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MBio Dec 2022Genetic exchange between different strains in the sand fly vector has been experimentally demonstrated and is supported by population genetic studies. In nature,...
Genetic exchange between different strains in the sand fly vector has been experimentally demonstrated and is supported by population genetic studies. In nature, opportunities for interstrain mating are restricted to flies biting multiply infected hosts or through multiple bites of different hosts. In contrast, self-mating could occur in any infected sand fly. By crossing two recombinant lines derived from the same Leishmania major strain, each expressing a different drug-resistance marker, self-hybridization in L. major was confirmed in a natural sand fly vector, , and in frequencies comparable to interstrain crosses. We provide the first high resolution, whole-genome sequencing analysis of large numbers of selfing progeny, their parents, and parental subclones. Genetic exchange consistent with classical meiosis is supported by the biallelic inheritance of the rare homozygous single nucleotide polymorphisms (SNPs) that arose by mutation during the generation of the parental clones. In contrast, heterozygous SNPs largely failed to be transmitted in Mendelian ratios for reasons not understood. SNPs that were heterozygous in both parents, however, recombined to produce homozygous alleles in some hybrids. For trisomic chromosomes present in both parents, transmittal to the progeny was only altered by self-hybridization, involving a gain or loss of somy in frequencies predicted by a meiotic process. Whole-genome polyploidization was also observed in the selfing progeny. Thus, self-hybridization in , with its potential to occur in any infected sand fly, may be an important source of karyotype variation, loss of heterozygosity, and functional diversity. are parasitic protozoa that cause a wide spectrum of diseases collectively known as the leishmaniases. Sexual reproduction in has been proposed as an important source of genetic diversity and has been formally demonstrated to occur inside the sand fly vector midgut. Nevertheless, in the wild, opportunities for genetic exchange between different species or strains are restricted by the capacity of different strains to colonize the same sand fly. In this work, we report the first high resolution, whole-genome sequence analysis of intraclonal genetic exchange as a type of self-mating in Our data reveal that self-hybridization can occur with comparable frequency as interstrain mating under experimental lab conditions, leading to important genomic alterations that can potentially take place within every naturally infected sand fly.
Topics: Animals; Leishmania major; Phlebotomus; Psychodidae; Reproduction; Mutation
PubMed: 36394334
DOI: 10.1128/mbio.02858-22 -
Turkiye Parazitolojii Dergisi Jun 2021This study aimed to determine the differences between the gene expression profiles of and promastigotes through comparative analysis of gene expressions. (Comparative Study)
Comparative Study
OBJECTIVE
This study aimed to determine the differences between the gene expression profiles of and promastigotes through comparative analysis of gene expressions.
METHODS
Cell culture of (MHOM/IL/80) and (MHOM/MA/67/ITMAP/263) cell lines was performed. Afterwards, total RNA isolation and cDNA synthesis were performed and fold changes in the expression levels of 30 genes that play a role in metabolic pathways and nucleic acid synthesis and co-expressed in two species were evaluated by reverse transcriptase polymerase chain reaction. Functions of genes were determined using LeishDB and KEGG databases.
RESULTS
In this study, profiles of protein-coding 30 genes expressed in and promastigotes were evaluated and significant differences were found between the two species (p<0.001). There was a significant fold change in the expression levels of 29% of genes common in the two species. The expression levels of nine genes in were found to be markedly higher than those of (fold change >1). These genes include phosphoglycan beta 1.3 galactosyltransferase-like, lathosterol oxidase-like, fatty acid elongase, 3-oxo-5 alpha-steroid 4-dehydrogenase, calpain-like cysteine peptidase, acetyl-coA synthetase, 3'-nucleotidase/nuclease, 3'-nucleotidase/nuclease precursor and 3-ketoacyl-coA thiolase-like. When the functions of the proteins that correspond to the genes common in the two species were examined in detail using the databases, it was determined that these genes play role in lipid, protein, carbohydrate and nucleic acid metabolic functions of the parasite.
CONCLUSION
Alterations in the expression profiles of genes common to and species may cause differences in the virulence, pathogenesis, clinical features and treatment modality between these parasite species. In addition, evaluation of gene profiles is important in the selection of species-specific or common targets for vaccine and drug studies.
Topics: Animals; Humans; Leishmania infantum; Leishmania major; Life Cycle Stages; Protozoan Proteins; Species Specificity; Transcriptome
PubMed: 34103283
DOI: 10.4274/tpd.galenos.2021.66375 -
Turkiye Parazitolojii Dergisi Mar 2020The province of Khorasan-Razavi in the North East of Iran is an endemic area for anthroponotic cutaneous leishmaniasis (ACL caused mainly by ) and zoonotic cutaneous... (Review)
Review
The province of Khorasan-Razavi in the North East of Iran is an endemic area for anthroponotic cutaneous leishmaniasis (ACL caused mainly by ) and zoonotic cutaneous leishmaniasis (ZCL caused mainly by ). Based on clinical signs, some cities were considered as ACL foci while others were considered to be endemic for ZCL. This paper reviews studies performed on patients diagnosed with cutaneous leishmaniasis (CL) via the use of direct slide examination, ELISA, electrophoresis isoenzyme, RAPD PCR and PCR in Mashhad; the study also includes cases of CL in other cities of the Khorasan-Razavi province where only PCR used as a diagnostic tool. The data show that both and caused CL in most of the cities investigated. Our review shows that was found in areas where ACL is prevalent and was observed in areas with high incidence of ZCL. This distribution represents a major change in the epidemiological pattern of Leishmania in the Khorasan-Razavi province.
Topics: Adult; Animals; Electrophoresis; Enzyme-Linked Immunosorbent Assay; Humans; Incidence; Iran; Leishmania major; Leishmania tropica; Leishmaniasis, Cutaneous; Polymerase Chain Reaction; Prevalence; Random Amplified Polymorphic DNA Technique; Zoonoses
PubMed: 32212595
DOI: 10.4274/tpd.galenos.2019.6137 -
JCI Insight Jul 2023The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells....
The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major-infected (L. major-infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.
Topics: Leishmania major; Phagocytes; Apoptosis; Humans; Virulence; Mice, Inbred C57BL; Cells, Cultured; Mice; Animals
PubMed: 37310793
DOI: 10.1172/jci.insight.169020 -
Journal of Visualized Experiments : JoVE Oct 2022Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical...
Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major, offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.
Topics: Humans; Mice; Animals; Bacterial Toxins; Leishmania major; Membrane Lipids; Cell Membrane; Cholesterol; Mammals
PubMed: 36373947
DOI: 10.3791/64341 -
Antimicrobial Agents and Chemotherapy Feb 2020There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million...
There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of and Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and -infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of -infected macrophages.
Topics: Amphotericin B; Animals; Antiprotozoal Agents; Chitosan; Culture Media; Dose-Response Relationship, Drug; Female; Humans; Hydrogen-Ion Concentration; Leishmania major; Leishmania mexicana; Life Cycle Stages; Macrophages; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Molecular Weight; Nitric Oxide; Parasitic Sensitivity Tests; Pinocytosis; Primary Cell Culture; Reactive Oxygen Species; THP-1 Cells; Tumor Necrosis Factor-alpha
PubMed: 31871082
DOI: 10.1128/AAC.01772-19 -
Turkiye Parazitolojii Dergisi Jun 2021The relationship between drug resistance and the expression of hexokinase (HK) has been indicated in leishmaniasis. According to the prolonged treatment period in...
OBJECTIVE
The relationship between drug resistance and the expression of hexokinase (HK) has been indicated in leishmaniasis. According to the prolonged treatment period in cutaneous leishmaniasis (CL) patients co-infected with in Iran, this study aims to investigate the expression of HK in the proteome of and using a proteomic approach.
METHODS
A total of 205 samples were removed from the lesions of patients in Fars province, Iran, for the characterization of and using polymerase chain reaction (PCR). After protein extraction, two-dimensional gel electrophoresis was employed for protein separation. Several spots were isolated for HK determination in the proteomes of and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS).
RESULTS
The PCR results showed 5 positive cases for and 96 positive cases for . MALDI TOF/TOF MS indicated HK as a common protein in the proteome of and . HK was up-regulated in the proteome in comparison with the proteome.
CONCLUSION
Since a relationship between HK expression and drug resistance has been indicated in leishmaniasis, the overexpression of HK in might be related to the increased duration of the treatment period in CL patients co-infected with .
Topics: Coinfection; Crithidia; Drug Resistance; Euglenozoa Infections; Hexokinase; Humans; Iran; Leishmania major; Proteome; Proteomics
PubMed: 34103282
DOI: 10.4274/tpd.galenos.2020.6983 -
International Journal of Biological... Mar 2021Telomeres from different eukaryotes, including trypanosomatids, are transcribed into TERRA noncoding RNAs, crucial in regulating chromatin deposition and telomere...
Telomeres from different eukaryotes, including trypanosomatids, are transcribed into TERRA noncoding RNAs, crucial in regulating chromatin deposition and telomere length. TERRA is transcribed from the C-rich subtelomeric strand towards the 3'-ends of the telomeric array. Using bioinformatics, we confirmed the presence of subtelomeric splice acceptor sites at all L. major chromosome ends. Splice leader sequences positioned 5' upstream of L. major chromosomes subtelomeres were then mapped using SL-RNA-Seq libraries constructed from three independent parasite life stages and helped confirm TERRA expression from several chromosomes ends. Northern blots and RT-qPCR validated the results showing that L. major TERRA is processed by trans-splicing and polyadenylation coupled reactions. The number of transcripts varied with the parasite's life stage and continuous passages, being more abundant in the infective forms. However, no putative subtelomeric promoters involved in TERRA's transcriptional regulation were detected. In contrast, the observed changes in parasite's telomere length during development, suggest that differences in telomeric base J levels may control TERRA transcription in L. major. Also, TERRA-R loops' detection, mainly in the infective forms, was suggestive of TERRA's involvement in telomere protection. Therefore, Leishmania TERRA shares conserved features with other eukaryotes and advances new telomere specific functions in a Public Health-impacting parasite.
Topics: Cloning, Molecular; Databases, Genetic; Gene Expression Profiling; Gene Expression Regulation, Developmental; Leishmania major; Polyadenylation; Protozoan Proteins; RNA Splicing; Sequence Analysis, RNA; Telomere; Transcription Factors
PubMed: 33548324
DOI: 10.1016/j.ijbiomac.2021.01.192 -
Archives of Biochemistry and Biophysics May 2021ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing...
ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA+ proteins have a range of functions, from DNA replication to protein degradation. Rvbs, also known as RUVBLs, are AAA+ ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major, one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitro. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance.
Topics: Adenosine Triphosphatases; Amino Acid Sequence; DNA Helicases; Leishmania major; Protein Folding; Protein Multimerization; Protein Structure, Quaternary; Solutions
PubMed: 33775623
DOI: 10.1016/j.abb.2021.108841 -
Parasites & Vectors Mar 2017Infection and clinical disease associated with Leishmania major and Leishmania tropica, two common agents of human cutaneous leishmaniosis, have rarely been reported in... (Comparative Study)
Comparative Study
BACKGROUND
Infection and clinical disease associated with Leishmania major and Leishmania tropica, two common agents of human cutaneous leishmaniosis, have rarely been reported in dogs. This study describes dogs infected with these Leishmania spp. prevalent in the Middle East and North Africa, and compares the serological response of dogs infected with Leishmania infantum, L. major or L. tropica to whole promastigote antigen enzyme-linked immunosorbent assay (ELISA) of each species and to rK39 dipstick.
RESULTS
Leishmania major infection in a 5-month-old male dog was associated with alopecic and ulcerative periocular and limb skin lesions which responded to allopurinol treatment. Infection was detected by skin and blood polymerase chain reaction (PCR) and confirmed by DNA sequencing but the dog was seronegative. Leishmania tropica infection was detected in a 3-month-old female dog co-infected with Babesia vogeli and Anaplasma platys and with no skin lesions. PCR and DNA sequencing of the blood and parasite culture were positive for L. tropica. Sera from 11 dogs infected with L. infantum, L. major or L. tropica were reactive with all three Leishmania spp. antigens except for sera from a dog with L. major infection. No significant differences were found between reactivity of dog sera to the antigen of the infecting species, or to the other Leishmania spp. antigens. Sera from dogs infected with L. infantum and L. tropica were positive with the rK39 antigen kit, while dogs with L. major infection were seronegative.
CONCLUSIONS
Skin lesions in L. major infected dogs from this study and previous reports (n = 2) were ulcerative and located on the muzzle, feet and foot pads and not associated with generalized lymphadenomegaly and splenomegaly. In previous L. tropica infections, skin lesions were proliferative mucocutaneous in young dogs (n = 2), or associated with widespread dermatitis, lymphadenomegaly and splenomegaly in older dogs with similarity to L. infantum infection (n = 2). This study suggests that ELISA serology with whole promastigote antigen is not distinctive between L. infantum, L. major and L. tropica canine infections and that some L. major infections are not seropositive. PCR with DNA sequencing should be used to discriminate between canine infections with these three species.
Topics: Africa, Northern; Animals; Coinfection; Dog Diseases; Dogs; Enzyme-Linked Immunosorbent Assay; Female; Humans; Israel; Leishmania infantum; Leishmania major; Leishmania tropica; Leishmaniasis, Cutaneous; Male; Middle East; Polymerase Chain Reaction; Skin
PubMed: 28285601
DOI: 10.1186/s13071-017-2050-7