LinJ.31.2550
(LmjF.31.2461)
LinJ.22.0670
LinJ.36.2480
(LmjF.36.2350)
LmxM.22.0691 LmxM.22.0692 LmxM.36.2350
a
N/S, non-significant.
b
N/D, not determined.
c
Gene names in italics and in parentheses represent pseudogenes.
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Function
Impact on virulence
Refs
Introduction into L. major N/S a N/D N/D
Knockout in L. donovani N/D b N/D N/D
[80] [77,80] [77]
N/S N/D N/D N/D N/D N/D N/D N/D N/D N/S 11–14 increase N/D
N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 200 decrease N/D
[77,80] [77] [77] [77] [77,80] [77] [77,80] [77] [77] [80] [77,80] [77]
N/D
N/D
[77]
N/D
N/D
[77]
N/S
N/D
[77,80]
Hypothetical
N/S
N/D
[77,81]
Nucleotide sugar transporter, putative A2
18–20 increase
N/S
[80,88]
3 increase
25 decrease
[54,89]
Cytosolic glyceraldehyde 3-phosphate dehydrogenase
7–8 increase
4–10 decrease
[80]
Hypothetical Tuzin (putative) Cytochrome b5-like protein Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical protein, conserved BET1-like protein, putative Hypothetical protein conserved Hypothetical
Review non-metastatic laboratory clones was only maintained in two of the five clinical isolates [83]. 2D gel comparison of soluble proteins from L. major and L. donovani whole cell lysate found that there was little overlap in the results between the two, making comparison difficult [84]. Avenues to explore The factors determining disease phenotype are better understood for Leishmania than for T. cruzi or T. brucei, possibly due to the availability of short-term mouse models for leishmaniasis that can mimic human disease. However, even in the case of Leishmania, the factors already identified cannot individually account fully for the observed disease phenotype. Further investigation into parasite determinants of disease and into combinations of these factors is therefore still required, and these should be guided by recent technological advances, especially in omics techniques (Box 2). For example, Garcia et al. found that the large amount of DNA required to sequence T. brucei presented a major hurdle, leading to bias in favor of strains that can be easily recovered from patients and subsequently cultured [85]. However, methods have now been developed that radically decrease the amount of starting material required for analysis, up to and including single cell genomic sequencing [86]. Such techniques would eliminate the need for prior culture of clinical isolates, although cost remains a limiting issue. Most previous work on parasite factors involved in disease phenotype compared only a few strains or species, complicating the identification of the specific disease-determining factors. In addition, studies such as these require precise Box 2. Outstanding questions Trypanosomatid parasites Increased focus on hypothetical proteins and on species-specific genes. Increased focus on clones of a given strain or closely related strains that cause different disease phenotypes. Strain bank with detailed clinical history. T. cruzi Improved animal models. Comparison of biological properties, virulence, and tropism using multiple representatives of all six DTUs in a single study, with a focus on multiple human isolates with detailed patient history. Genome sequencing of strains from TcIV and TcV. Proteomic comparison of closely related strains. T. brucei Improved animal models, especially for T. b. gambiense. Sequencing additional T. b. gambiense and T. b. rhodesiense isolates. Large-scale proteomic comparison of T. b. gambiense and T. b. rhodesiense. Leishmania Sequencing and proteomic comparison of atypical L. donovani isolates. Sequencing and proteomic comparison of cutaneous and visceral L. tropica isolates. Further characterization of visceral L. amazonensis isolates. Sequencing of multiple Viannia subgenus parasites.
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and accurate patient clinical records for each isolate, and these are often lacking. Patient history before the isolation of the parasites and information on subsequent disease progression or response to treatment would be especially useful. The creation of a strain repository with detailed patient information would help researchers worldwide. Decreasing costs for sequencing and label-free proteomic approaches will allow genomic and proteomic comparisons of multiple clinical isolates simultaneously. Studying clones of a given strain that cause a range of disease phenotypes can help to pinpoint the specific determinants involved. This may be particularly important in the case of T. cruzi where thousands of genetic dissimilarities are observed between DTUs. However, any factor identified by these omics studies must still be validated, for instance by knocking out the identified factor and assessing the impact on parasite virulence. Finally, for all three trypanosomatids, parasite determinants strongly influence disease phenotype. However, even though these parasites are closely related with over 6000 shared genes, they cause disparate diseases. There should be increased investigation of genes specific to T. cruzi, Leishmania, and T. brucei. In addition, the ability to cause leishmaniasis, Chagas disease, or sleeping sickness is restricted to trypanosomatid parasites. As such, trypanosomatid-specific genes with no homology to genes found in other organisms may be key to disease pathogenesis. Further study of these genes and the development of tools to identify their functions will provide us with essential insight into the devastating disease caused by these parasites. Concluding remarks and future perspectives Almost 10 years after the publication of the first three trypanosomatid genomes, much knowledge is still lacking. Parasite characteristics are important determinants of disease phenotype caused by all three, but the specific factors are still often unknown, particularly for T. brucei and T. cruzi. Their identification will be facilitated by the application of large-scale omics techniques. Hopefully this work will help guide the drug development and disease elimination efforts that are underway. References 1 Rassi, A. et al. (2010) Chagas disease. Lancet 375, 1388–1402 2 Brun, R. et al. (2010) Human African trypanosomiasis. Lancet 375, 148–159 3 Murray, H.W. et al. (2005) Advances in leishmaniasis. Lancet 366, 1561–1577 4 Mathers, C.D. et al. (2007) Measuring the burden of neglected tropical diseases: the global burden of disease framework. PLoS Negl. Trop. Dis. 1, e114 5 Zingales, B. et al. (2012) The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect. Genet. Evol. 12, 240–253 6 Ramirez, J.D. et al. (2010) Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients. PLoS Negl. Trop. Dis. 4, e899 7 Vago, A.R. et al. (2000) Genetic characterization of Trypanosoma cruzi directly from tissues of patients with chronic Chagas disease: differential distribution of genetic types into diverse organs. Am. J. Pathol. 156, 1805–1809 8 Luquetti, A.O. et al. (1986) Trypanosoma cruzi: zymodemes associated with acute and chronic Chagas’ disease in central Brazil. Trans. R. Soc. Trop. Med. Hyg. 80, 462–470 347
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33 Grisard, E.C. et al. (2014) Trypanosoma cruzi clone Dm28c draft genome sequence. Genome Announc. 2, e01114-13 34 Teixeira, S.M. et al. (2012) Trypanosomatid comparative genomics: contributions to the study of parasite biology and different parasitic diseases. Genet. Mol. Biol. 35, 1–17 35 Minning, T.A. et al. (2011) Widespread, focal copy number variations (CNV) and whole chromosome aneuploidies in Trypanosoma cruzi strains revealed by array comparative genomic hybridization. BMC Genomics 12, 139 36 Telleria, J. et al. (2010) Phylogenetic character mapping of proteomic diversity shows high correlation with subspecific phylogenetic diversity in Trypanosoma cruzi. Proc. Natl. Acad. Sci. U.S.A. 107, 20411–20416 37 Urech, K. et al. (2011) Sleeping sickness in travelers – do they really sleep? PLoS Negl. Trop. Dis. 5, e1358 38 Maclean, L. et al. (2007) Spatially and genetically distinct African Trypanosome virulence variants defined by host interferon-gamma response. J. Infect. Dis. 196, 1620–1628 39 Truc, P. et al. (1997) Confirmation of two distinct classes of zymodemes of Trypanosoma brucei infecting man and wild mammals in Cote d’Ivoire: suspected difference in pathogenicity. Ann. Trop. Med. Parasitol. 91, 951–956 40 Inoue, N. et al. (1998) Susceptibility of severe combined immunodeficient (SCID) mice to Trypanosoma brucei gambiense and T. b. rhodesiense. Trop. Med. Int. Health 3, 408–412 41 Kieft, R. et al. (2010) Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor. Proc. Natl. Acad. Sci. U.S.A. 107, 16137–16141 42 Capewell, P. et al. (2011) Differences between Trypanosoma brucei gambiense groups 1 and 2 in their resistance to killing by trypanolytic factor 1. PLoS Negl. Trop. Dis. 5 43 Symula, R.E. et al. (2012) Trypanosoma brucei gambiense group 1 is distinguished by a unique amino acid substitution in the HpHb receptor implicated in human serum resistance. PLoS Negl. Trop. Dis. 6, e1728 44 Capewell, P. et al. (2013) The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense. Plos Pathog. 9, e1003686 45 Uzureau, P. et al. (2013) Mechanism of Trypanosoma brucei gambiense resistance to human serum. Nature 501, 430–434 46 Stephens, N.A. and Hajduk, S.L. (2011) Endosomal localization of the serum resistance-associated protein in African trypanosomes confers human infectivity. Eukaryot. Cell 10, 1023–1033 47 Goodhead, I. et al. (2013) Whole-genome sequencing of Trypanosoma brucei reveals introgression between subspecies that is associated with virulence. MBio 4 48 Jackson, A.P. et al. (2010) The genome sequence of Trypanosoma brucei gambiense, causative agent of chronic human african trypanosomiasis. PLoS Negl. Trop. Dis. 4, e658 49 Geiger, A. et al. (2010) Exocytosis and protein secretion in Trypanosoma. BMC Microbiol. 10, 20 50 Ganguly, S. et al. (2010) Post-kala-azar dermal leishmaniasis – an overview. Int. J. Dermatol. 49, 921–931 51 Sulahian, A. et al. (1997) Experimental pathogenicity of viscerotropic and dermotropic isolates of Leishmania infantum from immunocompromised and immunocompetent patients in a murine model. FEMS Immunol. Med. Microbiol. 17, 131–138 52 Nicolas, L. et al. (2000) Leishmania major reaches distant cutaneous sites where it persists transiently while persisting durably in the primary dermal site and its draining lymph node: a study with laboratory mice. Infect. Immun. 68, 6561–6566 53 Ahmed, S. et al. (2003) Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis. Infect. Immun. 71, 401–410 54 Zhang, W.W. and Matlashewski, G. (2001) Characterization of the A2A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection. Mol. Microbiol. 39, 935–948 55 Duarte, M.I.S. et al. (1988) Comparative-study of the biological behavior in hamster of 2 isolates of Leishmania characterized respectively as L-Major-like and L-Donovani. Rev. Inst. Med. Trop. Sao Paulo 30, 21–27 56 Hoyer, C. et al. (2001) Use of genetic complementation to identify gene(s) which specify species-specific organ tropism of Leishmania. Med. Microbiol. Immunol. 190, 43–46
Review 57 Callahan, H.L. et al. (1996) Leishmania spp: Temperature sensitivity of promastigotes in vitro as a model for tropism in vivo. Exp. Parasitol. 84, 400–409 58 McCall, L.I. and Matlashewski, G. (2010) Localization and induction of the A2 virulence factor in Leishmania: evidence that A2 is a stress response protein. Mol. Microbiol. 77, 518–530 59 Charest, H. and Matlashewski, G. (1994) Developmental GeneExpression in Leishmania-Donovani – differential cloning and analysis of an amastigote-stage-specific gene. Mol. Cell. Biol. 14, 2975–2984 60 McCall, L.I. and Matlashewski, G. (2012) Involvement of the Leishmania donovani virulence factor A2 in protection against heat and oxidative stress. Exp. Parasitol. 132, 109–115 61 Zhang, W.W. et al. (2003) Comparison of the A2 gene locus in Leishmania donovani and Leishmania major and its control over cutaneous infection. J. Biol. Chem. 278, 35508–35515 62 Reithinger, R. et al. (2007) Cutaneous leishmaniasis. Lancet Infect. Dis. 7, 581–596 63 Maretti-Mira, A.C. et al. (2012) Transcriptome patterns from primary cutaneous Leishmania braziliensis infections associate with eventual development of mucosal disease in humans. PLoS Negl. Trop. Dis. 6, e1816 64 Sakthianandeswaren, A. et al. (2009) The role of host genetics in leishmaniasis. Trends Parasitol. 25, 383–391 65 Acestor, N. et al. (2006) Resistance to oxidative stress is associated with metastasis in mucocutaneous leishmaniasis. J. Infect. Dis. 194, 1160– 1167 66 Ives, A. et al. (2011) Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science 331, 775–778 67 Zangger, H. et al. (2013) Detection of Leishmania RNA virus in Leishmania parasites. PLoS Negl. Trop. Dis. 7, e2006 68 Karunaweera, N.D. (2009) Leishmania donovani causing cutaneous leishmaniasis in Sri Lanka: a wolf in sheep’s clothing? Trends Parasitol. 25, 458–463 69 Sharma, N.L. et al. (2005) Localized cutaneous leishmaniasis due to Leishmania donovani and Leishmania tropica: Preliminary findings of the study of 161 new cases from a new endemic focus in Himachal Pradesh, India. Am. J. Trop. Med. Hyg. 72, 819–824 70 De Lima, H. et al. (2009) Cutaneous leishmaniasis due to Leishmania chagasi/Le. infantum in an endemic area of Guarico State, Venezuela. Trans. R. Soc. Trop. Med. Hyg. 103, 721–726 71 Khanra, S. et al. (2011) Characterization of the recent clinical isolates of Indian Kala-azar patients by RAPD-PCR method. J. Parasit. Dis. 35, 116–122 72 Lira, R. et al. (1998) Leishmania tropica: The identification and purification of metacyclic promastigotes and use in establishing mouse and hamster models of cutaneous and visceral disease. Exp. Parasitol. 89, 331–342 73 Barral, A. et al. (1986) Isolation of Leishmania-Mexicana-Amazonensis from the bone-marrow in a case of american visceral leishmaniasis. Am. J. Trop. Med. Hyg. 35, 732–734
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74 Barral, A. et al. (1991) Leishmaniasis in Bahia, Brazil – evidence That Leishmania-Amazonensis produces a wide spectrum of clinical-disease. Am. J. Trop. Med. Hyg. 44, 536–546 75 Aleixo, J.A. et al. (2006) Atypical American visceral leishmaniasis caused by disseminated Leishmania amazonensis infection presenting with hepatitis and adenopathy. Trans. R. Soc. Trop. Med. Hyg. 100, 79–82 76 Almeida, R.P. et al. (1996) Biological behavior of Leishmania amazonensis isolated from humans with cutaneous, mucosal, or visceral leishmaniasis in BALB/c mice. Am. J. Trop. Med. Hyg. 54, 178–184 77 Rogers, M.B. et al. (2011) Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania. Genome Res. 21, 2129–2142 78 Peacock, C.S. et al. (2007) Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat. Genet. 39, 839–847 79 Real, F. et al. (2013) The genome sequence of Leishmania (Leishmania) amazonensis: functional annotation and extended analysis of gene models. DNA Res. 20, 567–581 80 Zhang, W.W. and Matlashewski, G. (2010) Screening Leishmania donovani-specific genes required for visceral infection. Mol. Microbiol. 77, 505–517 81 Zhang, W.W. et al. (2008) A genomic-based approach combining in vivo selection in mice to identify a novel virulence gene in Leishmania. PLoS Negl. Trop. Dis. 2, e248 82 Downing, T. et al. (2011) Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance. Genome Res. 21, 2143–2156 83 Walker, J. et al. (2006) Comparative protein profiling identifies elongation factor-1 beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis. Mol. Biochem. Parasitol. 145, 254–264 84 Drummelsmith, J. et al. (2003) Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms. Mol. Cell. Proteomics 2, 146–155 85 Garcia, A. et al. (2006) Human African trypanosomiasis: connecting parasite and host genetics. Trends Parasitol. 22, 405–409 86 Shapiro, E. et al. (2013) Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat. Rev. Genet. 14, 618–630 87 Zingales, B. et al. (2009) A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem. Inst. Oswaldo Cruz 104, 1051–1054 88 Zhang, W.W. et al. (2011) Expression of a Leishmania donovani nucleotide sugar transporter in Leishmania major enhances survival in visceral organs. Exp. Parasitol. 129, 337–345 89 Zhang, W.W. and Matlashewski, G. (1997) Loss of virulence in Leishmania donovani deficient in an amastigote-specific protein, A2. Proc. Natl. Acad. Sci. U.S.A. 94, 8807–8811
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Determinants of disease phenotype in trypanosomatid parasites Laura-Isobel McCall and James H. McKerrow Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, California, USA
Trypanosomatid parasites infect over 21 million people worldwide, with a range of disease phenotypes. Trypanosoma cruzi causes American trypanosomiasis, wherein 30–40% of infected individuals develop disease manifestations, most commonly cardiomyopathy but also digestive megasyndromes. In the case of Trypanosoma brucei, the etiological agent of African trypanosomiasis, disease progression can be rapid or slow, with early or late central nervous system involvement. Finally, Leishmania species cause leishmaniasis, a disease that ranges from self-healing but scarring cutaneous lesions to fatal visceral leishmaniasis in which parasites disseminate to the liver, spleen, and bone marrow. This review highlights parasite factors involved in disease phenotype in all three trypanosomatid diseases, with a particular focus on recent advances using large-scale ‘omics’ techniques. Trypanosomatid parasites: spectrum of disease Leishmania spp, Trypanosoma cruzi, and Trypanosoma brucei are protozoan parasites transmitted to mammalian hosts via their insect vectors, sandflies, triatomines, and tsetse flies, respectively. During their lifecycles Leishmania and T. cruzi have an intracellular mammalian stage, whereas T. brucei remains extracellular. T. cruzi causes American trypanosomiasis, commonly referred to as Chagas disease, in Central and South America (see Glossary) [1]. T. brucei causes African trypanosomiasis, also known as sleeping sickness [2]. Leishmania are the causative agents of leishmaniasis and are found worldwide in tropical and subtropical regions [3]. Jointly, these diseases cause over 4 million disabilityadjusted life years (DALYs). Twenty-one million people are currently infected, with over 2 million new cases per year [4]. Chagas disease is divided into an acute, usually asymptomatic phase, followed by the chronic stage of the disease. 60– 70% of infected individuals remain asymptomatic throughout their lifetime. Patients who progress to chronic symptomatic Chagas disease usually present with cardiomyopathy, but can also present with megacolon and/or megaesophagus, with or without cardiomyopathy [1]. Sleeping sickness manifests initially with fever and headache, and progresses to sleep disturbances and neurological disorders. It is typically fatal. Corresponding author: McKerrow, J.H. ([email protected]). Keywords: trypanosomes; leishmaniasis; Chagas disease; sleeping sickness; virulence factors; omics techniques. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.05.001
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Disease can be acute when caused by Trypanosoma brucei rhodesiense or chronic in the case of Trypanosoma brucei gambiense infection [2]. Leishmaniasis is found in three main forms: (i) self-healing but scarring cutaneous lesions; (ii) mucocutaneous disease with parasite dissemination to the nose, mouth and throat; and (iii) potentially fatal visceral
Glossary Amastin: small surface glycoproteins in trypanosomatids, implicated in disease progression. Cardiomyopathy: deterioration of heart muscle function, usually leading to heart failure. Chagas disease: American trypanosomiasis; disease caused by T. cruzi parasites. Chagas disease progresses from an acute phase immediately following parasite infection to a chronic, lifelong phase. Both phases can be asymptomatic; only 30–40% of infected individuals develop clinically manifest chronic Chagas disease with cardiac, digestive, or combined cardiodigestive symptoms. Cutaneous leishmaniasis (CL): skin disease caused by Leishmania parasites, characterized by parasite replication and lesion formation at the site of vector bite. Lesions are usually self-limiting and can include papules, nodules, or ulcers. Discrete typing units (DTUs): subdivision used to classify T. cruzi strains. Strains within a DTU are genetically more similar to each other than to strains from other DTUs (Box 1). gp90 and gp35/50 mucins: surface glycoproteins in T. cruzi. Their levels relative to other T. cruzi surface glycoproteins modulate the ability of the parasite to invade host cells. Hemolymphatic stage: initial stage of African trypanosomiasis (sleeping sickness) in which parasites are found in the blood and lymph. Symptoms include fever and headaches. Isolate: refers here to clinical isolates. Parasites isolated from a patient. Megacolon: enlargement of the colon. Megaesophagus: enlargement of the esophagus. Meningoencephalitic stage: second stage of African trypanosomiasis in which parasites cross the blood–brain barrier and are found in the central nervous system. Symptoms include sleep disturbances and neurological manifestations. Mucocutaneous leishmaniasis: disease caused by metastasis of some Leishmania spp to facial mucosal tissues, leading to ulceration and destruction of mucosal tissues in the nose, mouth, and throat. Serum resistance-associated (SRA) gene: gene responsible for T. brucei rhodesiense resistance to human serum. The encoded protein promotes TLF1 degradation. Strain: genetically distinct variant of a given species. Surface antigens: markers found on the parasite surface that elicit a host immune response. Tropism: preferential colonization of specific tissues by the parasite. Trypanosome lytic factors (TLF): human serum high-density lipoproteins that cause lysis and killing of T. brucei parasites that infect animals. The toxic components of TLF1 are apolipoprotein L-1 and haptoglobin-related protein. Human-pathogenic T. brucei subspecies have evolved mechanisms to resist killing by these factors. Trypomastigote small surface antigen (TSSA): a surface glycoprotein expressed in the trypomastigote stage of the T. cruzi lifecycle and which is involved in host cell invasion. Visceral leishmaniasis (VL): disease caused by some Leishmania spp in which parasites disseminate to the liver, spleen, and bone marrow. Symptoms include high fever and enlargement of the liver and spleen.
Review leishmaniasis with liver and spleen parasitemia [3]. Although host and environmental factors clearly influence disease, a common link between all three trypanosomatids is that parasite factors are a key determinant of disease phenotype. Underlying genetic differences between parasite strains and species lead to changes in the structure, activity, or levels of these factors. They are usually proteins, but can also include carbohydrates and nucleic acids. Parasite determinants of disease manifestation in Chagas disease Differences in tropism between discrete typing units (DTUs) T. cruzi is now classified into six DTUs, T. cruzi I (TcI), TcII, TcIII, TcIV, TcV, and TcVI, with multiple strains within each DTU (Box 1, Table 1). DTUs vary with regard to main geographic location and ecological niche, host, and vector preference [5]. Digestive syndromes are more prevalent in areas where TcII, TcV, and TcVI parasites predominate, including Argentina, Brazil, Bolivia, and Chile, but are rarer in areas where TcI and TcIV are found [1,5], suggesting that there may be an association between infecting DTU and disease manifestation, although this has yet to be confirmed. The strongest link between DTUs and disease phenotype was observed in Colombia: infection with TcI was associated with increased prevalence of cardiac alterations compared to infection with TcII, even after controlling for the lower overall prevalence of TcII [6]. Different strains were also observed between heart and esophagus biopsy samples in Brazil. However, the heart and esophagus samples came from separate patients in all but two cases, making it difficult to distinguish between variations in patient exposure to parasite strains and variations in parasite tropism [7]. Similarly, a possible link was suggested between TcII, TcV, and TcVI and megacolon [8]. By contrast, a later Colombian study using cardiac tissue samples rather than peripheral blood was unable to find a correlation between strain and disease manifestation [9]. A similar lack of association was observed in Brazil [10] and Bolivia [11,12]. Overall, a major limitation is that many of these studies were performed with samples only from peripheral blood [6,8,10,11]. There are reports of variations between circulating and organ-associated parasites [13], suggesting that Box 1. T. cruzi classification into DTUs There is considerable genetic diversity and significant variations in disease manifestations and disease severity within the T. cruzi species. To address this, T. cruzi has recently been reclassified into six discrete typing units (DTUs), TcI to TcVI, in which TcV and TcVI are hybrids derived from TcII and TcIII. DTUs differ in their geographical distribution, ecological niche, and vector and reservoir hosts. Their associations with specific disease phenotypes are an important topic currently under investigation. Each DTU can include multiple parasite strains which are more closely related to each other than to strains from other DTUs. This is manifested by a greater genetic similarity within a DTU than between DTUs, such that members of one DTU can be distinguished from other DTUs by the presence of common molecular markers. However, strains within a DTU are not necessarily identical and can also be distinguished from each other within the DTU using other molecular markers. The concept of DTUs has recently been reviewed in detail by Zingales et al. [5].
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Table 1. Representative T. cruzi strains by DTU DTU TcI
TcII
TcIII TcIV TcV TcVI a
Ref [87]
[87]
[87] [87] [87] [87]
Unless otherwise specified, isolates are from a human source.
b c
Strain a CA-I Colombian Dm28cb Gb Sylvio X10 12SF 21SF Peruvian Y 3869 Can III NRcl3 CL-Brenerc Tulahuen
Isolated from reservoir.
Isolated from vector.
peripheral blood samples may not be representative of the situation within affected organs. This is further complicated by the fact that coinfection with multiple parasite strains may be common [6]. Finally, these studies are limited to associations between local parasites and disease, making it difficult to determine whether the absence of a specific DTU in patients with a given disease phenotype is due to parasite factors or to lack of patient exposure to this DTU. Variations in pathology between T. cruzi strains from TcI and TcII were maintained in separate mouse strains, demonstrating that parasite factors are involved in determining disease severity in mice [14]. This forms the basis for the comparison of disease tropism between T. cruzi strains in mice. With regard to studies of cardiac pathology, some TcII strains caused more severe lesions than TcI or TcVI strains, whereas other TcII strains were less virulent [14–16]. Inflammation during chronic infection caused by TcVI was higher than during TcI infections, whereas TcV did not cause any inflammation [17]. In organs other than the heart, higher parasitism of spleen, liver, smooth muscle, and bone marrow was observed for TcII than for TcVI [18]. A large-scale study of 25 T. cruzi strains from TcI or TcIV found a broader organ tropism for TcI, whereas TcIV inflammation was restricted to skeletal muscle [19]. By contrast, CA-I strain (TcI) infected only cardiac and skeletal muscle of immunocompetent mice, leading to cardiac lesions and cardiac failure that recapitulate the disease seen in human infections [20]. Generalized conclusions are difficult to form from these studies. There is significant intra-DTU variation [21]; most of these studies used a single isolate from each DTU, and none compared all six DTUs. In addition, many of these studies used single reference strains, and often detailed patient information is lacking. This makes it difficult to determine whether disease severity and phenotype in mouse models accurately reflect disease phenotype in humans. Parasite factors influencing disease phenotype The variations in tropism and virulence described above in murine models are the basis for investigations into factors responsible for this variation. The diversity of surface 343
Review antigens between DTUs is most likely responsible for the variability in the cell invasion process between strains [22]. For instance, lower levels of gp90 and gp35/50 mucins have been suggested to promote higher invasiveness of CLBrener (TcVI) and Y (TcII) than G (TcI) [23]. The trypomastigote small surface antigen (TSSA) varied between DTUs [24], and TSSA from CL-Brener but not Sylvio X10 (TcI) was involved in adhesion to host cells [25]. Amastin sequence and expression levels also differed between DTUs [26,27]. Cells expressing higher amastin levels were detected earlier in the liver, suggesting a possible role in tropism [26]. Variations in other virulence factors have been observed; in particular, alterations in the glutathione binding site and expression levels of Tc52, a secreted virulence factor that alters host immune responses, were observed between DTUs [28]. Variation in expression of antioxidant tryparedoxin peroxidases and in glucose-6-phosphate and 6-phosphogluconate dehydrogenases were associated with increased resistance of TcVI to hydrogen peroxide and peroxynitrite [29,30]. The contribution of omics studies The CL-Brener (TcVI) strain was the first T. cruzi genome to be sequenced. TcVI are hybrids of TcII and TcIII; thus, the TcVI genome was divided into TcII-like and TcIII-like haplotypes [31]. More recently, the Sylvio X10/1 (TcI) [32] and the Dm28c (TcI) genomes were sequenced [33], providing representative genomes from four DTUs. Sequencing efforts are ongoing for representative strains from TcIV and TcV [34]. Comparison of the Sylvio X10/1 and CLBrener genomes highlighted many differences, including 17791 non-synonymous changes leading to chemically dissimilar amino acids. In addition, there were 1861 coding insertions or deletions in 1271 genes. Six very short open reading frames (ORFs) found in CL-Brener were absent in Sylvio X10/1. Finally, multigene families are significantly expanded in CL-Brener compared to Sylvio X10/1 [32]. An additional 16 strains were compared by genomic DNA hybridization to arrays designed from the CL-Brener genome. This highlighted significant copy-number variations, especially in large gene families of surface proteins such as mucins and trans-sialidases, as well as in enzymes involved in glycan synthesis, and in mitogen-activated protein kinases [35]. With regard to proteomics studies, comparison of 28 strains from all six DTUs identified nine DTU-specific proteins, two for TcIII and one each from the remaining DTUs. Most interesting are the proteins representative of TcV and TcVI, heat shock protein 85 and elongation factor 2, respectively. The remaining DTU-specific proteins are tubulins, making the link with virulence and disease manifestations unclear [36]. Parasite determinants of disease manifestation in African trypanosomiasis Trypanosoma brucei rhodesiense versus Trypanosoma brucei gambiense virulence Sleeping sickness progresses through two phases: the first hemolymphatic stage, where trypanosomes are found in the peripheral circulation, followed by the second meningoencephalitic stage, involving the central nervous system. 344
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Patients infected with T. b. rhodesiense, endemic to East Africa, progress to the second stage in a matter of weeks; conversely, patients infected with T. b. gambiense, found in West and Central Africa, may take over 1 year to progress to the first stage. T. b. rhodesiense is therefore generally considered to cause acute disease, and T. b. gambiense chronic disease. Fever for the first stage of disease, and second-stage neurological disorders and tremors are more common for T. b. rhodesiense versus T. b. gambiense infections. Insomnia, neurological disorders, and hepatosplenomegaly are more common in travelers infected with T. b. gambiense, whereas psychiatric disorders are more common in those infected with T. b. rhodesiense [37]. However, there is also variation within a subspecies; most human studies have focused on variations between T. b. rhodesiense foci (e.g., [38]). Similarly, T. b. gambiense has been subdivided into two groups, with group 1 being milder and more chronic than group 2 [39]. Similarly to human infections, most T. b. gambienseinfected BALB/c mice developed chronic infections whereas all tested T. b. rhodesiense isolates caused acute disease in this model, with accelerated mortality and elevated parasitemia. As in humans, variability in disease manifestations were also observed, with some T. b. gambiense isolates having high virulence and others causing chronic or sub-chronic infections [40]. These animal experiments, however, indicate that the observed disparities in human disease phenotype are due at least in part to parasite factors. Parasite factors influencing disease phenotype Comparative studies of T. b. gambiense and T. b. rhodesiense have focused mainly on resistance to trypanosome lytic factors (TLFs) found in human serum. T. b. gambiense group 1 express lower levels of a lower-affinity mutant of the T. brucei gambiense haptoglobin–hemoglobin receptor (TbgHpHbR) involved in the uptake of TLF1 [41–43]. T. b. gambiense also expresses T. b. gambiense-specific glycoprotein TgsGP, which promotes parasite membrane stiffening, thereby limiting membrane insertion and killing potential of lytic factors [44,45]. By contrast, serum resistance in T. b. rhodesiense is via the serum resistanceassociated (SRA) gene that is found only in this subspecies and is absent in T. b. gambiense. SRA interacts with TLF1 and promotes its degradation [46]. The contribution of omics studies Although T. b. rhodesiense usually causes acute disease, some strains have been associated with chronic disease. Genomes of representative T. b. gambiense and T. b. rhodesiense isolates have now been sequenced [47,48]. Sites at which classical acute disease-causing T. b. rhodesiense strains differ from the reference T. b. gambiense may be especially interesting because they could represent genes required for the establishment of chronic or acute disease. These are found on several chromosomes, in particular chromosomes 1 and 3. However, there are many more sites at which the T. b. rhodesiense isolate associated with chronic disease diverged from T. b. gambiense and where the acute disease-causing T. b. rhodesiense isolate resembled T. b. gambiense. These include large segments
Review of chromosomes 5, 8, and 10. This suggests that the mechanisms involved in the establishment of chronic versus acute disease may vary between T. b. gambiense and T. b. rhodesiense, but awaits confirmation through whole-genome sequencing of more strains. The 4000–6500 homozygous mutations shared by T. b. rhodesiense strains may be more relevant to identify factors influencing disease phenotype in East Africa than factors influencing disease phenotype in general [47]. Unfortunately, there have been few proteomics studies comparing T. brucei endemic to East versus West Africa. Within T. b. gambiense, the secretomes of strains from groups 1 and 2 were compared. A few strain-specific proteins were identified, including 36 specific to group 1 and 14 specific to group 2, the latter of which include proteins involved in folding and degradation [49].
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Table 2. Leishmania species and disease phenotype Old World
Cutaneous a
Visceral a New World
Cutaneous a Cutaneousa with the ability to cause mucocutaneous leishmaniasis
Visceral a a
L. L. L. L. L. L. L. L.
major tropica b aethiopica c donovani d infantum d mexicana c peruviana braziliensis e
L. L. L. L.
guyanensis e panamensis amazonensis f chagasi (= L. infantum) d
Most common disease manifestation.
b
Parasite determinants of leishmaniasis: self-contained or disseminated In contrast to studies of American and African trypanosomiasis, the link between the infecting parasite and disease phenotype is much clearer for leishmaniasis. Members of the Leishmania donovani species complex, including L. donovani, Leishmania chagasi, and Leishmania infantum, almost invariably cause visceral leishmaniasis (VL), usually in the absence of cutaneous manifestations in immunocompetent humans, whereas the remaining humaninfective Leishmania spp usually cause cutaneous leishmaniasis (CL), with varying degrees of severity: localized cutaneous, diffuse cutaneous, and disseminated or mucocutaneous leishmaniasis [3]. Cure of VL can be followed by post-kala-azar dermal leishmaniasis (PKDL), but this considered to be influenced mainly by host rather than parasite factors [50], and as such will not be discussed further here. Visceral versus cutaneous disease phenotype Most of the comparisons between CL- and VL-causing species have focused on the Leishmania major– L. donovani dichotomy. Importantly, disease phenotype can be replicated in mouse models; L. major causes progressive footpad swelling in susceptible mouse strains such as BALB/c following subcutaneous injection [51] and persisted at higher levels at intradermal injection sites [52]. Intradermal or visceral injection of L. donovani is associated with high visceral organ parasite levels [53,54]. Similarly, in hamsters, parasite load is much higher in the spleen and liver in L. donovani infection compared to L. major infection [55]. The ability to colonize internal organs and cause VL has been tied to thermotolerance [56,57]. The A2 family of proteins, expressed in L. donovani but not in L. major, promote resistance to the warmer temperatures found in these organs [58]. Originally identified as an amastigote (mammalian stage)-specific gene [59], A2 was subsequently shown to be stress-inducible and to protect against heat shock and oxidative stress [58,60], although the exact mechanism remains to be resolved. Introduction of the L. donovani A2 gene into L. major improves its ability to infect visceral organs [54] while decreasing its ability to cause footpad swelling [61].
Also causes visceral, viscerotropic leishmaniasis and lupoid leishmaniasis (leishmaniasis recidivans).
c
May be associated with diffuse cutaneous leishmaniasis.
d e f
Some reports of cutaneous leishmaniasis.
May cause disseminated leishmaniasis.
Some reports of visceral leishmaniasis.
Cutaneous versus mucocutaneous disease phenotype Mucocutaneous leishmaniasis is caused by parasites from the Viannia subgenus (Leishmania Viannia guyanensis, Leishmania Viannia panamensis, and Leishmania Viannia braziliensis) and from the Leishmania amazonensis species (Table 2) [62]. Much work has focused on comparing the host response in primary cutaneous lesions to mucocutaneous lesions (e.g., [63]) and on the role of host genetic background [64]. However, parasite factors are also involved. In L. guyanensis, metastatic ability has been tied to elevated peroxidase activity and increased resistance to oxidative stress [65]. Infection of L. guyanensis [66] or L. braziliensis [67] with the Leishmania RNA virus, promoting an exacerbated proinflammatory host response, may also promote mucosal disease [66]. Atypical strains: potential to identify additional determinants of disease There are several reports of L. donovani causing CL rather than VL. Sri Lanka is a major focus of CL caused by L. donovani [68], but atypical leishmaniasis cases caused by L. donovani or L. infantum have been described worldwide (e.g., [69]). Symptoms for these cases may be dissimilar from classical cutaneous leishmaniasis, for instance with regards to age of patients, parasite burden, and increased frequency of non-ulcerative lesions [70]. Characterization of L. donovani in Sri Lanka is currently ongoing. The reverse situation has also been identified. For example, Leishmania tropica was found to cause VL (e.g., [71]). L. tropica parasites isolated from patients with VL were associated with higher visceral parasite burden in hamsters infected by the intracardiac route than L. tropica isolated from CL patients. Increased visceralization was also observed for the former following subcutaneous footpad infection in hamsters [72]. However, these parasites have not been further characterized. L. amazonensis has also been reported to cause VL in the New World [73–75], 345
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but comparison of L. amazonensis isolates from CL and VL patients has so far led to conflicting results in animal models [73,76] and therefore awaits further investigation. The contribution of omics studies Genomic sequencing of L. infantum, L. major, L. braziliensis, and Leishmania mexicana highlighted 19 L. infantumspecific genes, two L. mexicana-specific genes, 14 L. majorspecific genes, and 67 L. braziliensis-specific genes [77,78]. Recently, the genome sequence of another member of the L. mexicana species complex, L. amazonensis, was also published, allowing the identification of factors characteristic of this species complex. These include a 30 -nucleotidase/ nuclease, expansion of the aminotransferase gene family, and the presence of amastin clades distinct from those found in other Leishmania species [79]. Genes present in L. infantum but absent in L. major were ectopically expressed in L. major. Three of these, the hypothetical LinJ.28.0340, the nucleotide sugar transporter LinJ.15.0900, and the cytosolic glyceraldehyde-3-phosphate dehydrogenase LinJ.36.2480, were able to increase L. major liver and spleen parasite burden, indicating a possible role in the ability to cause VL [80,81]. However,
LinJ.15.0900 and LinJ.36.2480 are present in L. mexicana (Table 3). Significant differences in chromosome ploidy have been identified between Leishmania strains and species [77,79,82], possibly leading to gene dosage effects. Similarly, multigene family loci were amplified in certain Leishmania spp [79]. However, the role of these in disease phenotype has not been examined; mutations in key genes may also be particularly important. Given the evolutionary distance between L. major and L. donovani, the identification of the key disease-determining mutations is difficult using L. major and L. donovani genome sequences. Sequencing of atypical clinical isolates, such as L. donovani isolates that cause CL rather than VL, may be able to identify such key mutations. Sequencing of multiple clinical isolates of metastatic and non-metastatic subgenus Viannia parasites may also help to identify additional factors that promote mucocutaneous disease. With regards to proteomic studies, comparison of metastatic and non-metastatic L. guyanensis identified variations in expression patterns for elongation factor 1b (EF-1b) and cytosolic tryparedoxin peroxidase (TXNPx). However, the pattern observed in the metastatic versus
Table 3. Leishmania species-specific genes L. infantum-specific genes L. infantum
L. major
L. mexicana
L. braziliensis
LinJ.02.0720 LinJ.08.0750 LinJ.09.1580
(LmjF.08.0795) c (LmjF.09.1500)
(LmxM.08_29.0795) (LmxM.09.1500)
(LbrM.09.1560)
LinJ.10.1170 LinJ.15.1660 LinJ.18.1030 LinJ.19.1120 LinJ.20.1200 LinJ.21.1810 LinJ.22.0300 LinJ.22.1570 LinJ.24.0450 LinJ.24.2130 LinJ.28.0340 LinJ.29.0320
(LmjF.10.1081) (LmjF.20.1175) (LmjF.24.2045) (LmjF.28.0420)
(LmxM.10.1085) (LmxM.20.1175) (LmxM.28.0420)
(LbrM.10.1190) (LbrM.28.0430)
LinJ.29.0650
LinJ.31.2120
LmxM.30.2465 (not pseudogene) (LmxM.31.1505) (LbrM.32.1680) LinJ.32.1580 Visceralizing genes absent in L. major but present in L. mexicana LinJ.15.0900 (LmjF.15.0840) LmxM.15.0840 LinJ.31.2550
(LmjF.31.2461)
LinJ.22.0670
LinJ.36.2480
(LmjF.36.2350)
LmxM.22.0691 LmxM.22.0692 LmxM.36.2350
a
N/S, non-significant.
b
N/D, not determined.
c
Gene names in italics and in parentheses represent pseudogenes.
346
Function
Impact on virulence
Refs
Introduction into L. major N/S a N/D N/D
Knockout in L. donovani N/D b N/D N/D
[80] [77,80] [77]
N/S N/D N/D N/D N/D N/D N/D N/D N/D N/S 11–14 increase N/D
N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 200 decrease N/D
[77,80] [77] [77] [77] [77,80] [77] [77,80] [77] [77] [80] [77,80] [77]
N/D
N/D
[77]
N/D
N/D
[77]
N/S
N/D
[77,80]
Hypothetical
N/S
N/D
[77,81]
Nucleotide sugar transporter, putative A2
18–20 increase
N/S
[80,88]
3 increase
25 decrease
[54,89]
Cytosolic glyceraldehyde 3-phosphate dehydrogenase
7–8 increase
4–10 decrease
[80]
Hypothetical Tuzin (putative) Cytochrome b5-like protein Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical Hypothetical protein, conserved BET1-like protein, putative Hypothetical protein conserved Hypothetical
Review non-metastatic laboratory clones was only maintained in two of the five clinical isolates [83]. 2D gel comparison of soluble proteins from L. major and L. donovani whole cell lysate found that there was little overlap in the results between the two, making comparison difficult [84]. Avenues to explore The factors determining disease phenotype are better understood for Leishmania than for T. cruzi or T. brucei, possibly due to the availability of short-term mouse models for leishmaniasis that can mimic human disease. However, even in the case of Leishmania, the factors already identified cannot individually account fully for the observed disease phenotype. Further investigation into parasite determinants of disease and into combinations of these factors is therefore still required, and these should be guided by recent technological advances, especially in omics techniques (Box 2). For example, Garcia et al. found that the large amount of DNA required to sequence T. brucei presented a major hurdle, leading to bias in favor of strains that can be easily recovered from patients and subsequently cultured [85]. However, methods have now been developed that radically decrease the amount of starting material required for analysis, up to and including single cell genomic sequencing [86]. Such techniques would eliminate the need for prior culture of clinical isolates, although cost remains a limiting issue. Most previous work on parasite factors involved in disease phenotype compared only a few strains or species, complicating the identification of the specific disease-determining factors. In addition, studies such as these require precise Box 2. Outstanding questions Trypanosomatid parasites Increased focus on hypothetical proteins and on species-specific genes. Increased focus on clones of a given strain or closely related strains that cause different disease phenotypes. Strain bank with detailed clinical history. T. cruzi Improved animal models. Comparison of biological properties, virulence, and tropism using multiple representatives of all six DTUs in a single study, with a focus on multiple human isolates with detailed patient history. Genome sequencing of strains from TcIV and TcV. Proteomic comparison of closely related strains. T. brucei Improved animal models, especially for T. b. gambiense. Sequencing additional T. b. gambiense and T. b. rhodesiense isolates. Large-scale proteomic comparison of T. b. gambiense and T. b. rhodesiense. Leishmania Sequencing and proteomic comparison of atypical L. donovani isolates. Sequencing and proteomic comparison of cutaneous and visceral L. tropica isolates. Further characterization of visceral L. amazonensis isolates. Sequencing of multiple Viannia subgenus parasites.
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and accurate patient clinical records for each isolate, and these are often lacking. Patient history before the isolation of the parasites and information on subsequent disease progression or response to treatment would be especially useful. The creation of a strain repository with detailed patient information would help researchers worldwide. Decreasing costs for sequencing and label-free proteomic approaches will allow genomic and proteomic comparisons of multiple clinical isolates simultaneously. Studying clones of a given strain that cause a range of disease phenotypes can help to pinpoint the specific determinants involved. This may be particularly important in the case of T. cruzi where thousands of genetic dissimilarities are observed between DTUs. However, any factor identified by these omics studies must still be validated, for instance by knocking out the identified factor and assessing the impact on parasite virulence. Finally, for all three trypanosomatids, parasite determinants strongly influence disease phenotype. However, even though these parasites are closely related with over 6000 shared genes, they cause disparate diseases. There should be increased investigation of genes specific to T. cruzi, Leishmania, and T. brucei. In addition, the ability to cause leishmaniasis, Chagas disease, or sleeping sickness is restricted to trypanosomatid parasites. As such, trypanosomatid-specific genes with no homology to genes found in other organisms may be key to disease pathogenesis. Further study of these genes and the development of tools to identify their functions will provide us with essential insight into the devastating disease caused by these parasites. Concluding remarks and future perspectives Almost 10 years after the publication of the first three trypanosomatid genomes, much knowledge is still lacking. Parasite characteristics are important determinants of disease phenotype caused by all three, but the specific factors are still often unknown, particularly for T. brucei and T. cruzi. Their identification will be facilitated by the application of large-scale omics techniques. Hopefully this work will help guide the drug development and disease elimination efforts that are underway. References 1 Rassi, A. et al. (2010) Chagas disease. Lancet 375, 1388–1402 2 Brun, R. et al. (2010) Human African trypanosomiasis. Lancet 375, 148–159 3 Murray, H.W. et al. (2005) Advances in leishmaniasis. Lancet 366, 1561–1577 4 Mathers, C.D. et al. (2007) Measuring the burden of neglected tropical diseases: the global burden of disease framework. PLoS Negl. Trop. Dis. 1, e114 5 Zingales, B. et al. (2012) The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect. Genet. Evol. 12, 240–253 6 Ramirez, J.D. et al. (2010) Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients. PLoS Negl. Trop. Dis. 4, e899 7 Vago, A.R. et al. (2000) Genetic characterization of Trypanosoma cruzi directly from tissues of patients with chronic Chagas disease: differential distribution of genetic types into diverse organs. Am. J. Pathol. 156, 1805–1809 8 Luquetti, A.O. et al. (1986) Trypanosoma cruzi: zymodemes associated with acute and chronic Chagas’ disease in central Brazil. Trans. R. Soc. Trop. Med. Hyg. 80, 462–470 347
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