Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

by Jeziel D. Damasceno, Jo ão Reis-Cunha, Kathryn Crouch, Dario Beraldi, Craig Lapsley, Luiz R. O. Tosi, Daniella Bartholomeu, Richard McCulloch Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome ofLeishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins inLeishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at su...
Source: PLoS Genetics - Category: Genetics & Stem Cells Authors: Source Type: research

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Protozoans of the genus Leishmania are the causative agents of an important neglected tropical disease referred to as leishmaniasis. During their lifecycle, the parasites can colonize the alimentary tract of the sand fly vector and the parasitophorous vacuole of the mammalian host, differentiating into distinct stages. Motile promastigotes are found in the sand fly vector and are transmitted to the mammalian host during the insect blood meal. Once in the vertebrate host, the parasites differentiate into amastigotes and multiply inside macrophages. To successfully establish infection in mammalian hosts, Leishmania parasites...
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research
Cutaneous leishmaniasis is a zoonotic infectious disease broadly distributed worldwide, causing a range of diseases with clinical outcomes ranging from self-healing infections to chronic disfiguring disease. The effective immune response to this infection is yet to be more comprehensively understood and is fundamental for developing drugs and vaccines. Thus, we used experimental models of susceptibility (BALB/c) and partial resistance (C57BL/6) to Leishmania amazonensis infection to investigate the local profile of mediators involved in the development of cutaneous leishmaniasis. We found worse disease outcome in BALB/c mi...
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research
American Tegumentary Leishmaniasis (ATL) is an endemic disease in Latin America, mainly caused in Brazil by Leishmania (Viannia) braziliensis. Clinical manifestations vary from mild, localized cutaneous leishmaniasis (CL) to aggressive mucosal disease. The host immune response strongly determines the outcome of infection and pattern of disease. However, the pathogenesis of ATL is not well understood, and host microRNAs (miRNAs) may have a role in this context. In the present study, miRNAs were quantified using qPCR arrays in human monocytic THP-1 cells infected in vitro with L. (V.) braziliensis promastigotes and in plasma...
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research
J Immunol Res. 2021 Mar 29;2021:6657785. doi: 10.1155/2021/6657785. eCollection 2021.ABSTRACTBACKGROUND: Cytokines and growth factors involved in the tissue inflammatory process influence the outcome of Leishmania infection. Insulin-like growth factor (IGF-I) constitutively present in the skin may participate in the inflammatory process and parasite-host interaction. Previous work has shown that preincubation of Leishmania (Leishmania) amazonensis with recombinant IGF-I induces accelerated lesion development. However, in human cutaneous leishmaniasis (CL) pathogenesis, it is more relevant to the persistent inflammatory pro...
Source: Journal of Immunology Research - Category: Allergy & Immunology Authors: Source Type: research
by Nofar Baron, Nitin Tupperwar, Irit Dahan, Uzi Hadad, Geula Davidov, Raz Zarivach, Michal ShapiraLeishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation inLeishmania is mostly post-transcriptional. TheLeishmania genome encodes six eIF4E candidates, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some o...
Source: PLoS Neglected Tropical Diseases - Category: Tropical Medicine Authors: Source Type: research
Leishmaniasis and trypanosomiasis are largely neglected diseases prevailing in tropical and subtropical conditions. These are an arthropod-borne zoonosis that affects humans and some animals and is caused by infection with protozoan of the genera Leishmania and Trypanosoma, respectively. These parasites present high genomic plasticity and are able to adapt themselves to adverse conditions like the attack of host cells or toxicity induced by drug exposure. Different mechanisms allow these adapting responses induced by stress, such as mutation, chromosomal rearrangements, establishment of mosaic ploidies, and gene expansion....
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research
Localized skin lesions are characteristic of cutaneous leishmaniasis (CL); however, Leishmania (Viannia) species, which are responsible for most CL cases in the Americas, can spread systemically, sometimes resulting in mucosal disease. Detection of Leishmania has been documented in healthy mucosal tissues (conjunctiva, tonsils, and nasal mucosa) and healthy skin of CL patients and in individuals with asymptomatic infection in areas of endemicity of L. (V.) panamensis and L. (V.) braziliensis transmission. However, the conditions and mechanisms that favor parasite persistence in healthy mucosal tissues are unknown. In this ...
Source: Infection and Immunity - Category: Infectious Diseases Authors: Tags: Fungal and Parasitic Infections Source Type: research
In this study, we first generated L. passim clone expressing fluorescent marker and then attempted to use CRISPR/Cas9 for the genome editing. We will discuss how these approaches can be used to better understand honey bee-trypanosomatid parasite interactions. Materials and Methods Culture of L. passim Lotmaria passim strain SF (PRA-403) was obtained from the American Type Culture Collection (ATCC) and cultured in the modified FP-FB medium (Salathe et al., 2012) at 25°C without CO2. To monitor the growth rate of L. passim, the parasites were first inoculated at 5 × 105/mL and their number during the culture was...
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research
In this study, we tested whether Arg1 is required for spontaneous healing of acute cutaneous leishmaniasis in C57BL/6 mice and for lifelong parasite persistence in draining lymph nodes (dLNs) of healed mice. In vitro, bone marrow-derived macrophages and lymphoid endothelial cells readily expressed Arg1 in response to IL-4 and/or IL-13, whereas skin or dLN fibroblasts failed to do so, even during hypoxia. In vivo, Arg1 was found in skin lesions and, to a much lower extent, also in dLNs of acutely infected C57BL/6 mice but became undetectable at both sites after healing. Deletion of Arg1 in hematopoietic and endothelial cell...
Source: Journal of Immunology - Category: Allergy & Immunology Authors: Tags: J Immunol Source Type: research
DNA polymerase β of Leishmania donovani is important for infectivity and it protects the parasite against oxidative damage. Int J Biol Macromol. 2018 Nov 16;: Authors: Khan MI, Mishra A, Jha PK, Abhishek K, Chaba R, Das P, Sinha KK Abstract The visceral leishmaniasis is caused by L. donovani, a neglected tropical disease with an estimated number of 500,000 cases worldwide. Apart from the absence of effective vaccine, the available drugs have limitations like toxic side effects and emergence of drug resistance. The genome of Leishmania is remarkably challenged by the oxidative stress present insid...
Source: International Journal of Biological Macromolecules - Category: Biochemistry Authors: Tags: Int J Biol Macromol Source Type: research
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