Leishmaniasis, is a neglected tropical disease of global importance with rising incidence due to climate change, limited therapeutic options, and increasing drug resistance. Novel strategies targeting parasite biology are urgently needed. In this study, we propose that the functionality and regulation of the parasite's sole SUMO-specific protease, poorly understood to date, represent a tractable biological target. Using an integrated approach combining bioinformatics, biochemical assays, SUMOylation reconstitution, proteomics, and infection models, we characterised the localisation, enzymatic properties, and phenotypic consequences of dysregulating this protease. GFP-tagged domain fragments localised predominantly to mitochondria with additional puncta at the nuclear periphery and exhibited canonical SUMO processing and deconjugation activity. An in vitro SUMOylation reconstitution assay demonstrated that both cysteine synthase (CS) and cystathionine β-synthase (CBS) are SUMOylated, with CS displaying a pronounced RanBP2-dependent enhancement of conjugation, confirming substrate-specific E3 ligase activity. Overexpression of the protease catalytic domain triggered profound cellular remodelling, including severe flagellar shortening (CD-GFP, 1.10 ± 1.05 μm and WT, 17.95 ± 4.35 μm, p < 0.05), disrupted vesicular trafficking (p < 0.05), and an ∼11-fold depletion of sterols (CD-GFP, 4.48 × 108 ± 2.90 × 108 AU and WT, 49.00 × 108 ± 59.3 × 108 AU, p < 0.05). Quantitative proteomics identified significant alterations in 135 proteins (76 upregulated, 59 downregulated; p < 0.05), encompassing pathways linked to energy metabolism, oxidative stress responses, ribosome biogenesis, and sterol biosynthesis. Parasite infectivity in mammalian macrophages at macrophage-to-parasite ratios of 1:10 and 1:20 were comparable (p > 0.05), with only a modest increase at 1:40 in CD-GFP-infected macrophages (p < 0.05). Collectively, our findings establish the SUMO protease as a key regulator of parasite morphology and metabolism. By revealing its broader roles in cellular adaptation and stress resilience, this work positions SUMO-dependent pathways as a promising regulatory axis for understanding, and ultimately disrupting, the transmission cycle of Leishmania and related kinetoplastid pathogens.

The global spread of high-risk clo1nes (HRCs) of multidrug-resistant (MDR) Pseudomonas aeruginosa has hindered infection control and treatment strategies worldwide. In Colombia, globally relevant HRCs such as ST235 and ST111 have been widely reported. In this study, we evaluated phenotypic and metabolic variations associated with intracellular survival and dissemination in P. aeruginosa. A total of 100 clinical isolates were collected from 22 hospitals in Colombia. The isolates had been previously characterized and classified as MDR or susceptible strains (SSs), and their sequence types (STs) had been earlier determined. Based on this prior characterization, isolates were grouped in this study as multidrug-resistant high-risk clones (HRC, n = 50; corresponding to sequence types ST235 and ST111), multidrug-resistant non-high-risk clones (NHRCs, n = 27; non-ST235/ST111), and susceptible strains (SS, n = 23; also, non-ST235/ST111). Phenotypic traits, including motility, spontaneous mutation frequency, biofilm formation, and pigment production, were evaluated. In addition, a subset of 30 isolates was assessed for intracellular survival in vitro and metabolomic profiling using liquid chromatography-mass spectrometry. HRC isolates exhibited significantly reduced motility compared with NHRC and SS isolates (swarming: HRC vs. NHRC, p = 0.0032; HRC vs. SS, p = 0.010; swimming: HRC vs. NHRC and SS, p < 0.0001; twitching: HRC vs. SS, p = 0.0004), as well as lower pigment production (pyocyanin: HRC vs. NHRC and SS, p < 0.0001; pyoverdine: HRC vs. NHRC, p < 0.0001). Metabolomic analysis revealed increased concentrations of metabolites associated with iron acquisition and siderophore-related pathways in HRC isolates. Overall, these findings suggest that P. aeruginosa HRCs display distinct phenotypic and metabolic patterns that may contribute to persistence and dissemination in clinical settings, contributing to their epidemiological success.

Inflammatory bowel disease (IBD) is a multifactorial disease, and patients frequently experience extraintestinal manifestations affecting multiple sites. Causes of systemic inflammation remain poorly understood, but molecules originating from the intestine likely play a role, with microbial and host small molecules polarizing host immune cells towards a pro- or anti-inflammatory phenotype. Using the dextran sodium sulfate (DSS) mouse model, which mimics the disrupted barrier function, microbial dysbiosis, and immune cell dysregulation of IBD, we investigated metabolomic and phenotypic changes at intestinal and systemic sites. Using spatial biology approaches, we mapped the distribution and relative abundance of molecules and cell types across a range of tissues, revealing significant changes in DSS-treated mice. Molecules identified as contributing to the statistical separation of treated from control mice were spatially localized within organs to determine their effects on cellular phenotypes through imaging mass cytometry. This spatial approach identified both intestinal and systemic molecular drivers of inflammation, including several not previously implicated in inflammation linked to IBD or the systemic effects of intestinal inflammation. Metabolic and inflammatory pathway interplay underpins systemic disease, and determining drivers at the molecular level may aid the development of new targeted therapies.

Cardiac viral diseases are among the major causes of economic losses in Atlantic salmon (Salmo salar L.) aquaculture. These include cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) and pancreas disease (PD) caused by Atlantic salmonid alphavirus (SAV). The resulting cardiomyopathies impact fish stock in terms of mortality, quality, growth performance and economic loss. Diagnosis of these diseases is currently based on clinical signs, histopathology and RT-qPCR. To identify putative biomarkers for use in the health assessment of Atlantic salmon, a quantitative proteomics investigation was undertaken with the aim of differentiating fish with CMS from healthy fish and fish with PD. Serum samples (n = 9/group) were collected during health assessment from pens where clinical CMS or PD were present and compared to serum from healthy Atlantic salmon. There were 34 differentially abundant proteins (DAPs) in CMS compared to healthy, 66 comparing CMS to PD, and 81 comparing PD to healthy. In relation to healthy samples, most DAPs were shared between CMS and PD, with higher relative abundances observed in PD. An exception to this was serum fibrinogen, which was identified as a putative biomarker for CMS, whereas differentiation of Atlantic salmon with CMS from those with PD was enhanced by the calculation of the ratio of fibrinogen to skeletal troponin C.

In plants, the apoplast contains a diverse set of proteins that underpin mechanisms for maintaining cell homeostasis, cell wall remodeling, cell signaling, and pathogen defense. Apoplast protein composition is highly regulated, primarily through the control of secretory traffic in response to endogenous and environmental factors. Dynamic changes in apoplast proteome facilitate plant survival in a changing climate. Even so, the apoplast proteome profiles in plants remain poorly characterized due to technological limitations. Recent progress in quantitative proteomics has significantly advanced the resolution of proteomic profiling in mammalian systems and has the potential for application in plant systems. In this protocol, we provide a detailed and efficient protocol for tandem mass tag (TMT)-based quantitative analysis of Arabidopsis thaliana secretory proteome to resolve dynamic changes in leaf apoplast proteome profiles. The protocol employs apoplast flush collection followed by protein cleaning using filter-aided sample preparation (FASP), protein digestion, TMT-labeling of peptides, and mass spectrometry (MS) analysis. Subsequent data analysis for peptide detection and quantification uses Proteome Discoverer software (PD) 3.0. Additionally, we have incorporated in silico-generated spectral libraries using PD 3.0, which enables rapid and efficient analysis of proteomic data. Our optimized protocol offers a robust framework for quantitative secretory proteomic analysis in plants, with potential applications in functional proteomics and the study of trafficking systems that impact plant growth, survival, and health. Key features • Rapid and high-purity collection of Arabidopsis thaliana leaf apoplast flush. • Use of filter-aided sample preparation (FASP) for protein cleaning to obtain high-quality data. • Use of in-house-generated theoretical spectral libraries for efficient and rapid analysis of MS data.

The gut microbiome has been strongly linked to health and disease, exerting its systemic effects through host and bacterial molecules that disseminate from the intestine. Understanding how these molecules may contribute to, exacerbate, or even improve specific health conditions is a key mechanistic challenge in microbiome research. Using the germ-free mouse model, we used a spatial biology approach to map the location of small molecules in intestinal and systemic tissues in addition to phenotyping cells in their vicinity. Significant differences were noted in molecular species across all tissues tested, including the ileum, colon, spleen, lung, liver, and kidney, with the greatest number of changes in the liver. Molecules putatively identified as phenol sulfate and 5-amino valeric acid betaine were noted to have significantly changed in abundance in the germ-free mouse intestine as well as systemically. Phenotypic characterization of germ-free mouse organs identified significant alterations in immune cell numbers indicative of an aberrant immune response, underlining the critical role of the microbiome in immune stimulation and priming, even at sites distal from the intestine. Our findings highlight the significant molecular and cellular changes that occur in the absence of a gut microbiota, identifying key microbiome-derived metabolites and host phenotypic signatures.

INTRODUCTION:Praziquantel (PZQ) is commonly used to treat schistosomiasis; however, there is considerable interindividual variability in its efficacy, partly because of genetic variation. Data on this relationship is scarce across Africa - where schistosomiasis is prevalent. This study aimed to investigate the pharmacokinetic/pharmacodynamic and pharmacogenetic relationship between PZQ and its metabolites in a Zimbabwean population infected with Schistosoma haematobium by leveraging dried blood spots (DBS) and mass spectrometry (MS). METHODS:DBS were obtained from 38 Zimbabwean participants on PZQ treatment at four-time points (0.5, 1.5, 2.5, and 4 h). We compared two extraction methods for recovering PZQ and its metabolites from the DBS cards and performed MS analysis to determine the concentrations. A random forest model was used to determine whether CYP1A2 , CYP2C9 , CYP2C19 , CYP2D6 , CYP3A4 , and CYP3A5 known variants were predictive of PZQ efficacy. The relationships between PZQ/metabolite concentration, metabolite ratio, and drug exposure with genotype were determined using a one-way analysis of variance. RESULTS:An acetonitrile and water (4 : 1) mixture was determined to be optimal for recovering PZQ and its metabolites from the DBS cards. Subsequent MS analysis identified PZQ and six metabolite compounds - including phase 1 metabolites (-2H)-O-PZQ, O2-PZQ, and 4-OH-PZQ. Pooled MS sampling was comparable to individual MS sampling for determining pharmacokinetic profiles at the 2.5 and 4-h time points. The (-2H)-O-PZQ and O2-PZQ metabolites had significantly higher concentrations in participants with CYP2C9*1/*9 and *9/*9 versus those with CYP2C9*1/*1. CYP1A2 rs2069514-A (formerly *1C ) and rs762551-A ( CYP1A2*30 ; formerly *1F ) were observed to alter PZQ pharmacokinetic profiles; however, differences in analyte concentrations across the corresponding genotypes were NS. CONCLUSION:We show that low-cost microsampling using DBS and MS is feasible for detecting and quantifying PZQ and its metabolites. Furthermore, our pharmacogenetics analysis elucidates the impact of known cytochrome P450 variants on PZQ drug response in an African setting.

Perturbation of the gut microbiota has been implicated in neurological diseases via communication across the microbiome-gut-brain axis. As a result, the discovery of mechanisms underlying interaction across this axis are becoming increasingly important. The germ-free (GF) mouse model has enabled an improved understanding of the influence of the gut microbiota on brain development and function. By utilising an advanced spatial profiling approach, we determined transcriptional changes in the brain, improving our understanding of how brain cells function and interact within their microenvironment in the absence of microbiome influence. Targeted regions of interest were selected based on brain regions implicated in neurological disease or reported structural differences between GF mouse brains and those of colonized mice. In the hippocampus 276 differentially expressed genes (DEGs) were identified, 345 DEGs in the thalamus, and 21 DEGs in pons. Contrastingly we identified only 2 DEGs in the midbrain and 4 in the medulla oblongata, with no DEGs in the cerebellum or corpus callosum. This data provides an overview of gut microbiota influence on gene expression in the brain, highlighting multiple genes of interest for further investigation in the context of microbiome influence on brain function and their potential relevance to neurological disease. ### Competing Interest Statement The authors have declared no competing interest.

In this study, we present a comprehensive characterization of a highly efficient desulfurizing bacterial isolate, SB1D. The isolate exhibited remarkable desulfurization of dibenzothiophene (DBT) and demonstrated the ability to metabolize benzothiophene (BT) and several of their alkylated derivatives. Genome-related index analyses, including 16S rRNA gene similarity (100%), Average Nucleotide Identity (ANI; 98.7%), digital DNA-DNA hybridization (dDDH; 88.7%), and phylogenomics, identified the strain as Rhodococcus qingshengii. Additionally, orthologous gene cluster analysis showed that SB1D shared the highest number of ortholog clusters (60) with R. qingshengii. The GC-MS analysis of the extracted metabolites identified 2-hydroxybiphenyl (2-HBP) and 4-methylhydroxybiphenyl (4-MHBP) as the major end-products of DBT and 4-methyldibenzothiophene (4-MDBT) desulfurization, respectively. The RAST genomic analysis revealed the presence of several organic-sulfur metabolism-related genes in the genome of SB1D. Together, these findings confirm that the isolate employs the sulfur-specific 4S pathway for the desulfurization of DBT and 4-MDBT. To our knowledge, this is the first report providing genome-based characterization and desulfurization pathway analysis of R. qingshengii SB1D, with the proven ability to desulfurize multiple thiophenic compounds found in diesel, and holds promise as a valuable biocatalyst for applications in biodesulfurization.

Purpose: This feasibility study investigated the practicability of collecting and analyzing tear proteins from preterm infants at risk of retinopathy of prematurity (ROP). We sought to identify any tear proteins which might be implicated in the pathophysiology of ROP as well as prognostic markers. Methods: Schirmer's test was used to obtain tear samples from premature babies, scheduled for ROP screening, after parental informed consent. Mass spectrometry was used for proteomic analysis. Results: Samples were collected from 12 infants, which were all adequate for protein analysis. Gestational age ranged from 25 + 6 to 31 + 1 weeks. Postnatal age at sampling ranged from 19 to 66 days. One infant developed self-limiting ROP. Seven hundred one proteins were identified; 261 proteins identified in the majority of tear samples, including several common tear proteins, were used for analyses. Increased risk of ROP as determined by the postnatal growth ROP (G-ROP) criteria was associated with an increase in lactate dehydrogenase B chain in tears. Older infants demonstrated increased concentration of immunoglobulin complexes within their tear samples and two sets of twins in the cohort showed exceptionally similar proteomes, supporting validity of the analysis. Conclusions: Tear sampling by Schirmer test strips and subsequent proteomic analysis by mass spectrometry in preterm infants is feasible. A larger study is required to investigate the potential use of tear proteomics in identification of ROP. Translational Relevance: Tear sampling and subsequent mass spectrometry in preterm infants is feasible. Investigation of the premature tear proteome may increase our understanding of retinal development and provide noninvasive biomarkers for identification of treatment -warranted ROP.

The gut microbiota exerts a significant influence on human health and disease. While compositional changes in the gut microbiota in specific diseases can easily be determined, we lack a detailed mechanistic understanding of how these changes exert effects at the cellular level. However, the putative local and systemic effects on human physiology that are attributed to the gut microbiota are clearly being mediated through molecular communication. Here, we determined the effects of gut microbiome-derived metabolites l-tryptophan, butyrate, trimethylamine (TMA), 3-methyl-4-(trimethylammonio)butanoate (3,4-TMAB), 4-(trimethylammonio)pentanoate (4-TMAP), ursodeoxycholic acid (UDCA), glycocholic acid (GCA) and benzoate on the first line of defence in the gut. Using in vitro models of intestinal barrier integrity and studying the interaction of macrophages with pathogenic and non-pathogenic bacteria, we could ascertain the influence of these metabolites at the cellular level at physiologically relevant concentrations. Nearly all metabolites exerted positive effects on barrier function, but butyrate prevented a reduction in transepithelial resistance in the presence of the pathogen Escherichia coli, despite inducing increased apoptosis and exerting increased cytotoxicity. Induction of IL-8 was unaffected by all metabolites, but GCA stimulated increased intra-macrophage growth of E. coli and tumour necrosis-alpha (TNF-α) release. Butyrate, 3,4-TMAB and benzoate all increased TNF-α release independent of bacterial replication. These findings reiterate the complexity of understanding microbiome effects on host physiology and underline that microbiome metabolites are crucial mediators of barrier function and the innate response to infection. Understanding these metabolites at the cellular level will allow us to move towards a better mechanistic understanding of microbiome influence over host physiology, a crucial step in advancing microbiome research.

Amphotericin B (AmB) is a potent antifungal and antiparasitic medication that exerts its action by disrupting the cell membrane of the leishmanial parasite, leading to its death. Understanding the genetic alterations induced by Amphotericin B is crucial for gaining insights into drug resistance mechanisms and developing more effective treatments against Leishmania infections. As a new Leishmania species, the molecular response of Leishmania orientalis to anti-leishmanial drugs has not been fully explored. In this study, Leishmania orientalis strain PCM2 culture was subjected to AmB exposure at a concentration of 0.03 uM over 72 hours compared to the control. The genomic alteration and transcriptomic changes were investigated by utilising the whole genome and RNA sequencing methods, followed by the analysis of single nucleotide polymorphisms (SNPs), differential gene expression, and chromosomal copy number variations (CNVs) assessed using read depth coverage (RDC) values across the entire genome. The chromosomal CNV analysis showed no significant difference between L. orientalis from the control and AmB-treated groups. The distribution of SNPs displayed notable variability, with higher SNP incidence in the control group compared to the AmB-treated group. Gene ontology analysis unveiled functions of the SNPs -associated genes involved in transporter function, genetic precursor synthesis, and purine nucleotide metabolism. Notably, the impact of AmB treatment on the L. orientalis gene expression profiles exhibited diverse expressional alterations, particularly the downregulation of pivotal genes such as the tubulin alpha chain gene. The intricate interplay between SNPs and gene expression alterations might underscore the complex regulatory networks underlying the AmB resistance of L. orientalis strain PCM2.

Despite advances in sequencing technologies that enable a greater understanding of mammalian gut microbiome composition, our ability to determine a role for individual strains is hampered by our inability to isolate, culture and study such microbes. Here we describe highly unusual Clostridium XIVa group strains isolated from the murine gut. Genome sequencing indicates that these strains, Clostridium symbiosum LM19B and LM19R and Clostridium clostridioforme LM41 and LM42, have significantly larger genomes than most closely related strains. Genomic evidence indicates that the isolated LM41 and LM42 strains diverge from most other Clostridium XIVa strains and supports reassignment of these groups at genus-level. We attribute increased C. clostridioforme LM41 and LM42 genome size to acquisition of mobile genetic elements including dozens of prophages, integrative elements, putative group II introns and numerous transposons including 29 identical copies of the IS66 transposase, and a very large 192 Kb plasmid. antiSmash analysis determines a greater number of biosynthetic gene clusters within LM41 and LM42 than in related strains, encoding a diverse array of potential novel antimicrobial compounds. Together these strains highlight the potential untapped microbial diversity that remains to be discovered within the gut microbiome and indicate that, despite our ability to get a top down view of microbial diversity, we remain significantly blinded to microbe capabilities at the strain level.

Certain factors hinder the commercialization of biodesulfurization process, including low substrate-specificity of the currently reported desulfurizing bacteria and restricted mass transfer of organic-sulfur compounds in biphasic systems. These obstacles must be addressed to clean organic-sulfur rich petro-fuels that pose serious environmental and health challenges. In current study, a dibenzothiophene desulfurizing strain, Gordonia rubripertincta W3S5 (source: oil contaminated soil) was systematically evaluated for its potential to remove sulfur from individual compounds and mixture of organic-sulfur compounds. Metabolic and genetic analyses confirmed that strain W3S5 desulfurized dibenzothiophene to 2-hydroxybiphenyl, suggesting that it follows the sulfur specific 4 S pathway. Furthermore, this strain demonstrated the ability to produce trehalose biosurfactants (with an EI 24 of 53%) in the presence of dibenzothiophene, as confirmed by TLC and FTIR analyses. Various genome annotation tools, such as ClassicRAST, BlastKOALA, BV-BRC, and NCBI-PGAP, predicted the presence of otsA , otsB , treY , treZ , treP , and Trehalose-monomycolate lipid synthesis genes in the genomic pool of strain W3S5, confirming the existence of the OtsAB, TreYZ, and TreP pathways. Overall, these results underscore the potential of strain W3S5 as a valuable candidate for enhancing desulfurization efficiency and addressing the mass transfer challenges essential for achieving a scaled-up scenario.

African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei, T. vivax and T. congolense, is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line (‘SUPKO’) lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3′-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC50s were similar for T. b. brucei, T. congolense, T. evansi and T. equiperdum but correlated less well with T. vivax. However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.

AbstractRNA-DNA hybrids are widespread epigenetic features of genomes that provide a growing range of activities in transcription, chromatin and DNA replication and repair. Understanding of these diverse functions has been advanced by characterising the proteins that interact with the hybrids, with all such studies revealing hundreds of potential interactors. However, all interaction analyses to date have focused on mammalian cells, and so it is unclear if a similar spectrum of RNA-DNA hybrid interactors is found in other eukaryotes, thus limiting our understanding of the conserved and lineage-specific activities linked to these genetic structures. The African trypanosome is a compelling organism in which to address these questions. As a divergent single-cell eukaryotic parasite of the Discoba grouping,Trypanosoma bruceidisplays substantial divergence in several aspects of core biology from its mammalian host and, unusually for a protist, has well-developed tools for molecular genetic analysis. For these reasons, we used DNA-RNA hybrid immunoprecipitation coupled with mass spectrometry to reveal 602 putative interactors inT. bruceimammal- or insect vector-infective stage cells. We show that the approach selects for a subset of the parasite proteome and reveals a range of predicted RNA-DNA hybrid associated activities, some overlapping with similar studies in mammals. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, impact onT. bruceinuclear RNA-DNA hybrid and DNA damage levels. Moreover, loss of each affects the operation of the crucial parasite immune survival mechanism of antigenic variation. Thus, our work reveals the broad range of activities contributed by RNA-DNA hybrids toT. bruceibiology, including new functions in host immune evasion as well as many conserved with mammals, and so likely fundamental to eukaryotic genome function.

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.

Mitochondrial DNAs (mtDNAs) appear in almost all eukaryotic species and are useful molecular markers for phylogenetic studies and species identification. Kinetoplast DNAs (kDNAs) are structurally complex circular mtDNA networks in kinetoplastids, divided into maxicircles and minicircles. Despite several kDNAs of many Leishmania species being examined, the kDNAs of the new species, Leishmania orientalis (formerly named Leishmania siamensis ) strain PCM2, have not been explored. This study aimed to investigate the maxicircle and minicircle DNAs of L. orientalis strain PCM2 using hybrid genome sequencing technologies and bioinformatic analyses. The kDNA sequences were isolated and assembled using the SPAdes hybrid assembler from the Illumina short-read and PacBio long-read data. Circular contigs of the maxicircle and minicircle DNAs were reconstructed and confirmed by BLASTn and rKOMICs programs. The kDNA genome was annotated by BLASTn before the genome comparison and phylogenetic analysis by progressiveMauve, MAFFT, and MEGA programs. The maxicircle of L. orientalis strain PCM2 (18,215 bp) showed 99.92% similarity and gene arrangement to Leishmania enriettii strain LEM3045 maxicircle with variation in the 12s rRNA gene and divergent region. Phylogenetics of the whole sequence, coding regions, divergent regions, and 12s rRNA gene also confirmed this relationship and subgenera separation. The identified 105 classes of minicircles (402–1177 bp) were clustered monophyletically and related to the Leishmania donovani minicircles. The kinetoplast maxicircle and minicircle DNAs of L. orientalis strain PCM2 contained a unique conserved region potentially useful for specific diagnosis of L. orientalis and further exploration of this parasite population genetics in Thailand and related regions.