Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand
Parasites & Vectors volume 15, Article number: 459 (2022)
The mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated.
This study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis.
We successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species.
This was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite.
Leishmaniasis is a sandfly-borne disease in tropical and subtropical regions of the world caused by protozoan parasites of the genus Leishmania. At least 54 species have been identified to date, which could be divided into two lineages: (1) Euleishmania consisting of four subgenera, Leishmania, Mundinia, Sauroleishmania, and Viannia; (2) Paraleishmania . In Thailand, leishmaniasis was considered to come from imported cases from patients who had visited the endemic areas since 1960; for example, an autochthonous visceral leishmaniasis case was detected in 1996 in a patient without underlying illness [2,3,4]. Leishmania infantum and L. donovani were frequently seen in these cases . Until 2008, a new species, Leishmania siamensis, from a Thai patient in the southern province, was described and later renamed Leishmania orientalis [3,4,5]. In the same year, a suspected case was reported as visceral leishmaniasis in another patient in the southern province of Thailand. The case was confirmed to be Leishmania martiniquensis, a member of the Leishmania enriettii complex [3, 6, 7]. Since then, more cases of these two Leishmania species have been observed. In 2015, one case of L. martiniquensis and 12 cases of L. orientalis were detected in 392 participants from the northern province who showed no symptoms . One Phlebotomus stantoni and one black rat also tested positive for L. martiniquensis. These have raised concerns about possible outbreaks of these Leishmania species and their impact on healthcare.
To better understand these parasites’ genetics, the chromosome-scale genomes of L. orientalis strain LSCM4 (34.19 Mbp) and L. martiniquensis strain LSCM1 (32.41 Mbp) isolated from patients in the northern province were published in 2021 [9,10,11]. Shortly after, in 2022, genomes of L. orientalis strain PCM2 (30.01 Mbp) and L. martiniquensis strains PCM3 (32.39 Mbp) and CU1 (30.80 Mbp) isolated from the southern provinces were released [12, 13]. Comparative genomic analysis of these Leishmania samples addressed the close relationship between the two L. martiniquensis strains, PCM3 and LSCM4, and further genetic distance between the two L. orientalis strains . Similarly, a genomic comparison of two L. martiniquesis strains, CU1 and LSCM1, identified 50 genes unique to strain CU1 and ten genes to the strain LSCM1. This nuclear genomic information has provided the basis for understanding the virulence and improving detection methods and drug development, e.g. identifying 16 species-specific protein therapeutic targets .
Kinetoplast DNA, or kDNA, is a substructure of complex DNA networks discovered within trypanosomatid mitochondria consisting of long maxicircles and minicircles [15,16,17,18,19]. The kDNAs of L. tarentolae have been widely used as a model for RNA editing studies [20,21,22]. A single mitochondrion of L. tarentolae is composed of 20–50 maxicircles (20–40 kb), which are equivalent to the mitochondrial DNA of other eukaryotes and 5,000–10,000 minicircles (0.5–2 kb) . Maxicircle sequences were homogeneous within the parasite species . The genes on the L. tarentolae maxicircle included four genes (9S rRNA, 12S rRNA, RPS12, and RPS3) that encoded for rRNA and protein components of the mitochondrial ribosome and 16 genes (ND1, ND3, ND4, ND5, ND7, ND8, ND9, COI, COII, COIII, Cyb, ATPase6, MURF1, MURF2, CR3, and CR4) involved in the electron transport chain complexes (GenBank; M10126.1). Many of the protein-coding maxicircle genes were transcribed as pre-mRNAs that must be modified post-transcriptionally via an extensive RNA editing process (i.e. insertion and deletion of uridine) to yield translatable mRNAs. The RNA editing process required several guide RNAs (gRNAs) encoded by classes of the minicircles with varying copy numbers to prevent the loss of particular types which could be lethal to the parasite . The maxicircle also contained the divergent region (DR), which was non-coding and enriched with unique repeat patterns [20, 24,25,26]. Camacho et al. compared kDNAs of L. major, L. infantum, and L. braziliensis with the maxicircle of L. tarentolae and found that the maxicircles of these three species were highly conserved. At the same time, all minicircles shared the conserved CSB boxes, but the copy number differed (97 for L. major, 49 for L. infantum, and 3 for L. braziliensis) . Ceccarelli et al. used the minicircle sequences to design PCR primers for distinguishing between L. infatum and Leishmania amazonensis . Thus, the maxicircle and minicircle DNAs were useful for examining inter- and intra-specific relationships between Leishmania species and strains. However, the maxicircle and minicircle DNAs of L. martiniquensis strain PCM3 had not been identified. This study aimed to extract these kDNA sequences from the short- and long-read whole-genome sequence data and apply bioinformatics analysis to explore these genomes with other Leishmania species. Results filled the missing knowledge gaps of the L. martiniquensis genomics and helped understand the genetic diversity and virulence of the parasite.
Materials and methods
Culture of L. martiniquensis strain PCM3
Leishmania martiniquensis strain PCM3 was maintained and provided by the Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand. The promastigotes were grown at 26 °C in RPMI 1640 modified with 13.3 mM glutamine, 2.5 mM arginine, 0.3 mM cysteine, 1.7 mM glutamate, 62.1 mM proline, 0.6 mM ornithine, 3.8 mM glucose, 2.2 mM fructose, 5.1 mM malate, 2.8 mM α-ketoglutarate, 0.5 mM fumarate, 0.5 mM succinate, 25 mM Hepes, 50 µg/ml gentamicin, 2X MEM vitamins (Gibco, US), and 20% heat-inactivated fetal bovine serum (HIFBS, Gibco, US).
Genomic DNA preparation
Genomic DNA was isolated from the promastigotes at the late logarithmic phase. After washing with ultrapure water, the promastigote pellet was suspended in 1 ml of lysis buffer (10 mM Tris, 10 mM KCl, 10 mM MgCl2, 0.5 M NaCl, 2 mM EDTA, and 0.5% SDS) and 20 µl of proteinase K solution (20 mg/ml). After 30 min, the samples were incubated with chloroform: isoamyl alcohol (24:1) at 56 °C and gently mixed vigorously for 10 min. The samples were centrifuged at 14,000 × g for 10 min at room temperature to obtain the upper aqueous phase. Ten microliters of RNAse solution (20 mg/ml) was added and incubated at room temperature for 3 min. Following RNase treatment, a 1:1 ratio of chloroform:isoamyl alcohol (24:1, v/v) was added and gently mixed vigorously for 10 min before centrifugation at 14,000×g for 10 min at room temperature. The upper aqueous phase was collected, and DNAs were precipitated overnight at − 70 °C in 200 µl of 4 M ammonium acetate and 800 µl of 100% ethanol. The precipitated samples were centrifuged at 14,000×g for 10 min at 4 °C and then washed twice with 70% ethanol. After 30 min at room temperature, DNA samples were air-dried and suspended in TE buffer [10 mM Tris–HCl (pH 8.0) and 0.1 mM EDTA]. The extracted genomic DNA for the long-read sequencing was further purified using the phenol/chloroform method. The quality and amount of DNA were determined by measuring absorbance at 260/280 nm and 260 nm using Nanodrop (Thermo Fisher Scientific, US). Electrophoresis on a 1% agarose gel was used to determine the genomic integrity. Before genome sequencing, the samples were maintained at − 70 °C.
Leishmania maxicircle and minicircle sequence extraction from raw sequence reads
For the short-read sequencing, a 101-bp pair-end read library was constructed for the whole genome sequencing using the Illumina HiSeq2000 platform (Illumina, USA). For the long-read sequencing, the 20-kb PacBio library was built according to the PacBio standard library preparation methods (Pacific Biosciences, CA, USA) using the SMRTbell Express Template Preparation kit 1.0. It was sequenced by the SMRTbell platform (Pacific Biosciences, USA). The Illumina reads were filtered using the BBTools program (http://jgi.doe.gov/data-and-tools/bb-tools) with the following parameter setting—ktrim = r [right-trimming (3′ adapters)] k = 23 mink = 11 (allows shorter kmers at the ends of the read) hdist = 1 (hamming distance) tpe (trim both reads) tbo qtrim = rl (trim the left and right sides) trimq = 10 (quality-trim to Q10) phiX (Illumina spikein)—to remove Illumina artifacts. The phiX sequences from bbtools library were used with 31-mer match (k = 31). To remove Illumina adapters, the UniVec database (https://www.ncbi.nlm.nih.gov/tools/vecscreen/univec/) was used as a reference for the adaptor sequences. These reads were used to generate hybrid assemblies for circular DNA by the SPAdes assembler version 3.15.3 [29, 30]; parameters were set as –pacbio–plasmid (assembly only circular DNA). Contigs were analyzed by BLASTn searches on the NCBI database. The BLAST results showed E-values < 0.01, and percentage of coverage > 75% was selected. The contigs sharing sequence homology with the complete maxicircle sequence of Leishmania was chosen for further analyses. For reference preparations, in-house python script was used to collect complete sequence of maxicircles, minicircles, and specific regions of Leishmania and Trypanosoma from the NCBI database. For the minicircles, the rKOMICS package was used for minicircle polishing, extending, and circularization , BLASTn scores were used to calculate the average nucleotide identity (ANI) using the pyani package , and percentage of similarity was represented by heatmap using the heatmaply package .
Annotation and visualization of maxicircle and minicircle sequences
The BLASTn alignment was used to find the most similar Leishmania maxicircle sequence in the NCBI nucleotide database [34,35,36,37,38,39,40,41,42]. The extracted maxicircle contigs were annotated using manual annotation. The gene order of the maxicircle DNAs (12S rRNA, 9S rRNA, ND8, ND9, MURF5, ND7, CO3, Cyb, ATPase 6, ND2, G3, ND1, CO2, MURF2, CO1, G4, ND4, G5 or ND3, RPS12, and ND5) of Leishmania arabica strain LEM1108 (GenBank: BK010878) and L. enriettii strain LEM3045 (GenBank: BK010880) were used as references to construct the complete circular maxicircle genome of L. martiniquensis strain PCM3. Pairwise comparison of the reference and L. martiniquensis maxicircle sequences was conducted by the EMBOSS Water program (https://www.ebi.ac.uk/Tools/psa/emboss_water/) using the following parameters—EDNAFULL for matrix, gap penalty of 10.0, and extend penalty of 0.5—to identify gene location. Visualization and exploration of the maxicircle genome were done by the CGview program on the Proksee server  and the progressiveMauve alignment program . Sequence read coverage of the maxicircle of L. martiniquensis strain PCM3 was analysed by using the samtools program version 1.15.1 [45, 46] and the SeqMonk program version 1.48.1 (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). For the minicircle, the L. martiniquensis minicircle sequences were compared against the NCBI database for identification of closely related minicircles in other Leishmania species. Results with the E-value < 0.001 and percentage of coverage > 20% were collected and illustrated by the Sankey diagram using the SankeyMATIC program .
Phylogenetic analysis of the obtained maxicircles and minicircles
Phylogenetic trees were built from the coding region of the maxicircle DNAs of available Leishmania and Trypanosoma species in the NCBI database compared with that of L. martiniquensis, similar to the method described by Solana et al. . For minicircles, only universal minicircle invariant (CSB-3, CSB-1 and CSB-2) regions from L. martiniquensis strain PCM3 were compared with those available from the NCBI database. MAFFT program version 7  on the Unipro UGENE version 44.0  was used to align the sequences using the DNA gap open penalty of 1.53 and the DNA gap extension penalty of 0.123, and the manual refinements were omitted. Phylogenetic relationships were inferred by using the randomized axelerated maximum likelihood methods with bootstrap values of 10,000 replicates using the RAxML tool version 8  and the approximately-maximum-likelihood method from the FastTree program version 2.1  with local-bootstrap support. The labels on the phylogenetic tree were modified using the MEGA 11 program .
Conserved motif analysis of the L. martiniquensis minicircles
The minicircle contigs from L. martiniquensis strain PCM3 were compared by multiple sequence alignment using the MAFFT program. The conserved motifs were annotated using data from previous studies and visualized by the WebLOGO3 program version 3.7 [54,55,56].
This study successfully extracted the complete mitochondrial genome (maxicircle and minicircles) of L. martiniquensis strain PCM3 isolated from the southern province of Thailand by utilising both short- and long-read sequence data. The de novo assembly of the maxicircle genome resulted in a complete circular DNA scaffold with a length of 19,008 base pairs. The L. martiniquensis maxicircle contigs were highly similar to the complete maxicircle sequences of Leishmania species within the same subgenus Mundinia including L. enriettii strain LEM3045 (GenBank: BK010880.1), which had a length of 17,999 base pairs with the e-value of 0.0, 86% sequence coverage, and 84.29% identity, and another subgenus including L. arabica strain LEM1108 (GenBank: BK010878.1), which had a length of 17,536 base pairs at the e-value of 0.0, 87% sequence coverage, and 82.17% identity, and L. tarentolae (GenBank: M10126.1), which had a length of 20,992 base pairs at the e-value of 0.0, 86% sequence coverage, and 83.32% identity (Fig. 1a). The pairwise alignment of the maxicircle sequences of L. martiniquensis strain PCM3 with the reference sequences of L. arabica strain LEM1108, L. enriettii strain LEM3045, and L. tarentolae gave similar percentage of identity (79.2, 80.6, and 79.2%) and similarity (79.92, 80.6, and 79.2%) with 8.5, 7.0, and 7.2% gaps. The same rRNA and coding genes (CDS) were well conserved in the maxicircles of these three Leishmania species, whereas the divergent region (DR or repeat region) varied in sequence composition and length with a few fragments shared between the three species. The completeness of the maxicircle DNA of L. martiniquensis strain PCM3 was clearly showed by the high level of sequence coverage and read depth after re-mapping of the long-read data back to the assembled contigs (Fig. 1b). The sequence read coverage of 100% also verified validity of the maxicircle of L. martiniquensis strain PCM3 (Fig. 1b).
The gene arrangement on the maxicircle of L. martiniquensis strain PCM3 was compared with those of L. enriettii strain LEM3045, L. arabica strain LEM1108, and L. tarentolae (Fig. 2). The four Leishmania species shared conserved gene arrangement of 20 coding genes ordered as 12S rRNA, 9S rRNA, ND8, ND9, MURF5, ND7, COIII, Cyb, ATPase 6, ND2, G3, ND1, COII, MURF2, COI, G4, ND4, ND3, RpS12, and ND5. The maxicircle variation could be observed across the four Leishmania species, particularly in the intergenic and DR regions.
Coding region-based phylogenetic analysis of the maxicircle of L. martiniquensis strain PCM3 compared with 28 complete maxicircles (22 Leishmania and 6 Trypanosoma samples) showed that this region could be used to categorize Leishmania samples into four subgenera: Viennia, Mundinia, Sauroleishmania, and Leishmania, separately from the genus Trypanosoma including subgenera Dutonella, Trypanozoon, Herpetosoma, and Schizotrypanum (Fig. 3). The maxicircle of L. matiniquensis strain PCM3 was correctly grouped with that of L. enriettii in the same subgenus Mundinia. This study also explored the phylogenetic relationship using the highly variable DR region, and the result was inconclusive.
For the minicircles, this study identified 214 minicircles (Fig. 4a) from L. martiniquensis strain PCM3 with length from 400 to 3385 base pairs (an average length of 717 base pairs). These were divided into 32 complete circular and 182 partial minicircles. The comparison of the average nucleotide identity (ANI) values showed that 53 minicircles were highly conserved in the examined Leishmania species (Fig. 4a, b), and 161 minicircles differed considerably from 40 minicircle sequences of 12 other Leishmania species and Herpetomonas samuelpessoai from the NCBI nucleotide database (Fig. 4a, c). The blastn comparison of these 214 minicircles showed that 175 contigs were similar to Leishmania sp. SA-2000 (GenBank: AF275904.1), which was the unclassified Leishmania species, six contigs were matched to L. infantum (AJS-IPTPS) minicircles (GenBank: Z35273.1), and four contigs were similar to those of L. naiffi (Fig. 5). A few contigs were similar to eight other Leishmania species and strains including L. amazonensis (1), L. donovani (2), L. guyanensis (1), L. lainsoni (2), L. major (4), L. tropica (1), L. braziliensis (1), and L. enriettii (1). Interestingly, 16 minicircles were unknown and could be specific to L. martiniquensis.
Sequence comparison of 214 L. martiniquensis minicircles showed two major conserved regions (Fig. 6a) and the hypervariable regions. The conserved regions I and II contained three highly conserved sequence boxes, which were characteristic of all examined minicircles (Fig. 6b). These CSBs included 10-bp CSB-1 (AgGGGCGTTC), 8-bp CSB-2 (cCCCGTNC), and 12-bp CSB-3 (GGGGTTGGTGTA) (Fig. 6c). The presence of these motifs and their order confirmed the correct minicircle identification. Phylogenetic comparison was made of the conserved regions of 214 minicircle sequences of L. martiniquensis strain PCM3 with 156 Leishmania and one Herpetomonas samuelpessoai minicircles from the NCBI database. The constructed tree revealed certain degrees of minicircle conservation compared with other Leishmania species (Fig. 7). Most L. martiniquensis minicircles were grouped separately into their own clades (five main clades highlighted as blue tips of the tree in Fig. 7), and some were within the minor clades of L. tropica, L. tarentolae, L. panamensis, L. lainsoni, L. guyanensis, and L. infantum, similar to the result showed in Figs. 4 and 5. As 22 minicircles of L. martiniquensis strain PCM3 had conserved CSB regions I and II, phylogenetic analysis of these regions showed that all of them were dissimilar and distantly separated (Fig. 7).
The mitochondrial genome or kinetoplastid DNA (one maxicircle and 214 minicircles) of L. martiniquensis strain PCM3 was obtained from the hybrid assembly of the short- and long-read sequence data. Due to the length of Leishmania maxicircles between 20 and 40 kilobases , the use of both long- and short-read sequence data facilitated better reconstruction of the complete maxicircle and multiple minicircle DNA (including some complete minicircles). The DR region contains repetitive sequences and is known to be difficult during the assembly process [20, 57, 58]. The use of long-read data provided larger coverage of the entire DR region, thus resolving the complexity issue in assembling this region and assisting the maxicircle completion (Fig. 1b).
This finding provided a complete overview of this parasite genomics and complemented previously published nuclear genomic data of L. martiniquensis strain PCM3 . The complete and partial maxicircle DNA (i.e. selected genes, all coding regions) could be used as genetic markers to identify Leishmania species and strains and to depict phylogenetic relatedness [59,60,61,62]. For example, Kaufer et al. used a variation on the Leishmania maxicircle sequences (ND7 gene) to design PCR–RFLP assays to discriminate nine Leishmania species . A recent study identified the origin of the intra-species L. donovani hybrid from Himachal Pradesh, India, that was derived from two independent L. donovani parents of the ISC1-Yeti clade from the Nepalese highlands using maxicircle phylogenetics, suggesting the usefulness for examination of intra-species diversity . Similarly, this study showed that the L. martiniquensis maxicircle was highly homologous to L. enriettii and L. arabica (Fig. 1), and the gene arrangement of the L. martiniquensis maxicircle supported the genetic relationship with L. enriettii, suggesting the use of maxicircles to explore the genetic diversity of these Leishmania species (Fig. 2) . This was also clearly supported by the close phylogenetic relationship of the maxicircle of L. martiniquensis strain PCM3 and L. enriettii strain LEM3045 from the same subgenus Mundinia (Fig. 3), consistent with the work of Solana et al., which also used the coding regions of maxicircle DNA ; however, the maxicircle sequences of subgenus Mundinia in the database remained limited and required further data gathering. Although the divergent region of L. martiniquensis maxicircle might not be suitable for the phylogenetic inference, the distinctiveness of this region was helpful in identification of Leishmania species and exploring intra-species variation. Therefore, the maxicircle data would benefit from further screening of L. martiniquensis isolates and other Mundinia members in areas of concern such as the Southeast Asian region where L. martiniquensis cases have been detected and observed more frequently than in the past (unpublished data).
Leishmania martiniquensis strain PCM3 had 214 minicircles which were considerably higher than those of L. major (97 classes), L. infantum (49 classes), and L. braziliensis (3 classes) and were lower than those of Trypanosoma cruzi (286 classes) . These minicircles shared three conserved sequence blocks (CSB1-3), which acted as replication origins to encode short guide RNAs essential for editing encoded transcripts from their maxicircle [56, 67]. The similarity of most L. martiniquensis minicircles to the unclassified Leishmania species (Leishmania sp. SA-2000) would suggest that their phenotypic and genetic relationship should be further examined (Figs. 4, 5) and the proposed possibility that Leishmania sp. SA-2000 would be another strain of L. martiniquensis. Novel 16 minicircles were unique and could be specific to L. martiniquensis or particularly the PCM3 strain. These unique minicircles could be associated with virulence and drug resistance, e.g. the resistant L. infantum strain ZK47 had a specific minicircle pattern and increased copy number . Furthermore, the presence of conserved regions I and II (Fig. 6), which contained two sets of the CSB-1, CSB-2, and CSB-3 conserved sequences in L. martiniquensis strain PCM3, was consistent with the previous observation of the conserved domain number variation in Trypanosomatid minicircles, which may be associated with the maturation of the maxicircle-encoded genes and the parasite adaptation [66, 69]. The phylogenetic analysis of the L. matiniquensis minicircles showed that most minicircles were classified separately into different clades and clustered distinctively from those of other Leishmania species, suggesting their paraphyletic origins. The results suggested potential usage of minicircle DNA sequence and number in identification of the L. martiniquensis species. For instance, the minicircles were also used to investigate the origin of the hybrid L. braziliensis × Leishmania peruviana isolates and found that their minicircles were derived from both parental species, while the maxicircle was uniparental . Another study designed PCR primers specific to the conserved region of minicircle DNA coupled with high-throughput sequencing and identified different minicircle numbers, i.e. 62 for L. amazonensis, 133 for L. braziliensis, 196 for L. guyanensis, 94 for L. infantum, 97 for L. lainsoni and 88 for L. naiffi, which were used for inter- and intra-species genetic distance calculation . Their minicircle patterns were applied to examine unknown Leishmani samples, and they correctly identified 10 out of 18 samples as L. amazonensis, L. infantum, L. lainsoni, and L. naiffi. Hence, not only the L. martiniquensis maxicircle, but also the sequence and number of minicircles would assist further diagnostics and screening of this parasite variant and population similar to previous studies in human patients and animals [72, 73].
The kDNAs of L. martiniquensis strain PCM3 in this study could provide the basis for understanding drug resistance phenotype. The alteration of kDNA by non-DNA-binding drugs such as sodium arsenite, tunicamycin, and pentamidine could lead to a phenomenon known as “transkinetoplastidy,” referring to dramatic changes in the population of maxicircles and minicircles [74, 75]. The kinetoplast study in Trypanosoma congolense revealed that mitochondrial membrane potential appeared to be involved in phenanthridine uptake, presumably via driving the accumulation of cationic compounds. When drug selection pressure was applied, populations with a lower mitochondrial electrical potential had an advantage because of isometamidium accumulation and lower toxicity . It has been demonstrated that trypanocidal chemotherapies and their related chemicals also accumulated in the mitochondrion and interacted with the kDNAs . Previous research also suggested that the Leishmania kinetoplast network may play a role in Leishmania and Trypanosoma survival [78,79,80,81]. Therefore, the kDNAs of L. martiniquensis strain PCM3 will benefit further experimental design to examine the association with drug-resistant and virulent phenotypes.
This study reported the maxicircle and minicircle sequences of L. martiniquensis strain PCM3 isolated from the southern province of Thailand. Analysis of the short- and long-read sequence data revealed conserved structure and gene arrangement of the maxicircle, while 214 minicircles were mostly similar to the unclassified Leishmania species. Variation of these minicircles could be essential to the parasite’s survival and adaptation. These kDNAs have completed the gaps in understanding of the L. martiniquensis genetics and are helpful for inter- and intra-species identification.
Availability of data and materials
The data supporting this study’s findings are submitted to the NCBI GenBank database and will be openly available in the NCBI GenBank database, the BioProject ID: PRJNA863057 and the locus tag prefix: NP941.
Akhoundi M, Downing T, Votýpka J, Kuhls K, Lukeš J, Cannet A, et al. Leishmania infections: molecular targets and diagnosis. Mol Aspects Med. 2017;57:1–29.
Thisyakorn U, Jongwutiwes S, Vanichsetakul P, Lertsapcharoen P. Visceral leishmaniasis: the first indigenous case report in Thailand. Trans R Soc Trop Med Hyg. 1999;93:23–4.
Leelayoova S, Siripattanapipong S, Manomat J, Piyaraj P, Tan-Ariya P, Bualert L, et al. Leishmaniasis in Thailand: a review of causative agents and situations. Am J Trop Med Hyg. 2017;96:534–42.
Leelayoova S, Siripattanapipong S, Hitakarun A, Kato H, Tan-ariya P, Siriyasatien P, et al. Multilocus characterization and phylogenetic analysis of Leishmania siamensis isolated from autochthonous visceral leishmaniasis cases, southern Thailand. BMC Microbiol. 2013;13:1–7.
Jariyapan N, Daroontum T, Jaiwong K, Chanmol W, Intakhan N, Sor-Suwan S, et al. Leishmania (Mundinia) orientalis n. sp.(Trypanosomatidae), a parasite from Thailand responsible for localised cutaneous leishmaniasis. Parasites Vectors. 2018;11(1):1–9.
Pothirat T, Tantiworawit A, Chaiwarith R, Jariyapan N, Wannasan A, Siriyasatien P, et al. First isolation of Leishmania from Northern Thailand: case report, identification as Leishmania martiniquensis and phylogenetic position within the Leishmania enriettii complex. PLoS Negl Trop Dis. 2014;8:e3339.
Sukmee T, Siripattanapipong S, Mungthin M, Worapong J, Rangsin R, Samung Y, et al. A suspected new species of Leishmania, the causative agent of visceral leishmaniasis in a Thai patient. Int J Parasitol. 2008;38:617–22.
Sriwongpan P, Nedsuwan S, Manomat J, Charoensakulchai S, Lacharojana K, Sankwan J, et al. Prevalence and associated risk factors of Leishmania infection among immunocompetent hosts, a community-based study in Chiang Rai, Thailand. PLoS Negl Trop Dis. 2021;15:e0009545.
Almutairi H, Urbaniak MD, Bates MD, Jariyapan N, Kwakye-Nuako G, Thomaz-Soccol V, et al. LGAAP: Leishmaniinae genome assembly and annotation pipeline. Microbiol Resource Announcem. 2021;10:e00439-e521.
Almutairi H, Urbaniak MD, Bates MD, Jariyapan N, Kwakye-Nuako G, Thomaz Soccol V, et al. Chromosome-scale genome sequencing, assembly and annotation of six genomes from subfamily Leishmaniinae. Sci Data. 2021;8:1–9.
Almutairi H, Urbaniak MD, Bates MD, Jariyapan N, Al-Salem WS, Dillon RJ, et al. Chromosome-scale assembly of the complete genome sequence of Leishmania (Mundinia) martiniquensis, isolate LSCM1, strain LV760. Microbiol Resource Announce. 2021;10:e00058-e121.
Anuntasomboon P, Siripattanapipong S, Unajak S, Choowongkomon K, Burchmore R, Leelayoova S, et al. Comparative draft genomes of Leishmania orientalis isolate PCM2 (formerly named Leishmania siamensis) and Leishmania martiniquensis isolate PCM3 from the southern province of Thailand. Biology. 2022;11:515.
Anuntakarun S, Phumee A, Sawaswong V, Praianantathavorn K, Poomipak W, Jitvaropas R, et al. Genome assembly and genome annotation of Leishmania martiniquensis isolated from a Leishmaniasis patient in Thailand. J Parasitol Res. 2022;2022:1–7.
Krobthong S, Yingchutrakul Y, Samutrtai P, Hitakarun A, Siripattanapipong S, Leelayoova S, et al. Utilizing quantitative proteomics to identify species-specific protein therapeutic targets for the treatment of Leishmaniasis. ACS Omega. 2022;7:12580–8.
Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M, et al. The genome of the kinetoplastid parasite, Leishmania major. Science. 2005;309:436–42.
Simpson L. The mitochondrial genome of kinetoplastid protozoa: genomic organization, transcription, replication, and evolution. Annu Rev Microbiol. 1987;41:363–80.
Maslov DA, Avila HA, Lake JA, Simpson L. Evolution of RNA editing in kinetoplastid protozoa. Nature. 1994;368:345–8.
Cavalcanti DP, de Souza W. The kinetoplast of trypanosomatids: from early studies of electron microscopy to recent advances in atomic force microscopy. Scanning. 2018;2018:9603051.
Jensen RE, Englund PT. Network news: the replication of kinetoplast DNA. Annu Rev Microbiol. 2012;66:473–91.
Muhich ML, Neckelmann N, Simpson L. The divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA contains a diverse set of repetitive sequences. Nucleic Acids Res. 1985;13:3241–60.
Alfonzo JD, Blanc V, Estevez AM, Rubio MAT, Simpson L. C to U editing of the anticodon of imported mitochondrial tRNATrp allows decoding of the UGA stop codon in Leishmania tarentolae. EMBO J. 1999;18:7056–62.
Sturm NR, Maslov DA, Blum B, Simpson L. Generation of unexpected editing patterns in Leishmania tarentolae mitochondrial mRNAs: misediting produced by misguiding. Cell. 1992;70:469–76.
Stuart K. Kinetoplast DNA, mitochondria DNA with a difference. Mol Biochem Parasitol. 1983;9:93–104.
Simpson L, Douglass SM, Lake JA, Pellegrini M, Li F. Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae. PLoS Negl Trop Dis. 2015;9:e0003841.
Flegontov PN, Strelkova MV, Kolesnikov AA. The Leishmania major maxicircle divergent region is variable in different isolates and cell types. Mol Biochem Parasitol. 2006;146:173–9.
Gerasimov ES, Zamyatnina KA, Matveeva NS, Rudenskaya YA, Kraeva N, Kolesnikov AA, et al. Common structural patterns in the maxicircle divergent region of Trypanosomatidae. Pathogens. 2020;9:100.
Camacho E, Rastrojo A, Sanchiz Á, González-De la Fuente S, Aguado B, Requena JM. Leishmania mitochondrial genomes: Maxicircle structure and heterogeneity of minicircles. Genes. 2019;10(10):758.
Ceccarelli M, Galluzzi L, Diotallevi A, Andreoni F, Fowler H, Petersen C, et al. The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis. Parasites Vectors. 2017;10(1):1–10.
Antipov D, Korobeynikov A, McLean JS, Pevzner PA. hybridSPAdes: an algorithm for hybrid assembly of short and long reads. Bioinformatics. 2016;32:1009–15.
Antipov D, Hartwick N, Shen M, Raiko M, Lapidus A, Pevzner PA. plasmidSPAdes: assembling plasmids from whole genome sequencing data. Bioinformatics. 2016;32:3380–7.
Geerts M, Schnaufer A, Van den Broeck F. rKOMICS: an R package for processing mitochondrial minicircle assemblies in population-scale genome projects. BMC Bioinform. 2021;22:1–14.
Pritchard L, Glover RH, Humphris S, Elphinstone JG, Toth IK. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. Anal Methods. 2016;8:12–24.
Galili T, O’Callaghan A, Sidi J, Sievert C. heatmaply: an R package for creating interactive cluster heatmaps for online publishing. Bioinformatics. 2018;34:1600–2.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402.
Madden TL, Tatusov RL, Zhang J.  Applications of network BLAST server. Methods Enzymol. 1996;266:131–41.
Zhang J, Madden TL. PowerBLAST: a new network BLAST application for interactive or automated sequence analysis and annotation. Genome Res. 1997;7:649–56.
Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7:203–14.
Morgulis A, Coulouris G, Raytselis Y, Madden TL, Agarwala R, Schäffer AA. Database indexing for production MegaBLAST searches. Bioinformatics. 2008;24:1757–64.
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinform. 2009;10:1–9.
Boratyn GM, Schäffer AA, Agarwala R, Altschul SF, Lipman DJ, Madden TL. Domain enhanced lookup time accelerated BLAST. Biol Direct. 2012;7:1–14.
Boratyn GM, Thierry-Mieg J, Thierry-Mieg D, Busby B, Madden TL. Magic-BLAST, an accurate RNA-seq aligner for long and short reads. BMC Bioinform. 2019;20:1–19.
Grant JR, Stothard P. The CGView Server: a comparative genomics tool for circular genomes. Nucleic Acids Res. 2008;36(Suppl_2):W181–W4.
Darling AC, Mau B, Blattner FR, Perna NT. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14:1394–403.
Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10(2):giab008.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9.
Bogart S. SankeyMATIC. A Sankey diagram builder for everyone Available online at: http://sankeymatic.com. 2020.
Solana JC, Chicharro C, García E, Aguado B, Moreno J, Requena JM. Assembly of a Large collection of Maxicircle sequences and their usefulness for Leishmania taxonomy and strain typing. Genes. 2022;13:1070.
Katoh K, Misawa K, Kuma Ki, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Re. 2002;30(14):3059–66.
Okonechnikov K, Golosova O, Fursov M, Team U. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012;28(8):1166-7.
Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–3.
Price MN, Dehal PS, Arkin AP. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol. 2009;26:1641–50.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547.
Crooks GE, Hon G, Chandonia J-M, Brenner SE. WebLogo: a sequence logo generator. Genome Res. 2004;14:1188–90.
Schneider TD, Stephens RM. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 1990;18:6097–100.
Ray DS. Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes. Mol Cell Biol. 1989;9:1365–7.
Sloof P, de Haan A, Eier W, van Iersel M, Boel E, van Steeg H, et al. The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA. Mol Biochem Parasitol. 1992;56:289–99.
Lee ST, Liu HY, Chu T, Lin SY. Specific A+ T-rich repetitive DNA-sequences in maxicircles from wildtype Leishmania mexicana amazonensis and variants with DNA amplification. Exp Parasitol. 1994;79:29–40.
Teles CBG, Basano SA, Zagonel-Oliveira M, Campos JJ, Oliveira AFJd, Freitas RAd, et al. Epidemiological aspects of American cutaneous leishmaniasis and phlebotomine sandfly population, in the municipality of Monte Negro, State of Rondônia, Brazil. Rev Soc Brasil Med Trop. 2013;46(1):60–6.
Ghasemian M, Maraghi S, Samarbafzadeh A, Jelowdar A, Kalantari M. The PCR-based detection and identification of the parasites causing human cutaneous leishmaniasis in the Iranian city of Ahvaz. Ann Trop Med Parasitol. 2011;105:209–15.
Fata A, Khamesipour A, Mohajeri M, Hosseininezhad Z, Afzal AM, Berenji F, et al. Whatman paper (FTA cards) for storing and transferring Leishmania DNA for PCR examination. Iran J Parasitol. 2009;4:37–42.
Fazaeli A, Fouladi B, Hashemi SS, Sharifi I. Clinical features of cutaneous leishmaniasis and direct PCR based identification of parasite species in a new focus in Southeast of Iran. Iranian J Public Health. 2008;37:44–51.
Kaufer A, Ellis J, Stark D. Identification of clinical infections of Leishmania imported into Australia: revising speciation with polymerase chain reaction-RFLP of the kinetoplast maxicircle. Am J Trop Med Hyg. 2019;101:590.
Lypaczewski P, Thakur L, Jain A, Kumari S, Paulini K, Matlashewski G, et al. An intraspecies Leishmania donovani hybrid from the Indian subcontinent is associated with an atypical phenotype of cutaneous disease. Iscience. 2022;25:103802.
Kaufer A, Barratt J, Stark D, Ellis J. The complete coding region of the maxicircle as a superior phylogenetic marker for exploring evolutionary relationships between members of the Leishmaniinae. Infect Genet Evol. 2019;70:90–100.
Callejas-Hernández F, Herreros-Cabello A, del Moral-Salmoral J, Fresno M, Gironès N. The complete mitochondrial DNA of Trypanosoma cruzi: Maxicircles and minicircles. Front Cellular Infect Microbiol. 2021;11.
Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol. 2006;44:1435–9.
Bussotti G, Benkahla A, Jeddi F, Souiaï O, Aoun K, Späth GF, et al. Nuclear and mitochondrial genome sequencing of North-African Leishmania infantum isolates from cured and relapsed visceral leishmaniasis patients reveals variations correlating with geography and phenotype. Microb Genomics. 2020;6(10).
Botero A, Kapeller I, Cooper C, Clode PL, Shlomai J, Thompson RA. The kinetoplast DNA of the Australian trypanosome, Trypanosoma copemani, shares features with Trypanosoma cruzi and Trypanosoma lewisi. Int J Parasitol. 2018;48:691–700.
Van den Broeck F, Savill NJ, Imamura H, Sanders M, Maes I, Cooper S, et al. Ecological divergence and hybridization of Neotropical Leishmania parasites. Proc Natl Acad Sci. 2020;117:25159–68.
Kocher A, Valiere S, Banuls A-L, Murienne J. High-throughput sequencing of kDNA amplicons for the analysis of Leishmania minicircles and identification of Neotropical species. Parasitology. 2018;145:585–94.
Alanazi AD, Alouffi AS, Alyousif MS, Rahi AA, Ali MA, Abdullah HH, et al. Molecular characterization of Leishmania species from stray dogs and human patients in Saudi Arabia. Parasitol Res. 2021;120:4241–6.
Ortega‐García MV, Salguero FJ, García N, Domínguez M, Moreno I, Berrocal A. Equine infection with Leishmania spp. in Costa Rica: study of five cases. Veterin Med Sci. 2021;7(6):2234–9.
Lee SY, Lee ST, Chang KP. Transkinetoplastidy—a novel phenomenon involving bulk alterations of mitochondrion-kinetoplast DNA of a trypanosomatid protozoan 1, 2. J Protozool. 1992;39:190–6.
Basselin M, Badet-Denisot M-A, Robert-Gero M. Modification of kinetoplast DNA minicircle composition in pentamidine-resistant Leishmania. Acta Trop. 1998;70:43–61.
Wilkes JM, Mulugeta W, Wells C, Peregrine AS. Modulation of mitochondrial electrical potential: a candidate mechanism for drug resistance in African trypanosomes. Biochem J. 1997;326:755–61.
Lanteri CA, Tidwell RR, Meshnick SR. The mitochondrion is a site of trypanocidal action of the aromatic diamidine DB75 in bloodstream forms of Trypanosoma brucei. Antimicrob Agents Chemother. 2008;52:875–82.
Lee S-T, Tarn C, Wang C-Y. Characterization of sequence changes in kinetoplast DNA maxicircles of drug-resistant Leishmania. Mol Biochem Parasitol. 1992;56:197–207.
Bruhn DF, Sammartino MP, Klingbeil MM. Three mitochondrial DNA polymerases are essential for kinetoplast DNA replication and survival of bloodstream form Trypanosoma brucei. Eukaryot Cell. 2011;10:734–43.
Agbe A, Yielding KL. Kinetoplasts play an important role in the drug responses of Trypanosoma brucei. J Parasitol. 1995:968–73.
Schamber-Reis BLF, Nardelli S, Régis-Silva CG, Campos PC, Cerqueira PG, Lima SA, et al. DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions. Mol Biochem Parasitol. 2012;183:122–31.
The authors thank the Department of Parasitology, Phramongkutklao College of Medicine; the Department of Microbiology, Faculty of Science, Mahidol University; and the Department of Genetics, Faculty of Science, Kasetsart University, for supporting this project and the Faculty of Science, Kasetsart University for providing high-performance computing facilities.
This research was funded by the Kasetsart University Research and Development Institute (KURDI), grant no. FF(KU)6.64.
The authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Anuntasomboon, P., Siripattanapipong, S., Unajak, S. et al. Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand. Parasites Vectors 15, 459 (2022). https://doi.org/10.1186/s13071-022-05592-1
- Leishmania martiniquensis