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Chitinase from Basal Trypanosomatids and Its Relation to Marine Environment: New Insights on Leishmania Genus Evolutionary Theories
Written By
Felipe Trovalim Jordão, Aline Diniz Cabral, Felipe Baena Garcia, Edmar Silva Santos, Rodrigo Buzinaro Suzuki, Max Mario Fuhlendorf and Márcia Aparecida Sperança
Submitted: 12 November 2022Reviewed: 29 March 2023Published: 07 June 2023
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Leishmaniasis, an infectious disease that affects humans, domestic dogs, and wild animals, is caused by 20 of the 53 Leishmania genus species and is transmitted by sandflies. Despite its significant impact, the disease is often neglected. Leishmania genus, belong to Trypanosomatide Family and Kinetoplastida Order, are grouped in five subgroups according to biogeographic and evolution history of parasites and hosts. The GH18 Leishmania chitinase is encoded by a specie-specific single copy gene, conserved in basal groups of trypanosomatids, and is absent in the genus Trypanosoma. Preservation of the chitinase genomic locus in the aquatic free-living protozoan Bodo saltans, discloses a primitive common origin. Trypanosomatid chitinase amino acid sequence comparative analysis revealed high similarity with chitinase from sea living prokaryotes and protozoan microorganisms, indicating a probable marine origin. Amino acid sequence comparative analysis revealed that perhaps the trypanosomatid chitinase derived from a water living Kinetoplastida ancestor and its phylogenetic reconstruction corroborates the Supercontinent Origins theory for Leishmania. The chitinase-encoding gene was effective for differential molecular diagnosis among Leishmania clinical important species worldwide.
Federal University of ABC, Center for Natural and Human Sciences, São Bernardo do Campo, São Paulo, Brazil
Aline Diniz Cabral
Department of Parasitology, Federal University of Uberlandia, Uberlãndia, Brazil
Felipe Baena Garcia
Federal University of ABC, Center for Natural and Human Sciences, São Bernardo do Campo, São Paulo, Brazil
Edmar Silva Santos
Federal University of ABC, Center for Natural and Human Sciences, São Bernardo do Campo, São Paulo, Brazil
Rodrigo Buzinaro Suzuki
Marilia University, São Paulo, Brazil
Max Mario Fuhlendorf
Federal University of ABC, Center for Natural and Human Sciences, São Bernardo do Campo, São Paulo, Brazil
Márcia Aparecida Sperança*
Federal University of ABC, Center for Natural and Human Sciences, São Bernardo do Campo, São Paulo, Brazil
*Address all correspondence to: marcia.speranca@ufabc.edu.br
1. Introduction
Leishmania genus protozoan parasites are the causative agent of leishmaniases, a complex of diseases that affect the tegument or the viscera. Leishmania parasites are transmitted among humans, domestic dogs, and wild animal hosts by insect vectors of the Psycodidae Family (sandflies) as well as the Phlebotomus (Old World) and Lutzomyia genus (New World - the Americas) [1]. Up to now 53 Leishmania species were described which are divided into five groups, the subgenera Leishmania, Viannia, Sauroleishmania, Mundini, and Paraleishmania. Most of the Leishmania species are zoonotic and 20 are incriminated to cause disease in human [1].
The diseases caused by Leishmania parasites, named leishmaniases, can be divided into Tegumentar (TL) and Visceral (VL), depending on the species of infecting parasite and host immunity conditions. Leishmaniasis can range from mild tegumentar ulcerations to fatal visceral infection. Leishmania parasites are endemic in 98 countries distributed in all continents and its prevalence is estimated as 0.4 and 1.2 million cases of VL and TL, respectively [2]. In the Americas, Brazil accounts for the highest incidence of leishmaniasis with wide spreading of TL and VL in expansion [3].
The VL is in second place as to the highest impact on health population, just behind malaria, in India and in the Mediterranean countries being caused by L. (Leishmania) donovani and L. (Leishmania) infantum, respectively [4]. In the Americas VL is caused by L. infantum, where this species of parasite is not endemic and probably it entered the Americas by infected dogs brought by Mediterranean colonizers [5]. Seventy-five percent of TL new cases occur in Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, Sudan, Costa Rica, and Peru, causing morbidity and disfiguration in infected people [6]. In South America, TL is mainly caused by the most prevalent endemic Leishmania species, viz., L. (Viannia) braziliensis, L. (Leishmania) amazonensis, and L. (Leishmania) mexicana. In spite of much efforts, a precise diagnostic test and effective treatment for leishmaniasis are still unavailable [7]. Thus, a detailed understanding of all aspects of specific biology and host-parasite relationships are important prior to facilitating the formulation of innovative and effective drugs and diagnostic tests for developing adequate prevention and control strategies.
The Leishmania parasites to complete their life cycle, they must invade the digestive tract of sand-fly species, which requires the degradation of insect chitin by a specific parasite chitinase [8]. Chitinase catalyzes the β-1,4-glycoside bond hydrolysis reaction of N-acetylglucosamine of chitin and chitodextrins [9]. Amino acid sequence similarity analysis of Leishmania GH18 chitinase grouped these enzymes in the GH18 and GH19 glycosyl hydrolase families. Initial studies in Leishmania revealed that chitinase and N-acetylglucosaminidase activities were found in promastigote supernatant cultures of L. (Leishmania) major. Similar activity of both enzymes was observed in L. donovani, L. infantum, L. braziliensis, Leptomonas seymouri, Crithidia fasciculate, and Trypanosoma lewisi. The chitinolytic action was attributed to the parasite secretion and was not secreted through the sand fly gut [10, 11]. The gene encoding a GH18 chitinase was initially obtained for L. donovani (Ld Cht1) using molecular approach and biochemical characterization. Molecular genetic studies enabled the identification of a similar gene in several species of Leishmania genus (L. major, L. infantum, L. donovani, and L. braziliensis) [12].
The biological importance of the Leishmania GH18 chitinase in parasite life cycle was confirmed after homologous episomal overexpression of chitinase in both amastigotes and promastigotes of L. Mexicana. In the insect vector, overexpression of Leishmania chitinase resulted in an increase in transmission rate where in the vertebrate host ensued increased pathogenicity, thereby indicating that chitinase plays an important role in parasite development, survival, and transmission in mammalian hosts [13, 14]. Due to the biological properties and characteristics of Leishmania genus GH18 chitinase, including locus conservation, species-specific amino acid and nucleotide sequence, expression in all species of parasite developmental stages, and extracellular exportation, this investigation focused on the potential of the chitinase-encoding gene as a molecular diagnostic tool and a phylogenetic marker for studying basal trypanosomatids groups.
2. Chitinase genetic locus conservation among Kinetoplastida revealed its marine origin
Evaluation of the phylogenetic relationship of the GH18 family chitinase in Kinetoplastida was performed through comparative analysis by Basic Local Alignment Search Tool (BLAST) on chitinase amino acid and nucleotide sequences of trypanosomatids, available in public databanks (Figure 1) [15]. The results showed that the chitinase amino acid sequence is highly conserved among the species of the Leishmania genus, with identity variations ranging from 78 to 100%. Additionally, basal trypanosomatids such as Leptomonas, Strigomonas, and Angomonas also harbor a GH18 chitinase that is similar to that of Leishmania, with identity percentages of 60, 40, and 35%, respectively. This chitinase was found to be absent in parasites from Trypanosoma genus. A protein with identity of 32% with Leishmania genus chitinase was also present in the free-living acquatic protozoa from Kinetoplastida Order and Bodonidae Family, Bodo saltans, which is used as external group of Trypanosomatid in phylogenetic studies. These results suggest the occurrence of a homologous GH18 chitinase in a Kinetoplastida ancestor.
Figure 1.
Leishmania species CH18 chitinase locus obtained from available sequences in the Tritrypdb data base, a trypanosomatide genomic bank, using the software for genomic analysis (https://tritrypdb.org/a/jbrowse.jsp?loc=LinJ.16:276441..307814&data=/a/service/jbrowse/tracks/linfJPCM5&tracks=gene%2CSyntenic%20Sequences%20and%20Genes%20(Shaded%20by%20Orthology).
The TritrypDB genomic resource tools were utilized to analyze the locus of the chitinase-encoding gene in all available Kinetoplastida sequences, comprising Bodo saltans, Leptomonas, Angomonas, Strigomonas, and Leishmania. The results indicated a high degree of conservation, which further supports the hypothesis of a shared origin. In all organisms included in the analysis, the GH18 chitinase is a single copy gene (Figure 1).
BLAST analysis of the chitinase amino acid sequence from Bodo saltans showed identity of 38% with the chitinase of the marine microorganisms Perkinsus marinus and Micromonas pusilla, indicating that the GH18 chitinase of the trypanosomatids ancestor emerged from marine environment. These data are supported by the phylogenetic reconstruction of Kinetoplastida GH18 chitinase which grouped in a common cluster, separated from the GH18 chitinases of human and insects (Figure 2).
Figure 2.
Evolutionary relationships of GH18 chitinase amino acid complete sequence by neighbor-joining method. Phylogenetic reconstruction was conducted in MEGA4 and the figure corresponds to the optimal tree with the sum of branch length = 6.42769400. The bootstrap values (1000 replicates) are shown next to the branches. Evolutionary distances used to infer the phylogenetic tree are drawn to scale.
3. The phylogenetic relationship among basal trypanosomatids chitinase corroborated the supercontinent origin hypothesis for Leishmania genus
The barcode for phylogenic relationship among trypanosomatids corresponds to the variable region V4 (described also as V7 and V8) of the small ribosomal subunit (V4 rRNA SSU) [5, 16]. In order to investigate the potential of chitinase-encoding gene for trypanosomatids phylogenetic reconstruction, a partial 953 bp chitinase-encoding gene fragment and the corresponding amino acid sequence from trypanosomatids available in genomic databanks and generated by our research group was evaluated for phylogenetic reconstruction by Neighbor-Joining [17] method, using the MEGA4 software (Figure 3) (Table 1). The obtained trees were compared to the phylogenetic analysis performed by Neighbor-Joining method, with the trypanosomatid barcode of the same species (Figure 4). The phylogenetic reconstruction of Leishmania based on GH18 chitinase-encoding gene corroborated the thesis of the Supercontinent Origin for Leishmania genus [18] with higher accuracy, according to bootstrap results, when compared to the trypanosomatid barcode. The rRNA SSU V4 groups the subgenera Mundinia together with the subgenera Viannia (Figure 4), while the phylogenetic relationships based on the chitinase protein separate the species of all Leishmania subgenera and Paraleishmania group.
Figure 3.
Evolutionary relationships of Leishmania genus and ancient trypanosomatides by neighbor-joining method using the 292 amino acid residues corresponding to the 949 bp chitinase-encoding gene fragment. Phylogenetic reconstruction was conducted in MEGA4 and the figure corresponds to the optimal tree with the sum of branch length = 4.73393897. The bootstrap values (1000 replicates) are shown next to the branches. Evolutionary distances used to infer the phylogenetic tree are drawn to scale. Leishmania subgenus and Paraleishmania parasites are described at right.
Figure 4.
Evolutionary relationships of Leishmania genus and ancient trypanosomatides by neighbor-joining method using the 834 nucleotide residues corresponding to the small RNA subunit variable region 4 (V4 rRNASSU) encoding gene. Phylogenetic reconstruction was conducted in MEGA4 and the figure corresponds to the optimal tree with the sum of branch length = 0.80513269. The bootstrap values (1000 replicates) are shown next to the branches. Evolutionary distances used to infer the phylogenetic tree are drawn to scale. Leishmania subgenus and Paraleishmania parasites are described at right. Box: L.enrietti classified in subgenus Mundinia.
4. Conventional polymerase chain reaction (PCR) associated to restriction length polymorphism differentiated Leishmania subgenera of old and New World
In silico analysis of the Genbank Leishmania species chitinase, which belongs to the glycosil-hydrolase 18 (GH18) family, revealed that it is localized on chromosome 16 and encoded by a single copy gene. The analysis also indicated high inter-subgenera identity in all crucial putative domains and post-translational modifications [21, 22].
Detection and identification of Leishmania parasite subgenera was successfully obtained by employment of Pst I restriction analysis on the Leishmania species chitinase gene 953 bp fragment. Using Dde I restriction analysis was possible to separate viscerotropic and tegumentar species from Old World Leishmania subgenus and Sauroleishmania and Viannia subgenera (Table 2).
Subgenera
Species
Geographic distribution
Dde I fragments (bp)
Pst I fragments (bp)
Leishmania
L.donovani
Old World
608, 157,188
390, 113, 258, 192
Leishmania
L. infantum
New and Old World
608, 157,188
390, 113, 258, 192
Leishmania
L. turanica
Old World
608, 157,188
390, 113, 258, 192
Leishmania
L. gerbili
Old World
608, 157,188
390, 113, 258, 192
Leishmania
L. major
Old World
58, 550, 79, 78, 188
390, 113, 258, 192
Leishmania
L. mexicana
New World
602, 6, 157, 188
390, 113, 450
Leishmania
L. amazonensis
New World
602, 6, 157, 188
390, 113, 450
Viannia
L. braziliensis
New World
325, 284, 344
503, 450
Sauroleishmania
L. tarantolae
Old World
765, 188
503, 450
Mundinia
L. enrietti
New World
112,292, 180, 25, 344
503, 258, 192
Table 2.
Restriction fragment sizes of the 953 bp chitinase PCR amplicon digested with Dde I and Pst I.
Several regions, such as Brazil, are endemic to more than one species of Leishmania, with both VL (L. infantum) and TL (L. braziliensis, L. amazonensis). Additionally, there are parasites from the Trypanosoma genera (T. cruzi, T. rangeli) that show cross-reaction in various serological and molecular tests. Thus, the differential molecular diagnosis of Leishmania based on chitinase-encoding gene is highly sensitive and specific and becomes relevant in these multiple species of Leishmania and Trypanosoma endemic areas. Therefore, a diagnostic method capable of distinguishing between different Leishmania species in animal, human, and vector reservoirs will better guide leishmaniasis control.
Nucleic acid detection techniques in samples from people and/or animals infected with Leishmania, such as PCR, are used for detection and identification of the parasite since the 1980s. PCR includes the amplification of various fragments, including those of the gene that encodes the small ribosomal RNA subunit (SSU rDNA) [23], the transcribed internal ribosomal DNA spacer (ITS) [24], sequences that correspond to kinetoplast (kDNA) [25], and mini-exon [26], as well as the gene encoding the heat shock protein HSP70 [27], etc. Despite its high sensitivity and, depending on the molecular target, high specificity, PCR is more commonly utilized in epidemiological studies rather than as a routine diagnostic method. In addition, to achieve high sensitivity in the methodologies evaluated so far, PCR complementation with other techniques including nested PCR and hybridization is required. To identify Leishmania species, various methodologies are employed, including the analysis of restriction fragment sizes of PCR products. However, as most gene targets have multiple copies, interpretation of the results can increase the difficulty of using these techniques in clinical routine. In addition, false positives are possible due to contamination with other post-PCR amplified samples or DNA fragments and cross-reaction with other pathogens, including Trypanosoma [28].
The differential diagnosis of Leishmania subgenera based on chitinase-encoding gene offers certain advantages over other molecular methods. This is because it is encoded by a single copy gene, which is absent in the Trypanosoma genus, thereby allowing for specific detection of Leishmania parasites. Also, the sensitivity of the method, regarding the size of the amplified fragment, is high, supporting post-PCR analysis of a single reaction obtained directly from biological samples. Restriction analysis of the 953 bp Leishmania chitinase PCR fragment with PstI permitted the identification of medical important species in Latin America where three different Leishmania subgenera circulate in animal reservoirs, human, and sandflies [21]. Given the specificity of the Leishmania chitinase-encoding gene, the molecular diagnostic method can also be used to identify isolated parasites from biological samples, with high specificity, by restriction analysis and/or sequencing [29]. Also, using the restriction enzyme Dde I on the 953 bp chitinase PCR fragment, it is possible to differentiate L. major from all others Old World Leishmania subgenus species, which is of clinical importance in Oriental TL endemic countries (Table 2) [30].
The origins of parasites from Leishmania genus and its evolutionary relationships are investigated through phylogenetic reconstructions associated to data on biogeographic dispersion and evolution of their vertebrates and sandflies corresponding hosts, being a matter of discussion on conflicting information [1]. The revision by Akhoundi, et al. 2016, details the three principal theories proposed for Leishmania origin, a Palearctic, Neotropical, and a Neotropical/African/Multiple Origins. The most widely accepted theory regarding the origin of Leishmania is the Supercontinent hypothesis, a variation of the Multiple Origins hypothesis. This theory suggests that the Viannia and Leishmania subgenera evolved independently during the separation of South America from Africa. The Supercontinent hypothesis proposes that Leishmania originated on Gondwana and evolved from monoxenous parasites [18]. This theory is supported by biogeographic data and animal host migration patterns, and was developed through phylogenetic reconstruction using a large multi-gene dataset (over 200,000 informative sites) [18]. In this work, the phylogenetic reconstruction of Leishmania genus with the 953 bp partial chitinase-encoding gene sequence corroborates the Supercontinent hypothesis, grouping species of each Leishmania subgenera and the Paraleishmania group (Figure 4).
The genomic locus of GH18 chitinase-encoding gene is conserved among basal trypanosomatids, including the B. saltans. Besides, amino acid sequence comparison studies among GH18 chitinases from trypanosomatids using public genome databanks revealed 35% of identity of GH18 chitinases of marine protozoa and bacteria to the similar enzyme of B. saltans. These results strongly suggest that the GH18 chitinase from Kinetoplastida group derived from a common marine ancestor, harboring the primitive enzyme.
The phylogenetic position of subgenus Sauroleishmania, according to the Supercontinent hypothesis, indicates the switch of its Leishmania ancestors from mammalian to reptilian hosts [1]. In considering a probable marine environment emergence of the trypanosomatid GH18 chitinase, a highly conserved and unique gene in basal groups, including Leishmania, it is possible to explore that the Sauroleishmania subgenus could diverge from an ancestor before the rise of mammals, during the transition of animals from marine to the terrestrial environment. In this case, Leishmania-like parasites could be found in fish and amphibians. Considering the conservation of the chitinase-encoding gene in Leishmania group, the diagnostic method developed in this work can be used to investigate this hypothesis directly on biological samples, circumventing the isolation difficulties of unknown Leishmania-like parasites.
The barcode gene used for phylogenetic studies in trypanosomatids corresponds to the V4 rRNA SSU. However, for basal groups of trypanosomatids, the chitinase-encoding gene and amino acid sequence presented better results, as demonstrated through comparison between phylogenetic trees generated by Neighbor-joining method for both markers (Figures 3 and 4). Furthermore, the 953 bp chitinase fragment can be obtained by PCR directly from biological samples, while the V4 rRNA fragment is obtained preferentially from isolated parasites. These results indicate that the partial sequence of the gene encoding trypanosomatids chitinase can be used as a barcode to investigate the phylogenetic relationships among basal species of the group.
In silico prediction of the biochemical and molecular characteristics of Leishmania chitinase, such as high solubility and exportation of the native protein to the extracellular medium, associated to species-specific sequences, as well as the absence of a similar gene in the genus Trypanosoma, indicated its potential use as an antigen in accurate differential serological diagnosis for Leishmania species. Thus, in order to produce high amounts of Leishmania chitinase from the four different species representing the three major taxonomic groups from the subgenera Viannia (L. braziliensis) and Leishmania (L. amazonensis/L. mexicana and L. infantum), a conventional prokaryotic expression system was initially used. However, after tests under various expression conditions, the proteins remained insoluble, probably due to unfolding, the absence of the predicted N-glycosylation signal, and the formation of inclusion bodies [21]. The homologous chitinase of L. donovani was partially soluble in E. coli only when expressed in fusion with thioredoxin [31], thus indicating that even when using different strategies, the prokaryotic system is not appropriate for producing a correct folded Leishmania chitinase, essential in obtaining an effective antigen for serological testing, for example.
After observing the successful expression of L. major GP63, a glycosylated membrane protein, in insect cells using recombinant baculovirus [32], we obtained several baculovirus constructions of chitinase from the four Leishmania species by utilizing the Bac-to-Bac insect cell expression system. However, despite several attempts, it was found that this was not the case. This suggests that there may be intrinsic molecular characteristics associated with the insolubility of Leishmania species chitinase, such as post-translational signals that are incompatible with the organelle machinery of insect cells. Consequently, this may lead to the accumulation of unfolded recombinant proteins in cellular compartments. Investigation by way of in situ detection of recombinant proteins from constructions with N and C terminal histidine-tags, using Fluorescein Isothiocyanate (FITC) labeled anti-histidine antibody, revealed their accumulation in the plasma membrane [21]. This was confirmed by Thin Layer Chromatography (TLC) of insect cell membranes infected with L. infantum chitinase baculoviruses previously enriched with recombinant chitinase, and presenting lipid chemical signatures of plasma membrane. Recombinant forms of Leishmania species chitinase accumulation in plasma membrane, independent of differential physicochemical characteristics, presuppose the occurrence of a specific molecular feature necessary for precise Leishmania chitinase folding, such as chaperones.
Chaperones play an essential role in the regulation of biological functions by facilitating changes in the conformation of non-native protein. This is achieved through several mechanisms, including ribosome nascent polypeptide folding, organelle and cellular protein-membrane addressing, and the disassembly of macromolecular aggregates. Biochemical modifications, such as phosphorylation networks and hydrophobic interactions, also contribute to this process [33, 34]. There are several eukaryotically conserved and divergent chaperone families in Leishmania parasites that could explain the need for specific chitinase-chaperone interaction in correct folding [35], such as cyclophilin, a specific L. donovani chaperone, involved in reversing ADP-dependent inactive aggregates of adenosine kinase under physiological conditions, and a key enzyme in the leishmanial purine salvage pathway [36]. Further evidence of specific chitinase-chaperone interaction comprises the highly conserved Leishmania genus chitinase C-terminal ATP-dependent kinase domain (Data in Brief co-publication) since biochemical regulation of several chaperone functions is often associated to ATP-dependent phosphorylation [37]. Further studies of native Leishmania chitinase are required to investigate this hypothesis.
Leishmania chitinase is specific to basal groups of trypanosomatids genera, probably derived from an ancestor living in a marine environment, and unique in the human pathogen group. There are no Leishmania chitinase or homologous proteins described with a molecular structure associated to biochemical function. Considering biological importance and Leishmania genus chitinase specificity, novel molecular-based studies of native protein should shed light on its biochemical function, thereby facilitating its use not only in diagnosis, but also in drug and vaccine designs for controlling and treating leishmaniasis.
This work was funded by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) (Grant numbers 2016/14514-4 and 2012/20221-9; fellowships 2013/26096-4 and 2018/05133-2).
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Written By
Felipe Trovalim Jordão, Aline Diniz Cabral, Felipe Baena Garcia, Edmar Silva Santos, Rodrigo Buzinaro Suzuki, Max Mario Fuhlendorf and Márcia Aparecida Sperança
Submitted: 12 November 2022Reviewed: 29 March 2023Published: 07 June 2023
Open access peer-reviewed chapter
