Generation of RNAi plasmid corresponding to the m1l1 gene (ID 140500) belonging to myosin class i in the fungus Mucor circinelloides

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To investigate biological roles of Myosin class I in the morphology of M. circinelloides and identify good candidates for further analyses of Mucor pathogenesis, we are going to inhibit their expression using RNAi technique. We named the two myosin class I like genes as M1L1 (ID 140500) and M1L2 (ID 116620). In the present study, we report the results of generation of RNAi plasmid to inhibit the expression of M1L1 gene.

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Nội dung Text: Generation of RNAi plasmid corresponding to the m1l1 gene (ID 140500) belonging to myosin class i in the fungus Mucor circinelloides

HNUE JOURNAL OF SCIENCE Natural Sciences, 2020, Volume 65, Issue 4A, pp. 27-32 This paper is available online at http://stdb.hnue.edu.vn GENERATION OF RNAi PLASMID CORRESPONDING TO THE m1l1 GENE (ID 140500) BELONGING TO MYOSIN CLASS I IN THE FUNGUS Mucor circinelloides Le Ngoc Mai1, Le Phuong Thao1, Pham Thi Thu Trang 2 and Trieu Anh Trung1 1 Faculty of Biology, Hanoi National University of Education 2 Nguyen Trai Gifted High School, Hai Duong province Abstract. Mucormycosis has emerged as a second most common filamentous fungal infection in human caused by species belonging to order Mucorales, especially in immunocompromised patients, with a high mortality ratio. Our understanding of its virulence determinants is limited, leading to the lacking of efficient therapies. Dimorphism is one of the phenotypic characteristics that can be used as a marker for virulence screening. Mucor circinelloides, a dimorphic fungus, has become an attractive model for studies on various studies, including mucormycosis. Our previous study identified Mcmyo5 protein belonging to Myosin class V which plays an important role in Mucor pathogenesis. We have found three protein families with high similarities of amino acid sequences compared to Mcmyo5, including Myosin class I, II and V, in the Mucor genome. To investigate the possible roles of those genes in Mucor pathogenesis using RNAi techniques, we generated the RNAi plasmid which is corresponding to endogenous gene, m1l1 (ID 140500). In the present study, we show the results of isolation and cloning a fragment of the target gene m1l1 into the vector pMAT1812 to obtain RNAi plasmid. This recombinant RNAi plasmid was checked by using the digestion of restriction enzyme and DNA sequencing techniques. Keywords: mucormycosis, myosin, RNAi, Mucor circinelloides. 1. Introduction Mucormycosis (zygomycosis) is the rare but lethal fungal infection, especially in immunocompromised patients, such as those with diabetes, organ-transplanted, AIDS, hematologic malignancies, or trauma [1-3]. Mucormycosis caused by species belonging to order Mucorales [4]. The most common pathogenic species found in the families Rizopus, Mucor, Lichtheimia, Apophysomyces, Rhizomucor and Cunninghamella. Mucormycosis is more difficult to treat than more common mycoses due to restriction of efficient therapies by lacking of knowledge about the disease and the organisms that cause the infection [5]. Mucor circinelloides is an emerging opportunistic human pathogen that causes mucormycosis. It has become an outstanding model species for various studies, including RNAi mechanism [6], carotenogenesis [7], lipid production [8], and mucormycosis [9] due to its available for genetic transformation. We have applied RNAi techniques to investigate gene functions in this fungus, based on well-studied RNA silencing mechanisms. In our knowledge, there is no study on mucormycosis and RNAi mechanism in M. circinelloides in Vietnam. Received March 27, 2020. Revised May 4, 2020. Accepted May 11, 2020 Contact Trieu Anh Trung, e-mail address: trungta@hnue.edu.vn 27
Le Ngoc Mai, Le Phuong Thao, Pham Thi Thu Trang and Trieu Anh Trung Dimorphism is a process in which they can grow like hyphae or like yeast, depending on the environmental conditions. This yeast-hyphae transition contributes to fungal virulence. M. circinelloides is also a dimorphic fungus that grows as hyphal mycelium in aerobic condition and as yeast in anaerobic/high CO2 condition. The transition between those two forms in this fungus was demonstrated to be involved in virulence, as the yeast phase is not pathogenic [9]. In addition, the size of sporangiospores is also related to virulence in M. circinelloides, being pathogenic only the strains producing large multinucleated spores [10]. These results suggested that dimorphism and other phenotypic characteristics could be used as markers for a preliminary evaluation of virulence in this fungus. We have identified mcmyo5 gene (ID 51513), encoding Mcmyo5 protein belonging to Myosin class V, involved in morphogenesis and pathogenesis of fungus M. circinelloides [11]. Myosin class V is a widely conserved protein family that belongs to the myosin superfamily. Myosin superfamily contains actin-based motor proteins that play diverse functions in cells, including cytokinesis, cell adhesion, endocytosis, exocytosis, movement of mRNA, movement of pigment granules and cell motility [12]. As occurs in most systems, M. circinelloides seems to have several myosin genes, since we have found several proteins highly similar to Mcmyo5 identified in the Mucor genome, including Myosin class I, II and V proteins. Their high similarities in amino acid sequences suggested these genes also involved in cell morphology and dimorphism of Mucor. To investigate biological roles of Myosin class I in the morphology of M. circinelloides and identify good candidates for further analyses of Mucor pathogenesis, we are going to inhibit their expression using RNAi technique. We named the two myosin class I like genes as M1L1 (ID 140500) and M1L2 (ID 116620). In the present study, we report the results of generation of RNAi plasmid to inhibit the expression of M1L1 gene. 2. Content 2.1. Materials and methods 2.1.1. Materials The M. circinelloides R7B used as wild type strain and plasmid pMAT1812 were kindly provided by the Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Spain. The E. coli DH5α strain was used to generate the competent cells for the transformation experiments. Plasmid pMAT1812 (9,5kb) containing 2 opposite promoters flanking the MCS region could generates dsRNA molecules from the insert DNA fragment. There is a fragment of carB gene involved in carotenogenesis in Mucor fungi which is located between 2 promoters. This fragment was used as reporter gene in the plasmid pMAT1812 (Figure 1). Figure 1. Structure, size, location of primers and restriction enzymes of the vector pMAT1812 (A) and target gene m1l1 (B) 28
Generation of RNAi plasmid corresponding to the m1l1 gene (ID 140500) belonging to myosin class… 2.1.2. Methods Isolation of genomic DNA and plasmids Genomic DNA from the R7B strain was extracted using the the i-genomic BYF DNA Extraction Mini Kit, Code: 17361 (Intron, Korea) following provider’s recommendation. Plasmids from E. coli strains were isolated using the DNA-spin TM Plasmid DNA Purification Kit (Intron; Cat.No 17096) following provider’s instruction. PCR and electrophoresis To amplify a 2kb fragment of the M1L1 gene, we performed PCR reactions using 2x PCR Master Mix Solution kit - Code: REF 25028 (Intron, Korea) with a primer pairs M1F-SL1 and M1R-SL1 following running program of 94 oC (5 minutes); 35 cycles of (94oC - 20 seconds, 58oC - 30 seconds, 72oC - 2 minutes 15 seconds); end: 72oC (10 minutes). The sequences of primers, include: M1F-SL1: 5’-TCGGTCATGCCCTCCACATTGTA-3’, and M1R-SL1: 5’- GCAGACTGGAAAGAAGGCGTCA-3’ All DNA products were purified using the MEGAquick-spin TM plus Fragment DNA Purification Kit (Intron; Cat. No 17289) following provider’s recommendation. The DNA fragments in this study were observed under UV light by using 1% agarose electrophoresis. Cloning experiments In this study, a 1,2kb XhoI restriction fragment obtained from 2kb PCR products was ligated into linear vector pMAT1812 digested by XhoI using T4 DNA ligase (Thermo Sciencetific) following protocol: ligation mixture including 50ng linear vector, 25ng insert DNA, 1µl 10X T4 DNA ligase buffer, 1U T4 DNA ligase and H2O upto 10µl total volume. The mixture was incubated 15 minutes at room temperature, then used 5µl of the mixture for transformation of 50µl of competent cells. The competent cells of E. coli DH5α strain were prepared using cold CaCl 2 and MgCl2 [13]. Plasmids were transformed into competent cells using heat shock method at 42oC in 45 seconds [14]. The transformed cells were grown on LB medium suplemented with ampicillin (100µg/ml). Selection of recombinant plasmids To identify the recombinant plasmids, ten E. coli colonies grown in LB supplemented with ampicillin were screened by colony PCR method using peuka1 (5’-CATGA AGTGTGAGACATTGCG-3’) and PU (5’-GTTGTAAAACGACGGCCAGT-3’) that located in the vector pMAT1812. The positive plasmids were extracted and checked by using restriction enzymes and sequencing (1st BASE DNA Sequencing Division, Malaysia). 2.2. Results and discussion 2.2.1. DNA extraction and PCR results Genomic DNA was extracted from the wild type strain R7B using the i-genomic BYF DNA Extraction Mini Kit (Intron) which has compatible components and protocol for isolation genomic DNA from fungi. The results of extracted genomic DNA using this kit were good enough for PCR reactions. The obtained genomic DNA were checked using the NanoVue Plus UV-Vis Spectrophotometer (GE Healthcare), its concentrations were around 500 ng/µl, ratio of 260nm/280nm wavelength around 1.7-2.0. The specific PCR products also were checked on agarose gel and purified using the DNA Purification Kit (Intron). The PCR fragments also had high quality after purification. 29
Le Ngoc Mai, Le Phuong Thao, Pham Thi Thu Trang and Trieu Anh Trung Figure 2. PCR result of the 2kb fragment from the genomic DNA of R7B strain 2.2.2. Ligation and transformation The 1,2kb XhoI restriction fragment was ligated into the linear vector pMAT1812 with the ratio insert/vector ~ 4/1 using the T4 DNA ligase. The ligation mixture was transformed into the competent E. coli DH5α cells using the heat shock method. As the result, we obtained nearly 50 colonies grown in the LB plates supplemented by ampicillin. The ratio of successful ligation was around 20%, which was estimated by the ratio of positive colony PCR results (Figure 3). 2.2.3. Colony PCR Conlony PCR was performed to screen positive colonies which might contain expected plasmids. In this study, we used two primers peuka1 and PU which located at the flanking positions of the MCS regions. Therefore, the negative and positive results were 2kb and 3,2kb fragments, respectively (see Figures 1A and 4B). Results of colony PCR identified 2 positive samples, at the lanes 1 and 6 (Figure 3). Plasmids 1 and 6 were named as pAT151 and pAT152, repectively. Figure 3. Colony PCR. M: Marker Sizer TM-1000 DNA Marker (Intron, Cat.No 24074). Lanes 1 to 10: PCR results obtained from random slected colonies 2.2.4. Confirmation of RNAi plasmids using restriction enzymes Plasmids pAT151 and pAT152 (Figure 3) were isolated to check their structure. Both of those plasmids were checked by digestion using two different restriction enzymes, XhoI and PstI (Figure 4). 30
Generation of RNAi plasmid corresponding to the m1l1 gene (ID 140500) belonging to myosin class… A B Figure 4. (A) Checking plasmids by using restriction enzymes. M: Marker SizerTM -1000 DNA Marker (Intron, Cat.No 24074). Lanes 1 and 2: plasmids pAT151 and pAT152 were digested by XhoI, respectively. Lanes 3 and 4: plasmids pAT151 and pAT152 were digested by PstI, respectively. (B) Schematic representation of RNAi plasmid with the position of restriction enzymes and primers As expectation, recombinant plasmids generate 2 bands (9,5kb and 1,2kb) when digested by XhoI; 5 bands (4,5kb, 4,4kb, 0,9kb, 0,5kb and 0,4kb) when digested by PstI. The agarose electrophoresis results obtained in Figure 3 were confirmed that both plasmids pAT151 and pAT152 are expected recombinant plasmids. Both plasmids pAT151 and pAT152 digested by XhoI generated 2 bands, around 9,5kb and 1,2kb. When digested by PstI, bands 4,5kb and 4,4kb seemed to be so close that difficult to distinguish them. However, the 3 bands remaining showed the expected sizes (Figure 4). 2.2.5. Confirmation of RNAi plasmids by sequencing Figure 5. A part of alignment result between original genomic DNA sequence and the insert sequence obtained from sequencing of plasmid pAT151 with primer peuka1. 31
Le Ngoc Mai, Le Phuong Thao, Pham Thi Thu Trang and Trieu Anh Trung To check the sequencing of plasmid, we sequenced plasmid pAT151 using primer peuka1. This sequence and the original DNA fragment were compared using Clustal Omega (1.2.4) multiple sequence alignment (EBI, https://www.ebi.ac.uk). As the result, the insert fragment into plasmid pMAT1812 had the same sequence as the original DNA fragment obtained from M. circinelloides genomic DNA (Figure 5). 3. Conclusions Taken together, those results confirmed that we obtained the recombinant plasmids, pAT151 and pAT152, that successfully cloned 1,2kb fragment of the M1L1 gene into plasmid pMAT1812. Those plasmids were already to transform into protoplasts of the wild type M. circinelloides fungal strain to investigate the role of the corresponding gene. Acknowledgement. This research is funded by Ministry of Education and Training, Vietnam under grant number B2019-SPH-562-12. REFERENCES [1] K. A. Marr, R. A. Carter, F. Crippa, A. Wald, and L. Corey, 2002. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin Infect Dis, Vol. 34, No. 7, pp. 909-917. [2] M. M. Roden et al., 2005. Epidemiology and outcome of zygomycosis: a review of 929 reported cases. Clin Infect Dis, Vol. 41, No. 5, pp. 634-653. [3] R. Neblett Fanfair et al., 2012. Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri in 2011. N. Engl J. Med, Vol. 367, No. 23, pp. 2214-2225. [4] J. A. Ribes, C. L. Vanover-Sams, and D. J. Baker, 2000. Zygomycetes in human disease. Clinical Microbiology Reviews, Vol. 13, No. 2. pp. 236-301. [5] L. Lopez-Fernandez et al., 2018. Understanding Mucor circinelloides pathogenesis by comparative genomics and phenotypical studies. Virulence, Vol. 9, No. 1, pp. 707-720. [6] F. E. Nicolás, S. Torres-Martínez, and R. M. Ruiz-Vázquez, 2003. Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes. EMBO J., Vol. 22, No. 15, pp. 3983-3991. [7] S. Torres-Martínez, R. M. Ruiz-Vázquez, V. Garre, S. López-García, E. Navarro, and A. Vila, 2012. Molecular tools for carotenogenesis analysis in the zygomycete mucor circinelloides. Methods Mol. Biol. [8] G. S. S. Andrade, A. K. F. Carvalho, C. M. Romero, P. C. Oliveira, and H. F. De Castro, 2014. Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil. Bioprocess Biosyst. Eng., Vol. 37, pp. 2539-2548. [9] S. C. Lee, A. Li, S. Calo, and J. Heitman, 2013. Calcineurin Plays Key Roles in the Dimorphic Transition and Virulence of the Human Pathogenic Zygomycete Mucor circinelloides. PLoS Pathog., Vol. 9, No. 9. [10] C. H. Li et al., 2011. Sporangiospore size dimorphism is linked to virulence of Mucor circinelloides. PLoS Pathog., Vol. 7, No. 6. [11] T. A. Trieu et al., 2017. RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis. PLoS Pathog, Vol. 13, No. 1. [12] J. A. Hammer and J. R. Sellers, 2011. Walking to work: Roles for class v myosins as cargo transporters. Nature Reviews Molecular Cell Biology, Vol. 13(1), pp. 13-26. [13] D. A. Morrison, 1977. Transformation in Escherichia coli: cryogenic preservation of competent cells. J. Bacteriol., Vol. 132(1), pp. 349-351. [14] A. Froger and J. E. Hall, 2007. Transformation of Plasmid DNA into E. Coli using the heat shock method. J. Vis. Exp., (6) 253. 32