scholarly journals Generation of a CRISPR/Cas9-Based Vector Specific for Gene Manipulation in Leishmania major

2019 ◽  
Author(s):  
Ghodratollah SALEHI SANGANI ◽  
Vahid JAJARMI ◽  
Ali KHAMESIPOUR ◽  
Mahmoud MAHMOUDI ◽  
Abdolmajid FATA ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Leishmania Major ◽  
Gene Knockout ◽  
Ease Of Use ◽  
Selection Marker ◽  
Gene Manipulation ◽  
Resistance Proteins ◽  
U6 Promoter ◽  
Overlap Extension ◽  
Leishmania Parasites

Background: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. Results: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

scholarly journals RamA, a transcriptional regulator conferring florfenicol resistance in Leclercia adecarboxylata R25

2020 ◽  
Vol 65 (6) ◽  
pp. 1051-1060
Author(s):  
Cong Cheng ◽  
Yuanyuan Ying ◽  
Danying Zhou ◽  
Licheng Zhu ◽  
Junwan Lu ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Efflux Pump ◽  
Gene Knockout ◽  
Regulatory Gene ◽  
Resistance Mechanism ◽  
Agricultural Practice ◽  
The Novel ◽  
Inappropriate Use ◽  
Rnd Efflux Pump ◽  
High Level

AbstractDue to the inappropriate use of florfenicol in agricultural practice, florfenicol resistance has become increasingly serious. In this work, we studied the novel florfenicol resistance mechanism of an animal-derived Leclercia adecarboxylata strain R25 with high-level florfenicol resistance. A random genomic DNA library was constructed to screen the novel florfenicol resistance gene. Gene cloning, gene knockout, and complementation combined with the minimum inhibitory concentration (MIC) detection were conducted to determine the function of the resistance-related gene. Sequencing and bioinformatics methods were applied to analyze the structure of the resistance gene-related sequences. Finally, we obtained a regulatory gene of an RND (resistance-nodulation-cell division) system, ramA, that confers resistance to florfenicol and other antibiotics. The ramA-deleted variant (LA-R25ΔramA) decreased the level of resistance against florfenicol and several other antibiotics, while a ramA-complemented strain (pUCP24-prom-ramA/LA-R25ΔramA) restored the drug resistance. The whole-genome sequencing revealed that there were five RND efflux pump genes (mdtABC, acrAB, acrD, acrEF, and acrAB-like) encoded over the chromosome, and ramA located upstream of the acrAB-like genes. The results of this work suggest that ramA confers resistance to florfenicol and other structurally unrelated antibiotics, presumably by regulating the RND efflux pump genes in L. adecarboxylata R25.

LB Broth-lyophilized Rabbit Anti-sheep Cell Haemolysin as a Simple Culture Medium for Cultivation of Leishmania Major Promastigotes

2019 ◽  
Author(s):  
Vahid Nasiri ◽  
Farnoosh Jameie ◽  
Habibollah Paykari
Keyword(s):  
Blood Cell ◽  
Culture Medium ◽  
Leishmania Major ◽  
Low Cost ◽  
Protozoan Parasites ◽  
Causative Agents ◽  
Genus Leishmania ◽  
Leishmania Parasites ◽  
Growth Ability

Background and Aims: The protozoan parasites of the genus Leishmania are the causative agents of various clinical diseases. Different methods of cultivation of Leishmania parasites are available. In the present study, the efficacy of the LB broth with rabbit lyophilized anti-sheep red blood cell haemolysin was evaluated in the cultivation of promastigotes of Leishmania major. Materials and Methods: Conventional LB broth medium was prepared and autoclaved for 15 min at 121°C and then lyophilized rabbit anti-sheep cell haemolysin was added at 1-10% final concentrations. The efficacy of the medium was evaluated by assessing the growth ability and replication patterns of the promastigotes of Leishmania major. Results: Medium with 1-10% lyophilized rabbit haemolysin supported the growth of the parasites and can be used for cultivation of Leishmania parasites with acceptable In vivo infectivity for research purpose. Conclusions: The ability of the parasites to survive and proliferate in the presence of lyophilized rabbit haemolysin indicates that this material is a good nutritional source. This study opens a new way to make low-cost medium that can be used in cultivation of Leishmania parasites

scholarly journals Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

2011 ◽  
Vol 78 (2) ◽  
pp. 568-574 ◽  
Author(s):  
Tao Zheng ◽  
Qihong Huang ◽  
Changyi Zhang ◽  
Jinfeng Ni ◽  
Qunxin She ◽  
...  
Keyword(s):  
Selectable Marker ◽  
Gene Knockout ◽  
Plasmid Vector ◽  
Selection Marker ◽  
Content Type ◽  
Sulfolobus Islandicus ◽  
Gene Coding ◽  
High Level ◽  
Coa Reductase

ABSTRACTWe report here a novel selectable marker for the hyperthermophilic crenarchaeonSulfolobus islandicus. The marker cassette is composed of thesac7dpromoter and thehmggene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Psac7d-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting thepyrEFgene of the expression vector pSeSD for Psac7d-hmgwith which theSulfolobusexpression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization ofSulfolobustransformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His6-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEFandhmg) was subsequently exploited for genetic analysis. AherAmerodiploid strain ofS. islandicuswas constructed usingpyrEFmarker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from theherAmerodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability ofS. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.

scholarly journals Molecular Detection of Leishmania major in Hemiechinus auritus, A Potential Reservoir of Zoonotic Cutaneous Leishmaniasis in Damghan, Iran

2019 ◽  
Author(s):  
Behrad Pourmohammadi ◽  
Sadegh Mohammadi-Azni
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Leishmania Major ◽  
Natural Infection ◽  
Leishmania Infection ◽  
Northern Iran ◽  
Zoonotic Cutaneous Leishmaniasis ◽  
Potential Reservoir ◽  
Leishmania Spp ◽  
First Time ◽  
Leishmania Parasites

Background: Zoonotic cutaneous leishmaniasis caused by Leishmania major is endemic in 17 of 31 Iranian provinces. Various species of rodents have been introduced as the main reservoirs of the disease. This study was conducted to de­termine the natural infection of hedgehogs with Leishmania spp. in an endemic area of the disease, northern Iran. Methods: Fifteen long-eared hedgehogs were captured alive during 18 months study period, from Apr 2015 to Sep 2016, in Damghan City, Semnan Province, Iran. The animals were identified using apparent characteristics and to de­termine the Leishmania infection, impression smears were prepared from their ear lobes, hind feet, livers, and spleens. Micro­scopic examination and semi-nested PCR were applied to determine the infection and to identify the parasites species respectively. Results: All examined animals were identified as Hemiechinus auritus (Family: Erinaceidae). In microscopic examina­tion, 8 (53.3%) samples were shown to be infected with Leishmania parasites. The higher and lower rate of the infection was observed in the ears as well as the feet and in the liver specimens, 53.3%, and 33.3% respectively. Forty percent (6/ 15) of the samples were molecularly positive and all were identified as L. major parasites. All the examined animals in au­tumn and 50% of them in summer were shown to be infected with Leishmania parasites. Conclusion: This study demonstrated the natural infection of H. auritus with L. major for the first time in Damghan City and introduced these mammals as new potential reservoirs of ZCL in the study area.

scholarly journals A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Prem P. Kandel ◽  
Hongyu Chen ◽  
Leonardo De La Fuente
Keyword(s):  
Natural Transformation ◽  
Gene Knockout ◽  
Xylella Fastidiosa ◽  
Twitching Motility ◽  
Wild Type ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Type Iv ◽  
Overlap Extension ◽  
Pilin Subunit

ABSTRACT Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2. IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

scholarly journals Innate Resistance to Leishmania amazonensis Infection in Rat Is Dependent on NOS2

2021 ◽  
Vol 12 ◽  
Author(s):  
Yun-Fu Chen ◽  
Si-Fei Yu ◽  
Chang-You Wu ◽  
Na Wu ◽  
Jia Shen ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Gene Knockout ◽  
Inbred Mouse ◽  
Clinical Manifestations ◽  
Parasite Load ◽  
Leishmania Amazonensis ◽  
Leishmania Infection ◽  
Mouse Strains ◽  
Innate Resistance ◽  
Key Factor

Leishmania infection causes diverse clinical manifestations in humans. The disease outcome is complicated by the combination of many host and parasite factors. Inbred mouse strains vary in resistance to Leishmania major but are highly susceptible to Leishmania amazonensis infection. However, rats are highly resistant to L. amazonensis infection due to unknown mechanisms. We use the inducible nitric oxide synthase (Nos2) gene knockout rat model (Nos2−/− rat) to investigate the role of NOS2 against leishmania infection in rats. Our results demonstrated that diversion toward the NOS2 pathway is the key factor explaining the resistance of rats against L. amazonensis infection. Rats deficient in NOS2 are susceptible to L. amazonensis infection even though their immune response to infection is still strong. Moreover, adoptive transfer of NOS2 competent macrophages into Nos2−/− rats significantly reduced disease development and parasite load. Thus, we conclude that the distinct L-arginine metabolism, observed in rat macrophages, is the basis of the strong innate resistance to Leishmania. These data highlight that macrophages from different hosts possess distinctive properties and produce different outcomes in innate immunity to Leishmania infections.

scholarly journals Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena

Gene ◽  
2014 ◽  
Vol 534 (2) ◽  
pp. 249-255 ◽  
Author(s):  
Masaaki Iwamoto ◽  
Chie Mori ◽  
Yasushi Hiraoka ◽  
Tokuko Haraguchi
Keyword(s):  
Resistance Gene ◽  
Selection Marker ◽  
Effective Selection ◽  
Puromycin Resistance Gene

scholarly journals The Isolation and Mapping of Disease Resistance Gene Analogs in Maize

1998 ◽  
Vol 11 (10) ◽  
pp. 968-978 ◽  
Author(s):  
N. C. Collins ◽  
C. A. Webb ◽  
S. Seah ◽  
J. G. Ellis ◽  
S. H. Hulbert ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Plant Disease Resistance ◽  
Amino Acid Identity ◽  
Disease Resistance Gene ◽  
Resistance Gene Analogs ◽  
Resistance Proteins ◽  
Genomic Regions ◽  
Leucine Rich Repeats

Many of the plant disease resistance genes that have been isolated encode proteins with a putative nucleotide binding site and leucine-rich repeats (NBS-LRR resistance genes). Oligonucleotide primers based on conserved motifs in and around the NBS of known NBS-LRR resistance proteins were used to amplify sequences from maize genomic DNA by polymerase chain reaction (PCR). Eleven classes of non-cross-hybridizing sequences were obtained that had predicted products with high levels of amino acid identity to NBS-LRR resistance proteins. These maize resistance gene analogs (RGAs) and one RGA clone obtained previously from wheat were used as probes to map 20 restriction fragment length polymorphism (RFLP) loci in maize. Some RFLPs were shown to map to genomic regions containing virus and fungus resistance genes. Perfect co-segregation was observed between RGA loci and the rust resistance loci rp1 and rp3. The RGA probe associated with rp1 also detected deletion events in several rp1 mutants. These data strongly suggest that some of the RGA clones may hybridize to resistance genes.

scholarly journals A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

2017 ◽  
Vol 4 (5) ◽  
pp. 170095 ◽  
Author(s):  
Tom Beneke ◽  
Ross Madden ◽  
Laura Makin ◽  
Jessica Valli ◽  
Jack Sunter ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Leishmania Major ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Gene Knockout ◽  
Guide Rna ◽  
Functional Studies

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

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