Tinospora crispa Ethanolic Extract Downregulates Protein Kinase Genes Expression and Activity during Toxoplasma gondii Infection: A Prospective Drug Target for Lytic Cycle Inhibition
DOI:
https://doi.org/10.48048/tis.2023.6538Keywords:
Tinospora crispa, Toxoplasma gondii, Vero cells, Protein kinase, Lytic cycle, Gene expressionAbstract
Infection with Toxoplasma gondii remains widespread among humans and animals as water, soil and food continue to serve as the major carriers of the sporulated oocyst. The infection is poorly controlled due to lack of a potent vaccine against the parasite, and the current medication presents severe side effects on the host, less efficacy on the parasite and is accompanied by the potential development of resistance by the parasite. The aim of this study was to evaluate the in vitro activities of ethanolic extract of Tinospora crispa (EETC) on protein kinases involved in the lytic cycle of T. gondii infection in Vero cell line. The EETC was obtained through the maceration of dried stem powder. Vero cells infected with the RH strain of T. gondii were used to evaluate the inhibitory effect of EETC against T. gondii calcium dependent protein kinase (CDPK) genes and microneme proteins (MIC) that are essential for host cell invasion and intracellular replication of the tachyzoite. Gene expression profiling of CDPK genes was determined through quantitative real-time PCR (qPCR) after 24 h of treatment. The expression of microneme protein was determined through western blot technique. The RT-qPCR revealed downregulation of most protein kinase (PK) genes after treatment with EETC. The expression of CDPK1, PKG, CDPK3, CDPK6, CDPK7 genes that participate in the lytic cycle of T. gondii infection was downregulated. The expression of the TgMIC1 and TgMIC2 proteins were observed to have decreased in both 4 and 24 h post-infection treatment models. This study showed that EETC contains promising drug candidates effective against T. gondii that can target the protein kinase genes involved in the lytic cycle of the parasite to prevent disease progression.
HIGHLIGHTS
- This study explored the possible mechanism of action of phytochemicals on Toxoplasma gondii parasite-host cell invasion and intracellular replication
- The study evaluated the effects of ethanolic extract of Tinospora crispa Miers on protein kinase genes that are involved in host cell invasion
- The target protein kinase genes were downregulated which prevents the secretion of microneme proteins
- Inhibition of microneme secretion prevents host cell invasion and intracellular replication by Toxoplasma gondi
GRAPHICAL ABSTRACT
Downloads
Metrics
References
S Lourido, J Shuman, C Zhang, KM Shokat, R Hui and LD Sibley. Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 2010; 465, 359-62.
J Morlon-Guyot, L Berry, CT Chen, MJ Gubbels, M Lebrun and W Daher. The Toxoplasma gondii calcium-dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival. Cell. Microbiol. 2014; 16, 95-114.
K Kato, T Sugi, H Takemae, R Takano, H Gong and A Ishiwa. Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog. Parasites Vectors 2016; 9, 405.
O Billker, S Lourido and LD Sibley. Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 2009; 5, 612-22.
JM McCoy, L Whitehead, GG Dooren and CJ Tonkin. TgCDPK3 regulates calcium-dependent egress of Toxoplasma gondii from host cells. PLoS Pathog. 2012; 8, e1003066
E Garrison, M Treeck, E Ehret, H Butz, T Garbuz and BP Oswald. A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma. PLoS Pathog. 2012; 8, e1003049
S Long, Q Wang and LD Sibley. Analysis of noncanonical calciumdependent protein kinases in Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9. Infect. Immun. 2016; 84, 1262-73.
AM Gurnett, PA Liberator, PM Dulski, SP Salowe, RGK Donald, JW Anderson, J Wiltsie, CA Diaz, G Harris, B Chang, SJ Darkin-Rattray, B Nare, T Crumley, PS Blum, AS Misura, T Tamas, MK Sardana, J Yuan, T Biftu and DM Schmatz. Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites: A novel chemotherapeutic target. J. Biol. Chem. 2002; 277, 15913-22.
IJ Blader, BI Coleman, CT Chen and MJ Gubbels. Lytic cycle of Toxoplasma gondii: 15 years later. Annu. Rev. Microbiol. 2015; 69, 463-85.
SA Abdullahi, NZ Unyah, N Nordin, R Basir, WM Nasir and AA Alapid. Therapeutic Targets on Toxoplasma gondii parasite in combatting Toxoplasmosis. Annu. Res. Rev. Biol. 2019; 32, 1-15.
HR Goulart, EA Kimura, VJ Peres, AS Couto, FAA Duarte and AM Katzin. Terpenes arrest parasite development and inhibit biosynthesis of isoprenoids in Plasmodium falciparum. Antimicrob. Agents Chemother. 2004; 48, 2502-9.
D Kerboeuf, M Riou and F Guegnard. Flavonoids and related compounds in parasitic disease control. Mini Rev. Med Chem. 2008; 8, 116-28.
B Guerra and OG Issinger. Natural compounds and derivatives as Ser/Thr protein kinase modulators and inhibitors. Pharmaceuticals 2019; 12, 4.
M Dormeyer, Y Adams, B Kramer, S Chakravorty, MT Tse, S Pegoraro, L Whittaker, M Lanzer and A Craig. Rational design of anticytoadherence inhibitors for Plasmodium falciparum based on the crystal structure of human intercellular adhesion molecule 1. Antimicrob. Agents Chemother. 2006; 50, 724-30.
JR Mead and N McNair. Antiparasitic activity of flavonoids and isoflavones against Cryptosporidium parvum and Encephalitozoon intestinalis. FEMS Microbiol. Lett. 2006; 259, 153-7.
RA Ferreira, ABD Oliveira, SA Gualberto, JMMD Corral, RT Fujiwara, PHG Guimarães and RWDA Vitor. New naphthoquinones and an alkaloid with in vitro activity against Toxoplasma gondii RH and EGS strains. Exp. Parasitol. 2012; 132, 450-7.
WC Lee, R Mahmud, R Noordin, SP Piaru, S Perumal and S Ismail. Alkaloids content, cytotoxicity and anti-Toxoplasma gondii activity of Psidium guajava L. and Tinospora crispa. Bangladesh J. Pharmacol. 2012; 7, 272-6.
ML Alomar, FAO Rasse-Suriani, A Ganuza, VM Cóceres, FM Cabrerizo and SO Angel. In vitro evaluation of β-carboline alkaloids as potential anti-Toxoplasma agents. BMC Res. Notes 2013; 6, 193.
M Wink and MF Roberts. Compartmentation of alkaloid synthesis, transport, and storage. In: M Wink and MF Roberts (Eds.). Alkaloids. Springer, New York, 1998, p. 239-62.
Y Li, F Liang, W Jiang, F Yu, R Cao, Q Ma, X Dai, J Jiang, Y Wang and S Si. DH334, a β-carboline anti-cancer drug, inhibits the CDK activity of budding yeast. Cancer Biol. Ther. 2007; 6, 1204-10.
J Gershenzon and N Dudareva. The function of terpene natural products in the natural world. Nat. Chem. Biol. 2007; 3, 408-14.
M Antczak, K Dzitko and H Długońska. Human toxoplasmosis: Searching for novel chemotherapeutics. Biomed. Pharmacother. 2016; 82, 677-84.
M Montazeri, A Daryani, M Ebrahimzadeh, E Ahmadpour, M Sharif and S Sarvi. Effect of propranolol alone and in combination with pyrimethamine on acute murine toxoplasmosis. Jundishapur J. Microbiol. 2015; 8, e22572.
AA Sharif, NZ Unyah, N Nordin, R Basir, MN Wana, AA Ahmad, T Mustapha and RA Majid. Susceptibility of Toxoplasma gondii to ethanolic extract of Tinospora crispa in vero cells. Evid. based Compl. Altern. Med. 2019; 2019, 2916547.
SX Chen, L Wu, XG Jiang, YY Feng and JP Cao. Anti-Toxoplasma gondii activity of GAS in vitro. J. Ethnopharmacol. 2008; 118, 503-7.
KK Ojo, ET Larson, KR Keyloun, LJ Castaneda, AE Derocher, KK Inampudi, JE Kim, TL Arakaki, RC Murphy, L Zhang, AJ Napuli, DJ Maly, CLMJ Verlinde, FS Buckner, M Parsons, WGJ Hol, EA Merritt and WCV Voorhis. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors. Nat. Struct. Mol. Biol. 2010; 17, 602-7.
Z Zhang, KK Ojo, SM Johnson, ET Larson, P He, JA Geiger, A Castellanos-Gonzalez, AC White Jr, M Parsons, EA Merritt, DJ Maly, CLMJ Verlinde, WCV Voorhis and E Fan. Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1. Bioorg. Med. Chem. Lett. 2012; 22, 5264-7.
F Wei, W Wang and Q Liu. Protein kinases of Toxoplasma gondii: Functions and drug targets. Parasitol. Res. 2013; 112, 2121-9.
E Moine, I Dimier-Poisson, C Enguehard-Gueiffier, C Logé, M Pénichon, N Moiré, C Delehouzé, B Foll-Josselin, S Ruchaud, S Bach, A Gueiffier, F Debierre-Grockiego and C Denevault-Sabourin. Development of new highly potent imidazo[1,2-b[pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1. Eur J Med Chem. 2015; 105, 80-105.
J Liu, Y Hu, DL Waller, J Wang and Q Liu. Natural products as kinase inhibitors. Nat. Prod. Rep. 2012; 29, 392-403.
HJ Youn, J Lakritz, GE Rottinghaus, HS Seo, DY Kim, Cho MH and AE Marsh. Anti-protozoal efficacy of high performance liquid chromatography fractions of Torilis japonica and Sophora flavescens extracts on Neospora caninum and Toxoplasma gondii. Vet. Parasitol. 2004; 125, 409-14.
KM Choi, J Gang and J Yun. Anti-Toxoplasma gondii RH strain activity of herbal extracts used in traditional medicine. Int. J. Antimicrob. Agents 2008; 32, 360-2.
TCD Oliveira, DAO Silva, C Rostkowska, SR Béla, EAV Ferro, PM Magalhães and JR Mineo. Toxoplasma gondii: Effects of Artemisia annua L. on susceptibility to infection in experimental models in vitro and in vivo. Exp. Parasitol. 2009; 122, 233-41.
LMD Pablos, G Gonza and R Rodrigues. Action of a pentacyclic triterpenoid, maslinic acid, against Toxoplasma gondii. J. Nat. Prod. 2010; 73, 831-4.
E Melo, K Vilela and C Carvalho. Effects of aqueous leaf extracts of Azadirachta indica A. Juss. (neem) and Melia azedarach L. (Santa Barbara or cinnamon) on the intracellular development of Toxoplasma gondii. Rev. Bras. Plantas Med. 2011; 13, 215-22.
N Kavitha, R Noordin, KL Chan and S Sasidharan. In vitro Anti-Toxoplasma gondii activity of root extract/fractions of Eurycoma longifolia Jack. BMC Complement Altern. Med. 2012; 12, 1102.
W Choi, M Jiang and J Chu. Antiparasitic effects of Zingiber officinale (Ginger) extract against Toxoplasma gondii. J. Appl. Biomed. 2013; 11, 15-26.
CBS Oliveira, YSR Meurer, MG Oliveira, WMTQ Medeiros, FON Silva and ACF Brito. Comparative study on the antioxidant and anti-Toxoplasma activities of vanillin and its resorcinarene derivative. Molecules 2014; 19, 5898-912.
JS Doggett, KK Ojo, E Fan, DJ Maly and WCV Voorhis. Bumped kinase inhibitor 1294 treats established toxoplasma gondii infection. Antimicrob. Agents Chemother. 2014; 58, 3547-9.
TB Kuhlenschmidt, FU Rutaganira, S Long, K Tang, KM Shokat and MS Kuhlenschmidt. Inhibition of Calcium-Dependent Protein Kinase 1 (CDPK1) in vitro by pyrazolopyrimidine derivatives does not correlate with sensitivity of Cryptosporidium parvum growth in cell culture. Antimicrob. Agents Chemother. 2016; 60, 570-9.
JL Wang, SY Huang, NZ Zhang, J Chen and XQ Zhu. Genome-wide expression patterns of calcium-dependent protein kinases in Toxoplasma gondii. Parasites Vectors 2015; 8, 1-7.
P Winzer, J Müller, A Aguado-Martínez, M Rahman, V Balmer and V Manser. In vitro and in vivo effects of the bumped kinase inhibitor 1294 in the related cyst-forming apicomplexans Toxoplasma gondii and Neospora caninum. Antimicrob Agents Chemother. 2015; 59, 6361-74.
MA Hulverson, R Choi, SLM Arnold, DA Schaefer, A Hemphill, McCloskey, DP Betzer, J Müller, RSR Vidadala, GR Whitman, KL Rivas, LK Barrett, RC Hackman, MS Love, CW McNamara, TK Shaughnessy, A Kondratiuk, M Kurnick, PN Banfor, JJ Lynch, GM Freiberg, …, WCV Voorhis. Advances in bumped kinase inhibitors for human and animal therapy for cryptosporidiosis. Int. J. Parasitol. 2017; 47, 753-63.
MA Hulverson, S Vinayak, R Choi, DA Schaefer, A Castellanos-Gonzalez, RSR Vidadala, CF Brooks, GT Herbert, DP Betzer, GR Whitman, HN Sparks, SLM Arnold, KL Rivas, LK Barrett, AC White Jr, DJ Maly, MW Riggs, B Striepen, WCV Voorhis and KK Ojo. Bumped-kinase inhibitors for cryptosporidiosis therapy. J. Infect. Dis. 2017; 215, 1275-84.
S Lourido, K Tang and LD Sibley. Distinct signalling pathways control Toxoplasma egress and host-cell invasion. EMBO J. 2012; 31, 4524-34.
F Robert-Gangneux, C Creuzet, J Dupouy-Camet and MP Roisin. Involvement of the mitogen-activated protein (MAP) kinase signalling pathway in host cell invasion by Toxoplasma gondii. Parasite. 2000; 7, 95-101.
S Kumar and AK Pandey. Chemistry and biological activities of flavonoids: An overview. Sci. World J. 2013; 2013, 162750.
M Lebrun, VB Carruthers and MF Cesbron-Delauw. Toxoplasma secretory proteins and their roles in cell invasion and intracellular survival. In: LM Weiss and K Kim (Eds.). Toxoplasma Gondii. 2nd eds. Academic Press, Massachusetts, 2013.
D Jacot and D Soldati-Favre. Does protein phosphorylation govern host cell entry and egress by the Apicomplexa? Int. J. Med. Microbiol. 2012; 302, 195-202.
EV Lourenço, SR Pereira, VM Faça, AAM Coelho-Castelo, JR Mineo and MC Roque-Barreira MC. Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin. Glycobiology 2001; 11, 541-7.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2023 Walailak University
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
