A Therapeutic Approach Against Leishmania donovani by Predicting RNAi Molecules Against the Surface Protein, gp63

Author(s): Farhana T. Chowdhury , Mohammad U.S. Shohan , Tasmia Islam , Taisha T. Mimu , Parag Palit* .

Journal Name: Current Bioinformatics

Volume 14 , Issue 6 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Background: Leishmaniasis is a disease caused by the Leishmania sp. and can be classified into two major types: cutaneous and visceral leismaniasis. Visceral leishmaniasis is the deadlier type and is mediated by Leishmania donovani and involves the establishment of persistent infection and causes damage to the liver, spleen and bone marrow. With no vaccine yet available against leishmaniasis and the current therapeutic drugs of leishmaniasis being toxic and expensive; an alternative treatment is necessary.

Objective: Surface glycocalyx protein gp63, plays a major role in the virulence and resulting pathogenicity associated with the disease. Henceforth, silencing the gp63 mRNA through the RNA interference system was the aim of this study.

Methods: In this study two competent siRNAs and three miRNAs have been designed against gp63 for five different strains of L. donovani by using various computational methods. Target specific siRNAs were designed using siDirect 2.0 and to design possible miRNA, another tool named IDT (IntegratedDNA Technology). Screening for off-target similarity was done by BLAST and the GC contents and the secondary structures of the designed RNAs were determined. RNA-RNA interaction was calculated by RNAcofold and IntraRNA, followed by the determination of heat capacity and the concentration of duplex by DNAmelt web server.

Results: The selected RNAi molecules; two siRNA and three miRNA had no off-target in human genome and the ones with lower GC content were selected for efficient RNAi function. The selected ones showed proper thermodynamic characteristics to suppress the expression of the pathogenic gene of gp63.

Keywords: Leishmaniasis, RNAi, siRNA, miRNA, gene silencing, computational method.

Herwaldt BL. Leishmaniasis. Lancet 1999; 354(9185): 1191-9.
Alvar J, Vélez ID, Bern C, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 2012; 7(5)e35671
Torres-Guerrero E, Quintanilla-Cedillo MR, Ruiz-Esmenjaud J, Arenas R. Leishmaniasis: A review. F1000 Res 2017; 6: 750.
Steverding D. The history of leishmaniasis. Parasit Vectors 2017; 10(1): 82.
Desjeux P. Leishmaniasis. Public health aspects and control. Clin Dermatol 1996; 14(5): 417-23.
Liévin-Le Moal V, Loiseau PM. Leishmania hijacking of the macrophage intracellular compartments. FEBS J 2016; 283(4): 598-607.
Handler MZ, Patel PA, Kapila R, Al-Qubati Y, Schwartz RA. Cutaneous and mucocutaneous leishmaniasis: Differential diagnosis, diagnosis, histopathology, and management. J Am Acad Dermatol 2015; 73(6): 911-926, 927-928.
de Freitas EO, Leoratti FM, Freire-de-Lima CG, et al. The contribution of immune evasive mechanisms to parasite persistence in visceral leishmaniasis. Front Immunol 2016; 7: 153.
Monzote L. Current treatment of leishmaniasis: A review. The Open Antimicrobial Agents J 2009; 1(1): 9-19.
Murray HW, Berman JD, Davies CR, Saravia NG. Advances in leishmaniasis. Lancet 2005; 366(9496): 1561-77.
Pandey BD, Pun SB, Kaneko O, Pandey K, Hirayama K. Case report: Expansion of visceral leishmaniasis to the western hilly part of Nepal. Am J Trop Med Hyg 2011; 84(1): 107-8.
Copeland NK, Aronson NE. Leishmaniasis: Treatment updates and clinical practice guidelines review. Curr Opin Infect Dis 2015; 28(5): 426-37.
Brittingham A, Morrison CJ, McMaster WR, McGwire BS, Chang K-P, Mosser DM. Role of the Leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis. J Immunol 1995; 155(6): 3102-11.
Joshi PB, Kelly BL, Kamhawi S, Sacks DL, McMaster WR. Targeted gene deletion in Leishmania major identifies leishmanolysin (GP63) as a virulence factor. Mol Biochem Parasitol 2002; 120(1): 33-40.
Lieke T, Nylén S, Eidsmo L, et al. Leishmania surface protein gp63 binds directly to human natural killer cells and inhibits proliferation. Clin Exp Immunol 2008; 153(2): 221-30.
Isnard A, Shio MT, Olivier M. Impact of Leishmania metalloprotease GP63 on macrophage signaling. Front Cell Infect Microbiol 2012; 2: 72.
Tosi MF. Innate immune responses to infection. J Allergy Clin Immunol 2005; 116(2): 241-9.
Russell DG. The macrophage-attachment glycoprotein gp63 is the predominant C3-acceptor site on Leishmania mexicana promastigotes. Eur J Biochem 1987; 164(1): 213-21.
Chaudhuri G, Chaudhuri M, Pan A, Chang KP. Surface acid proteinase (gp63) of Leishmania mexicana. A metalloenzyme capable of protecting liposome-encapsulated proteins from phagolysosomal degradation by macrophages. J Biol Chem 1989; 264(13): 7483-9.
Kulkarni MM, McMaster WR, Kamysz E, Kamysz W, Engman DM, McGwire BS. The major surface-metalloprotease of the parasitic protozoan, Leishmania, protects against antimicrobial peptide-induced apoptotic killing. Mol Microbiol 2006; 62(5): 1484-97.
Hallé M, Gomez MA, Stuible M, et al. The Leishmania surface protease GP63 cleaves multiple intracellular proteins and actively participates in p38 mitogen-activated protein kinase inactivation. J Biol Chem 2009; 284(11): 6893-908.
Contreras I, Gómez MA, Nguyen O, Shio MT, McMaster RW, Olivier M. Leishmania-induced inactivation of the macrophage transcription factor AP-1 is mediated by the parasite metalloprotease GP63. PLoS Pathog 2010; 6(10)e1001148
Olivier M, Gregory DJ, Forget G. Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view. Clin Microbiol Rev 2005; 18(2): 293-305.
Thiakaki M, Kolli B, Chang K-P, Soteriadou K. Down-regulation of gp63 level in Leishmania amazonensis promastigotes reduces their infectivity in BALB/c mice. Microbes Infect 2006; 8(6): 1455-63.
Akhoundi M, Downing T, Votýpka J, et al. Leishmania infections: Molecular targets and diagnosis. Mol Aspects Med 2017; 57: 1-29.
Chen D-Q, Kolli BK, Yadava N, et al. Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 level in promastigotes and their infection of macrophages in vitro. Infect Immun 2000; 68(1): 80-6.
Hassani K, Shio MT, Martel C, Faubert D, Olivier M. Absence of metalloprotease GP63 alters the protein content of Leishmania exosomes. PLoS One 2014; 9(4)e95007
Aagaard L, Rossi JJ. RNAi therapeutics: Principles, prospects and challenges. Adv Drug Deliv Rev 2007; 59(2-3): 75-86.
Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999; 286(5441): 950-2.
Lam JK, Chow MY, Zhang Y, Leung SW. siRNA versus miRNA as therapeutics for gene silencing. Mol Ther Nucleic Acids 2015; 4e252
Wilson RC, Doudna JA. Molecular mechanisms of RNA interference. Annu Rev Biophys 2013; 42: 217-39.
Gavrilov K, Saltzman WM. Therapeutic siRNA: Principles, challenges, and strategies. Yale J Biol Med 2012; 85(2): 187-200.
Dyawanapelly S, Ghodke SB, Vishwanathan R, Dandekar P, Jain R. RNA interference-based therapeutics: molecular platforms for infectious diseases. J Biomed Nanotechnol 2014; 10(9): 1998-2037.
Wu J, Liu B, Wu H, et al. A gold nanoparticle platform for the delivery of functional TGF-β1 siRNA into cancer cells. J Biomed Nanotechnol 2016; 12(4): 800-10.
Schwarz DS, Hutvágner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003; 115(2): 199-208.
Heale BS, Soifer HS, Bowers C, Rossi JJ. siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res 2005; 33(3)e30
Patzel V, Rutz S, Dietrich I, Köberle C, Scheffold A, Kaufmann SH. Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency. Nat Biotechnol 2005; 23(11): 1440-4.
Wheeler DL, Barrett T, Benson DA, et al. Database resources of the national center for biotechnology information. Nucleic Acids Res 2006; 35(Suppl. 1): D5-D12.
Sievers F, Wilm A, Dineen D, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 2011; 7(1): 539.
Naito Y, Yoshimura J, Morishita S, Ui-Tei K. siDirect 2.0: Updated software for designing functional siRNA with reduced seed-dependent off-target effect. BMC Bioinformatics 2009; 10(1): 392.
Ui-Tei K, Naito Y, Takahashi F, et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 2004; 32(3): 936-48.
Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol 2004; 22(3): 326-30.
Amarzguioui M, Prydz H. An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun 2004; 316(4): 1050-8.
Ui-Tei K, Naito Y, Nishi K, Juni A, Saigo K. Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect. Nucleic Acids Res 2008; 36(22): 7100-9.
Ahmed F, Ansari HR, Raghava GP. Prediction of guide strand of microRNAs from its sequence and secondary structure. BMC Bioinformatics 2009; 10(1): 105.
Karlin S, Altschul SF. Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci USA 1990; 87(6): 2264-8.
Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. NCBI BLAST: A better web interface. Nucleic Acids Res 2008; 36(Suppl. 2): W5-9.
Kibbe WA. OligoCalc: An online oligonucleotide properties calculator. Nucleic Acids Res 2007; 35(Suppl. 2): W43-6.
Gruber AR, Lorenz R, Bernhart SH, Neuböck R, Hofacker IL. The vienna RNA websuite. Nucleic Acids Res 2008; 36(Suppl. 2): W70-4.
Mann M, Wright PR, Backofen R. IntaRNA 2.0: Enhanced and customizable prediction of RNA-RNA interactions. Nucleic Acids Res 2017; 45(W1)W435-9
Markham NR, Zuker M. DINAMelt web server for nucleic acid melting prediction Nucleic Acids Res 2005 33(Web Server issue): W577-81.
Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003; 31(13): 3406-15.
Chitsaz H, Salari R, Sahinalp SC, Backofen R. A partition function algorithm for interacting nucleic acid strands. Bioinformatics 2009; 25(12): i365-73.
Zheng C, Zheng M, Gong P, et al. Polypeptide cationic micelles mediated co-delivery of docetaxel and siRNA for synergistic tumor therapy. Biomaterials 2013; 34(13): 3431-8.
Mathews DH. Predicting a set of minimal free energy RNA secondary structures common to two sequences. Bioinformatics 2005; 21(10): 2246-53.
Mückstein U, Tafer H, Hackermüller J, Bernhart SH, Stadler PF, Hofacker IL. Thermodynamics of RNA-RNA binding. Bioinformatics 2006; 22(10): 1177-82.
Bernhart SH, Tafer H, Mückstein U, Flamm C, Stadler PF, Hofacker IL. Partition function and base pairing probabilities of RNA heterodimers. Algorithms Mol Biol 2006; 1(1): 3.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [541 - 550]
Pages: 10
DOI: 10.2174/1574893613666180828095737
Price: $58

Article Metrics

PDF: 25