Specific human ATR and ATM inhibitors modulate single strand DNA formation in Leishmania major exposed to oxidative agent

Leishmania parasites are the causative agents of a group of neglected tropical diseases known as leishmaniasis. The molecular mechanisms employed by these parasites to adapt to the adverse conditions found in their hosts are not yet completely understood. DNA repair pathways can be used by Leishmania to enable survival in the interior of macrophages, where the parasite is constantly exposed to oxygen reactive species. In higher eukaryotes, DNA repair pathways are coordinated by the central protein kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). The enzyme Exonuclease-1 (EXO1) plays important roles in DNA replication, repair, and recombination, and it can be regulated by ATM- and ATR-mediated signaling pathways. In this study, the DNA damage response pathways in promastigote forms of L. major were investigated using bioinformatics tools, exposure of lineages to oxidizing agents and radiation damage, treatment of cells with ATM and ATR inhibitors, and flow cytometry analysis. We demonstrated high structural and important residue conservation for the catalytic activity of the putative LmjEXO1. The overexpression of putative LmjEXO1 made L. major cells more susceptible to genotoxic damage, most likely due to the nuclease activity of this enzyme and the occurrence of hyper-resection of DNA strands. These cells could be rescued by the addition of caffeine or a selective ATM inhibitor. In contrast, ATR-specific inhibition made the control cells more susceptible to oxidative damage in an LmjEXO1 overexpression-like manner. We demonstrated that ATR-specific inhibition results in the formation of extended single-stranded DNA, most likely due to EXO1 nucleasic activity. Antagonistically, ATM inhibition prevented single-strand DNA formation, which could explain the survival phenotype of lineages overexpressing LmjEXO1. These results suggest that an ATM homolog in Leishmania could act to promote end resection by putative LmjEXO1, and an ATR homologue could prevent hyper-resection, ensuring adequate repair of the parasite DNA

DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli.

Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4-110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(-). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(-) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts

Genetic variation of FcγRIIa induces higher uptake of Leishmania infantum and modulates cytokine production by adherent mononuclear cells in vitro

IntroductionSingle nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on their location and the type of nucleotide exchanged, an SNV can alter or inhibit the function of the gene in which it occurs. Immunoglobulin G (IgG) receptor genes have exhibited several polymorphisms, including rs1801274, which is found in the FcgRIIa gene. The replacement of A with T results in a Histidine (H) to Arginine (R) substitution, altering the affinity of the IgG receptor for IgG subtypes and C-reactive protein (CRP). In this study, we analyzed rs1801274 and its functional implications concerning L. Infantum uptake and cytokine production.MethodsWe genotyped 201 individuals from an endemic area for visceral leishmaniasis to assess the presence of rs1801274 using Taqman probes for a candidate gene study. Additionally, we included seventy individuals from a non-endemic area for a functional study. Subsequently, we isolated and cultivated one-week adherent mononuclear cells (AMCs) derived from the peripheral blood of participants residing in the non-endemic region in the presence of L. infantum promastigotes, with and without antigen-specific IgG and/or CRP. We analyzed the rate of phagocytosis and the production of nitric oxide (NO), tumor necrosis factor (TNF)-a, interleukin (IL)-10, IL-12 p70, IL-1b, IL- 6, and IL-8 in the culture supernatants.Results and discussionIn participants from the endemic region, the A/G (H/R isoform) heterozygous genotype was significantly associated with susceptibility to the disease. Furthermore, SNVs induced a change in the phagocytosis rate in an opsonin-dependent manner. Opsonization with IgG increased the production of IL-10, TNF-a, and IL-6 in AMCs with the H/R isoform, followed by a decrease in NO production. The results presented here suggest that the rs1801274 polymorphism is linked to a higher susceptibility to visceral leishmaniasis

Genetic identification <i>Trypanosoma cruzi</i> and <i>Trypanosoma rangeli</i> strains.

<p>Duplex PCR to detect specific subtelomeric sequences in <i>T</i>. <i>cruzi</i> and <i>T</i>. <i>rangeli</i>. The amplified products were observed in 2.0% agarose gel stained with ethidium bromide. <i>Trypanosoma rangeli</i> strains: P02; P07; P19; Cas4; SO48; LDG. <i>T</i>. <i>cruzi</i> strains and clone: Dm28c; JG; RN1; Hel; 3663; CLBr (CL Brener). MM: Molecular marker 100bp. NTC: No-template control.</p

Genetic characterization <i>Trypanosoma cruzi</i> and <i>Trypanosoma rangeli</i> strains.

<p>(A) kDNA analysis of <i>T</i>. <i>rangeli</i> strains in a silver stained 6% polyacrylamide gel containing PCR products obtained with primers S35/S36/KP1L. The presence of a 165-bp band indicates the presence of KP1 minicircles. (B) Detection of the 832-bp fragment of TcSC5D gene in <i>T</i>. <i>cruzi</i> strains and clone. (C) PCR–RFLP of TcSC5D products digested with <i>Hpa</i>I and <i>Sph</i>I enzymes. MM: Molecular marker 100bp. NTC: No-template control.</p

Discrimination between <i>T</i>. <i>cruzi</i> and <i>T</i>. <i>rangeli</i> by DNA content analysis.

<p>(A) DNA histogram of the clone CL Brener of <i>T</i>. <i>cruzi</i>, P07 strain (KP1+) of <i>T</i>. <i>rangeli</i>, and mixed CL Brener + P07. (B) DNA histogram of the clone CL Brener of <i>T</i>. <i>cruzi</i>, SO48 [KP1(−)] strain of <i>T</i>. <i>rangeli</i>, and mixed CL Brener + SO48.</p

Flow cytometric analysis of the relative DNA content in <i>Trypanosoma cruzi</i> and <i>Trypanosoma rangeli</i>.

<p>(A) DNA histogram showing superimposed traces of the clone CL Brener, <i>T</i>. <i>cruzi</i> strains and <i>L</i>. <i>major</i>. (B) DNA histogram showing superimposed traces of the clone CL Brener, <i>T</i>. <i>rangeli</i> strains and <i>L</i>. <i>major</i>.</p

Representative flow cytometric gating strategy for DNA content analysis of <i>Trypanosoma cruzi</i> CL-Brener and <i>Leishmania major</i> CC1 strains stained with propidium iodide (PI).

<p>Panels A and D represent relative size (FSC) and complexity (SSC). Panels B and E panels represent PI-A and PI-W parameters indicating single cells stained with PI. Panels C and F show DNA content histogram and G1-0 and G2-M peaks are pointed. Relative PI fluorescence intensity at G1-0 was used for DNA content estimation for all strains analyzed using <i>T</i>.<i>cruzi</i> CL-Brener as reference strain.</p

Image_1_Genetic variation of FcγRIIa induces higher uptake of Leishmania infantum and modulates cytokine production by adherent mononuclear cells in vitro.jpeg

IntroductionSingle nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on their location and the type of nucleotide exchanged, an SNV can alter or inhibit the function of the gene in which it occurs. Immunoglobulin G (IgG) receptor genes have exhibited several polymorphisms, including rs1801274, which is found in the FcgRIIa gene. The replacement of A with T results in a Histidine (H) to Arginine (R) substitution, altering the affinity of the IgG receptor for IgG subtypes and C-reactive protein (CRP). In this study, we analyzed rs1801274 and its functional implications concerning L. Infantum uptake and cytokine production.MethodsWe genotyped 201 individuals from an endemic area for visceral leishmaniasis to assess the presence of rs1801274 using Taqman probes for a candidate gene study. Additionally, we included seventy individuals from a non-endemic area for a functional study. Subsequently, we isolated and cultivated one-week adherent mononuclear cells (AMCs) derived from the peripheral blood of participants residing in the non-endemic region in the presence of L. infantum promastigotes, with and without antigen-specific IgG and/or CRP. We analyzed the rate of phagocytosis and the production of nitric oxide (NO), tumor necrosis factor (TNF)-a, interleukin (IL)-10, IL-12 p70, IL-1b, IL- 6, and IL-8 in the culture supernatants.Results and discussionIn participants from the endemic region, the A/G (H/R isoform) heterozygous genotype was significantly associated with susceptibility to the disease. Furthermore, SNVs induced a change in the phagocytosis rate in an opsonin-dependent manner. Opsonization with IgG increased the production of IL-10, TNF-a, and IL-6 in AMCs with the H/R isoform, followed by a decrease in NO production. The results presented here suggest that the rs1801274 polymorphism is linked to a higher susceptibility to visceral leishmaniasis.</p

Image_2_Genetic variation of FcγRIIa induces higher uptake of Leishmania infantum and modulates cytokine production by adherent mononuclear cells in vitro.jpeg

IntroductionSingle nucleotide variations (SNVs) are specific genetic variations that commonly occur in a population and often do not manifest phenotypically. However, depending on their location and the type of nucleotide exchanged, an SNV can alter or inhibit the function of the gene in which it occurs. Immunoglobulin G (IgG) receptor genes have exhibited several polymorphisms, including rs1801274, which is found in the FcgRIIa gene. The replacement of A with T results in a Histidine (H) to Arginine (R) substitution, altering the affinity of the IgG receptor for IgG subtypes and C-reactive protein (CRP). In this study, we analyzed rs1801274 and its functional implications concerning L. Infantum uptake and cytokine production.MethodsWe genotyped 201 individuals from an endemic area for visceral leishmaniasis to assess the presence of rs1801274 using Taqman probes for a candidate gene study. Additionally, we included seventy individuals from a non-endemic area for a functional study. Subsequently, we isolated and cultivated one-week adherent mononuclear cells (AMCs) derived from the peripheral blood of participants residing in the non-endemic region in the presence of L. infantum promastigotes, with and without antigen-specific IgG and/or CRP. We analyzed the rate of phagocytosis and the production of nitric oxide (NO), tumor necrosis factor (TNF)-a, interleukin (IL)-10, IL-12 p70, IL-1b, IL- 6, and IL-8 in the culture supernatants.Results and discussionIn participants from the endemic region, the A/G (H/R isoform) heterozygous genotype was significantly associated with susceptibility to the disease. Furthermore, SNVs induced a change in the phagocytosis rate in an opsonin-dependent manner. Opsonization with IgG increased the production of IL-10, TNF-a, and IL-6 in AMCs with the H/R isoform, followed by a decrease in NO production. The results presented here suggest that the rs1801274 polymorphism is linked to a higher susceptibility to visceral leishmaniasis.</p