Molecular and biochemical parasitology最新文献

{"title":"Stress-responsive AMP Kinase like protein regulates encystation of Entamoeba invadens","authors":"Piyali Goswami,&nbsp;Sintu Kumar Samanta ,&nbsp;Tarun Agarwal,&nbsp;Sudip K. Ghosh","doi":"10.1016/j.molbiopara.2022.111507","DOIUrl":"10.1016/j.molbiopara.2022.111507","url":null,"abstract":"<div><p><span>Starvation is always accompanied by an increase in the ratio of AMP/ATP followed by activation of AMPK<span>. It is one of the sensors for cellular energy status and is highly conserved across various species. Its role in the stage differentiation process of protozoan species like </span></span><span><em>Giardia</em></span>, <span><em>Plasmodium, </em><em>Trypanosome</em><em>,</em></span> and <span><em>Toxoplasma</em></span> has been reported. Since <span><em>Entamoeba</em></span> undergoes encystation in glucose-starved conditions; it intrigued us to investigate the existence and role of AMPK during the differentiation of trophozoites to the cyst. By employing <em>in silico</em> approaches, we have identified an AMPK homologue which is denominated here as EiAMPK (AMPK-like protein in <span><em>Entamoeba invadens</em></span><span><span>). Sequence and structural analysis indicate that EiAMPK is sequentially and structurally similar to the AMPK alpha subunit of other organisms. The recombinant form of EiAMPK was functionally active and in accordance, its activity was inhibited by an AMPK-specific inhibitor (eg. Compound C). The increased expression of EiAMPK during different stresses indicated that EiAMPK is a stress-responsive gene. To further investigate, whether EiAMPK has any role in encystation, we employed RNAi-mediated </span>gene silencing that demonstrated its active involvement in encystation. It is known that </span><em>Entamoeba</em><span><span> maintains a flow of glucose from the glycolytic pathway to chitin synthesis for cyst wall formation during encystation. It is conceivable that EiAMPK might have a command over such </span>glucose metabolism. As anticipated, the chitin synthesis was found greatly inhibited in both EiAMPK knockdown and Compound C treated cells, indicating that EiAMPK regulates the cyst wall chitin synthesis.</span></p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"251 ","pages":"Article 111507"},"PeriodicalIF":1.5,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40547739","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deleting ku80 improves the efficiency of targeted gene editing in Neospora caninum","authors":"Kaijian Wu ,&nbsp;Xingju Song ,&nbsp;Yayun Wu ,&nbsp;Xu Yang ,&nbsp;Jing Liu ,&nbsp;Qun Liu","doi":"10.1016/j.molbiopara.2022.111508","DOIUrl":"10.1016/j.molbiopara.2022.111508","url":null,"abstract":"<div><p>CRISPR/Cas9 technology has been widely used for gene editing in organisms. Gene deletion of the <em>ku80/ku70</em> complex can improve the efficiency of gene replacement in <em>Arabidopsis thaliana</em>, <em>Cryptococcus neoformans</em>, and <em>Toxoplasma gondii</em>, which remained elusive in <em>Neospora caninum</em>. Here, we knock out the <em>ku80</em> gene in Nc1 strain by using CRISPR/Cas9, detect the growth rate and virulence of <em>Nc</em>Δ<em>ku80</em>. Then we compare the efficiency of gene replacements between <em>Nc</em>Δ<em>ku80</em> and Nc1 strains by transfected with the same HA-tagged plasmids, and the percentage of HA-tagged parasites was investigated by IFA. The results showed that gene targeting efficiency was increased in the <em>Nc</em>Δ<em>ku80</em> strain via double crossover at several genetic loci, but its growth rate and virulence were unaffected. In conclusion, the <em>Nc</em>Δ<em>ku80</em> strain can be used as an effective strain for rapid gene editing of <em>N. caninum</em>.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"251 ","pages":"Article 111508"},"PeriodicalIF":1.5,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40610714","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Schistosomiasis related circulating cell-free DNA: A useful biomarker in diagnostics","authors":"Hanif Ullah ,&nbsp;Safia Arbab ,&nbsp;Ka Li ,&nbsp;Muhammad Inayat Ullah Khan ,&nbsp;Abdul Qadeer ,&nbsp;Nehaz Muhammad","doi":"10.1016/j.molbiopara.2022.111495","DOIUrl":"10.1016/j.molbiopara.2022.111495","url":null,"abstract":"<div><p><span><em>Schistosoma</em></span><span> is a genus of trematodes causing schistosomiasis, a major neglected tropical disease infecting more than 240 million people and with 700 million people at the risk of infection in the tropical and subtropical regions of the world, especially low-income countries. For the elimination of the disease, accurate diagnostic tools are needed. Besides allowing early treatment, early detection prevents environmental contamination and in turn ensures safe water sources in the endemic areas. Cell-free DNA (cfDNA) biomarker detection is a relatively new tool, used for the diagnosis of schistosomiasis in the early stages of infection from non-invasive clinical or experimental samples. cfDNA can be detected in </span><em>Schistosoma</em> infected host body fluids such as urine, serum, saliva and tissues, mainly in blood offering significant benefits for accurate diagnosis. In the current review, we described different characteristics of cfDNA, evidencing and supporting its potential uses in <em>Schistosoma</em> diagnosis and the improvement of treatment effectiveness.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"251 ","pages":"Article 111495"},"PeriodicalIF":1.5,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40522524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strongyloides spp. eliminate male-determining sperm post-meiotically","authors":"Alex Dulovic ,&nbsp;Iris Koch ,&nbsp;Katharina Hipp ,&nbsp;Adrian Streit","doi":"10.1016/j.molbiopara.2022.111509","DOIUrl":"10.1016/j.molbiopara.2022.111509","url":null,"abstract":"<div><p><span>If normal male meiosis occurs, it would be expected that 50 % of sperm lack an X chromosome (nullo X) and hence upon fertilisation, result in male progeny. However, for sexual reproduction within the free-living stages of </span><span><em>Strongyloides</em></span><span> spp. male offspring are absent. We had shown earlier by quantitative whole genome sequencing that within </span><em>Strongyloides</em> spp., nullo-X sperm are either absent (<em>S. papillosus</em>) or underrepresented (<em>S. ratti</em><span>) among mature sperm. To investigate how and when this elimination of male-determining sperm occurs, we characterised spermatogenesis<span><span> and the dynamic localisation of important molecular players such as tubulin, actin and major sperm protein by DIC microscopy, immunohistochemistry, and </span>fluorescent in situ hybridization (FISH) in </span></span><em>S. ratti</em>, <em>S. papillosus</em> and <em>Parastrongyloides trichosuri</em><span>. We found that meiotic divisions in these parasites proceeded as expected for organisms with XO males, resulting in four equally sized spermatocytes<span>, two with and two without an X chromosome. However, mature sperm were found to almost always contain an X chromosome. We also observed structures that contained protein constituents of sperm, such as actin and major sperm protein (MSP) but no DNA. These structures resemble </span></span><em>C. elegans</em> residual bodies in appearance and may assume their function. We hypothesize that spermatocytes without an X-chromosome undergo some form of programmed cell death and transform into these residual body-like structures. As in <em>C. elegans</em>, MSP is found in fibrous body-membranous organelles (FB-MOs). Knocking down MSP by RNAi showed that MSP is essential for fertility in <em>S. ratti</em>, as it is in <em>C. elegans</em>.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"251 ","pages":"Article 111509"},"PeriodicalIF":1.5,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40709198","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Is Strongyloides stercoralis hyperinfection induced by glucocorticoids a result of both suppressed host immunity and altered parasite genetics?","authors":"De'Broski R. Herbert ,&nbsp;Jonathan D.C. Stoltzfus ,&nbsp;Heather L. Rossi ,&nbsp;David Abraham","doi":"10.1016/j.molbiopara.2022.111511","DOIUrl":"10.1016/j.molbiopara.2022.111511","url":null,"abstract":"<div><p>The gastrointestinal (GI) nematode <em>Strongyloides stercoralis</em> (<em>S.s.</em>) causes human strongyloidiasis, a potentially life-threatening disease that currently affects over 600 million people globally. The uniquely pernicious aspect of <em>S.s.</em> infection, as compared to all other GI nematodes, is its autoinfective larval stage (L3a) that maintains a low-grade chronic infection, allowing undetectable persistence for decades. Infected individuals who are administered glucocorticoid therapy can develop a rapid and often lethal hyperinfection syndrome within days. Hyperinfection patients often present with dramatic increases in first- and second-stage larvae and L3a in their GI tract, with L3a widely disseminating throughout host organs leading to sepsis. How glucocorticoid administration drives hyperinfection remains a critical unanswered question; specifically, it is unknown whether these steroids promote hyperinfection through eliminating essential host protective mechanisms and/or through dysregulating parasite development. This current deficiency in understanding is largely due to the previous absence of a genetically defined mouse model that would support all <em>S.s.</em> life-cycle stages and the lack of successful approaches for <em>S.s</em>. genetic manipulation. However, there are currently new possibilities through the recent demonstration that immunodeficient NOD.Cg-<em>Prkdc</em>^{<em>scid</em>} <em>Il2rg</em>^{<em>tm1Wjl</em>}/SzJ (NSG) mice support sub-clinical infections that can be transformed to lethal hyperinfection syndrome following glucocorticoid administration. This is coupled with advances in transcriptomics, transgenesis, and gene inactivation strategies that now allow rigorous scientific inquiry into <em>S.s.</em> biology. We propose that combining in vivo manipulation of host immunity and deep immunoprofiling strategies with the latest advances in <em>S.s.</em> transcriptomics, <em>piggyBac</em> transposon-mediated transgene insertion, and CRISPR/Cas-9-mediated gene inactivation will facilitate new insights into the mechanisms that could be targeted to block lethality in humans with <em>S.s.</em> hyperinfection.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"251 ","pages":"Article 111511"},"PeriodicalIF":1.5,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0166685122000652/pdfft?md5=aad215055bfc5228af537b009938fce2&pid=1-s2.0-S0166685122000652-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40655719","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Increased iron uptake in the bladder wall of racemose cysts of Taenia solium","authors":"Miguel A. Orrego ,&nbsp;Carlos M. Vasquez ,&nbsp;Kayla Togneri ,&nbsp;Juan P. Laclette ,&nbsp;Hector H. Garcia ,&nbsp;Theodore E. Nash ,&nbsp;for the Cysticercosis Working Group in Peru","doi":"10.1016/j.molbiopara.2022.111496","DOIUrl":"10.1016/j.molbiopara.2022.111496","url":null,"abstract":"<div><p>Racemose neurocysticercosis is an aggressive infection caused by the aberrant expansion and proliferation of the bladder wall of the <em>Taenia solium</em> cyst within the subarachnoid spaces of the human brain. The parasite develops and proliferates in a microenvironment with low concentrations of growth factors and micronutrients compared to serum. Iron is important for essential biological processes, but its requirement for racemose cyst viability and proliferation has not been studied. The presence of iron in the bladder wall of racemose and normal univesicular <em>T. solium</em> cysts was determined using Prussian blue staining. Iron deposits were readily detected in the bladder wall of racemose cysts but were not detectable in the bladder wall of univesicular cysts. Consistent with this finding, the genes for two iron-binding proteins (<em>ferritin</em> and <em>melanotransferrin</em>) and <em>ribonucleotide reductase</em> were markedly overexpressed in the racemose cyst compared to univesicular cysts. The presence of iron in the bladder wall of racemose cysts may be due to its increased metabolic rate due to proliferation.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"251 ","pages":"Article 111496"},"PeriodicalIF":1.5,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9869405/pdf/nihms-1863889.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10505584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Aminoacyl-tRNA synthetase (AARS) as an attractive drug target in neglected tropical trypanosomatid diseases-Leishmaniasis, Human African Trypanosomiasis and Chagas disease","authors":"Vikas Kushwaha,&nbsp;Neena Capalash","doi":"10.1016/j.molbiopara.2022.111510","DOIUrl":"10.1016/j.molbiopara.2022.111510","url":null,"abstract":"<div><p><span><span>TriTryp diseases (Leishmaniasis, Human African Trypanosomiasis (HAT), and Chagas disease) are devastating parasitic neglected tropical diseases (NTDs) that affect billions of people in developing countries, cause high mortality in humans, and impose a large socio-economic burden. The current treatment options against tritryp diseases are suboptimal and challenging due to the emergence of resistance against available tritryp drugs. Hence, designing and developing effective anti-tritryp drugs with novel targets are required. Aminoacyl-tRNA synthetases (AARSs) involved in specific aminoacylation of transfer RNAs (tRNAs), interrupt </span>protein synthesis through inhibitors, and retard the parasite growth. AaRSs have long been studied as therapeutic targets in bacteria, and three </span>aaRS<span> inhibitors, mupirocin (against IleRS), tavaborole AN2690 (against LeuRS), and halofuginone (against ProRS), are already in clinical practice. The structural differences between tritryp and human aaRSs and the presence of unique sequences (N-terminal domain/C-terminal domain/catalytic domain) make them potential target for developing selective inhibitors. Drugs based on a single aaRS target developed by high-throughput screening (HTS) are less effective due to the emergence of resistance. However, designing multi-targeted drugs may be a better strategy for resistance development. In this perspective, we discuss the characteristics of tritryp aaRSs, sequence conservation in their orthologs and their peculiarities, recent advancements towards the single-target and multi-target aaRS inhibitors developed through rational design.</span></p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"251 ","pages":"Article 111510"},"PeriodicalIF":1.5,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40625827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation","authors":"Thanuja Thekke-Veetil ,&nbsp;Nancy K. McCoppin ,&nbsp;Leslie L. Domier ,&nbsp;M.R. Hajimorad ,&nbsp;Kris N. Lambert ,&nbsp;Hyoun-Sub Lim ,&nbsp;Glen L. Hartman","doi":"10.1016/j.molbiopara.2022.111489","DOIUrl":"10.1016/j.molbiopara.2022.111489","url":null,"abstract":"<div><p><span><span>Despite their economic significance in agricultural cropping systems, a lack of suitable molecular tools for manipulating gene expression has hindered progress in the functional genomics of plant </span>parasitic nematodes (PPN). Obligate sexual reproduction and the obligate nature of PPN-host interactions further complicate the development of </span><em>in vivo</em><span> gene delivery and expression systems in these pests. Methods such as microinjection<span> and microprojectile bombardment have been developed for introducing gene constructs into the free-living nematode, </span></span><span><em>Caenorhabditis elegans</em></span><span><span>. However, these procedures can be laborious and inefficient. Electroporation<span><span> has been used extensively to introduce macromolecules, including single-stranded </span>RNAs, into eukaryotic and </span></span>prokaryotic cells<span><span>. The technique has also been used for the delivery of DNA and double-stranded RNA constructs into nematodes by whole-animal electroporation. Here, we describe methods for the expression of a nematode-optimized NanoLuc </span>luciferase mRNA in the form of </span></span><em>in vitro</em> transcripts following whole-animal electroporation of <span><em>Heterodera glycines</em></span>, <span><em>Meloidogyne incognita</em></span>, and <em>C. elegans</em>. The ability to transiently express single-stranded RNA constructs in economically important PPN provides a rapid means to evaluate nematode and/or foreign genes for their biological significance and potential role in nematode management.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"250 ","pages":"Article 111489"},"PeriodicalIF":1.5,"publicationDate":"2022-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83851200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of nanoparticles with 5-fluorouracil and chloroquine on Acanthamoeba castellanii activity","authors":"Balsam Qubais Saeed ,&nbsp;Mutasem Rawas Qalaji ,&nbsp;Noor Akbar ,&nbsp;Ruqaiyyah Siddiqui ,&nbsp;Cagliani Roberta ,&nbsp;Shaista Manzoor ,&nbsp;Jibran Sualeh Muhammad ,&nbsp;Ahmed Omar Adrees ,&nbsp;Rula Al-Shahrabi ,&nbsp;Naveed Ahmed Khan","doi":"10.1016/j.molbiopara.2022.111492","DOIUrl":"10.1016/j.molbiopara.2022.111492","url":null,"abstract":"<div><p><span><em>Acanthamoeba</em></span> is opportunistic pathogens that cause vision-threatening <em>Acanthamoeba</em><span><span> keratitis (AK). Previous studies proposed the use of </span>chloroquine (CQ) and 5-fluorouracil (5FU) as anti-</span><em>Acanthamoeba</em> agents. The objective of this study was to determine the benefit of using 5FU and CQ nanoparticles (NP) formulations against <em>A. castellanii</em><span> that belonging to the T4 genotype and evaluate their anti-Acanthamoebic characteristic. Triplicate batches of 5FU nanoparticles (5FU-NP) were synthesized by using a modified nanoprecipitation method, while CQ nanoparticles (CQ-NP) synthesized using a modified double emulsion method. The synthesized nanoparticles were subjected to biological assays to investigate their amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects against </span><em>A. castellanii</em><span><span>, as well as cell cytotoxicity. Cytotoxicity assays were performed using human </span>keratinocyte<span><span> cells (HaCaT) to determine the effect of CQ and 5FU nanoformulations on host cells. 5FU-NP with a concentration of 60 µM showed significant inhibition to amoeba binding into human cell lines and remarkable prevention mainly during the encystation stage. Moreover, 5FU-NP resulted in less cytotoxicity and </span>pathogenicity when compared with the free 5FU. On the other hand, CQ and CQ-NP, at the same concentration, showed poor inhibition to amoeba binding into human cells and insignificant prevention to encystation stage. Moderate human cells damage was resulted following their treatment with CQ and CQ-NP. In conclusion, 5FU may have the potential as an antiamoebic agent against </span></span><em>Acanthamoeba</em> spp. preferably as a nanoformulation to enhance its activity and reduce its cytoxicity.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"250 ","pages":"Article 111492"},"PeriodicalIF":1.5,"publicationDate":"2022-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74473016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A single amino acid substitution alters activity and specificity in Plasmodium falciparum aspartyl & asparaginyl-tRNA synthetases","authors":"Vivek Kumar Sharma ,&nbsp;Swati Gupta ,&nbsp;Jyoti Chhibber-Goel ,&nbsp;Manickam Yogavel ,&nbsp;Amit Sharma","doi":"10.1016/j.molbiopara.2022.111488","DOIUrl":"10.1016/j.molbiopara.2022.111488","url":null,"abstract":"<div><p><span><span><span>The specificity of each aminoacyl-tRNA synthetase (aaRS) for its cognate amino acid ensures correct tRNA </span>esterification and allows fidelity in </span>protein synthesis. The aaRSs discriminate based on the chemical properties of their amino acid substrates and structural features of the binding pockets. In this study, we characterized aspartyl-(DRS) and asparaginyl-tRNA synthetase (NRS) from </span><span><em>Plasmodium falciparum</em></span> to determine the basis of their specificity towards <span>L</span>-asp and <span>L</span>-asn respectively. The negatively charged <span>L</span>-asp and its analogue <span>L</span>-asn differ only in their side-chain groups i.e., -OH and -NH₂<span>. Further, the amino acid binding sites are highly conserved within these two enzymes. Analysis of the substrate (</span><span>L</span>-asp/<span>L</span>-asn) binding sites across species revealed two highly conserved residues in <em>Pf</em><span>DRS (D408 and K372) and </span><em>Pf</em>NRS (E395 and L360) that are involved in recognition of the O^δ2/N^δ2 of <span>L</span>-asp/<span>L</span>-asn respectively. These residues were mutated and swapped between the D408→E in <em>Pf</em>DRS and the corresponding E395→D in <em>Pf</em>NRS. A similar approach was employed for residue number K372→L in <em>Pf</em>DRS and L360→K in <em>Pf</em>NRS. The mutated <em>Pf</em>DRS^D408E<span> retained its enzymatic activity<span> during step 1 of aminoacylation reaction towards </span></span><span>L</span>-asp and <span>L</span>-asn and esterified tRNA^Asp with <span>L</span>-asp like wild type enzyme, while the <em>Pf</em>DRS^K372L was rendered enzymatically inactive. The correspondingly mutated <em>Pf</em>NRS^E395D was enzymatically inactive. The mutated <em>Pf</em>NRS^L360K had an altered specificity and esterified tRNA^Asn with non-cognate amino acid <span>L</span>-asp and not <span>L</span>-asn. These data suggest that the residue K372 is crucial for the enzymatic activity of <em>Pf</em>DRS while the residue L360 in <em>Pf</em>NRS imparts specificity towards <span>L</span>-asn.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":"250 ","pages":"Article 111488"},"PeriodicalIF":1.5,"publicationDate":"2022-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86479495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}