rKOMICS: an R package for processing mitochondrial minicircle assemblies in population-scale genome projects

Background The advent of population-scale genome projects has revolutionized our biological understanding of parasitic protozoa. However, while hundreds to thousands of nuclear genomes of parasitic protozoa have been generated and analyzed, information about the diversity, structure and evolution of their mitochondrial genomes remains fragmentary, mainly because of their extraordinary complexity. Indeed, unicellular flagellates of the order Kinetoplastida contain structurally the most complex mitochondrial genome of all eukaryotes, organized as a giant network of homogeneous maxicircles and heterogeneous minicircles. We recently developed KOMICS, an analysis toolkit that automates the assembly and circularization of the mitochondrial genomes of Kinetoplastid parasites. While this tool overcomes the limitation of extracting mitochondrial assemblies from Next-Generation Sequencing datasets, interpreting and visualizing the genetic (dis)similarity within and between samples remains a time-consuming process. Results Here, we present a new analysis toolkit—rKOMICS—to streamline the analyses of minicircle sequence diversity in population-scale genome projects. rKOMICS is a user-friendly R package that has simple installation requirements and that is applicable to all 27 trypanosomatid genera. Once minicircle sequence alignments are generated, rKOMICS allows to examine, summarize and visualize minicircle sequence diversity within and between samples through the analyses of minicircle sequence clusters. We showcase the functionalities of the (r)KOMICS tool suite using a whole-genome sequencing dataset from a recently published study on the history of diversification of the Leishmania braziliensis species complex in Peru. Analyses of population diversity and structure highlighted differences in minicircle sequence richness and composition between Leishmania subspecies, and between subpopulations within subspecies. Conclusion The rKOMICS package establishes a critical framework to manipulate, explore and extract biologically relevant information from mitochondrial minicircle assemblies in tens to hundreds of samples simultaneously and efficiently. This should facilitate research that aims to develop new molecular markers for identifying species-specific minicircles, or to study the ancestry of parasites for complementary insights into their evolutionary history.

Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the ‘Mary kDNA PCR’ and a newly designed ‘LC kDNA PCR’ for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.

High-throughput sequencing of kDNA amplicons for the analysis ofLeishmaniaminicircles and identification of Neotropical species

SUMMARYLeishmaniakinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitiveLeishmaniadetection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization ofLeishmaniaminicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18Leishmaniastrains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis ofLeishmaniarelationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications ofLeishmaniaspp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications inLeishmaniaresearch.

Epidemiological survey of Lutzomyia longipalpis infected by Leishmania infantum in an endemic area of Brazil

The objective of this study was to perform an epidemiological survey to determine the areas at risk of visceral leishmaniasis through the detection and quantification of natural infection by Leishmania infantum in Lutzomyia longipalpis. The sandflies were captured between February 2009 and January 2010, at 21 sites in four regions of the Fortaleza municipality. Samples were screened for the presence of Leishmania DNA by Real Time PCR (qPCR), amplification of kDNA minicircle sequence. Out of the 123 pools of analyzed sandflies, 45 were positive for L.infantum, and the minimum infection rate was 3.7%. In the north, south, east and west regions, the pool screen assay predicted sand-fly infection prevalence of 3.4%, 4.7%, 4.9% and 8.4%, respectively. The parasite load ranged from 2.45 ± 0.96 to 2,820,246 ± 106,072. No statistical differences were found with respect to the frequency of sand-fly infection between the regions (P=0.3014), seasons (P = 0.3906) or trap locations (P = 0.8486). Statistical differences were found with respect to the frequency of sand-fly infection between the two seasons only in the west region (P=0.0152). The qPCR was able to detect and quantify L. infantum in L. longipalpis, therefore succeeding in identifying the areas of greatest risk of VL transmission.

Redox Potential Regulates Binding of Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin

ABSTRACT Kinetoplast DNA, the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a remarkable structure containing 5,000 topologically linked DNA minicircles. Their replication is initiated at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L- and H-strands, respectively. A UMS-binding protein (UMSBP), binds specifically the conserved origin sequences in their single stranded conformation. The five CCHC-type zinc knuckle motifs, predicted in UMSBP, fold into zinc-dependent structures capable of binding a single-stranded nucleic acid ligand. Zinc knuckles that are involved in the binding of DNA differ from those mediating protein-protein interactions that lead to the dimerization of UMSBP. Both UMSBP DNA binding and its dimerization are sensitive to redox potential. Oxidation of UMSBP results in the protein dimerization, mediated through its N-terminal domain, with a concomitant inhibition of its DNA-binding activity. UMSBP reduction yields monomers that are active in the binding of DNA through the protein C-terminal region. C. fasciculata trypanothione-dependent tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. The possibility that UMSBP binding at the minicircle replication origin is regulated in vivo by a redox potential-based mechanism is discussed.

Intramitochondrial Localization of Universal Minicircle Sequence-Binding Protein, a Trypanosomatid Protein That Binds Kinetoplast Minicircle Replication Origins

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase β, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.