Evaluation of Apoptotic Effect of Transgenic Leishmania tropica Expressing MLLO-Bax-Smac Fusion Gene in Infected Macrophages, an In Vitro Study
-
Maryam Aghaei
,
Hossein Khanahmad
,
Shahrzad Aghaei
,
Akram Jalali
,
Zabihollah Shahmoradi
,
Nooshin Hashemi
,
Akram Sarmadi
,
Shima Aboutalebian
,
Mohammad Ali Mohammadi
,
Sayed Mohsen Hosseini
,
Seyed Hossein Hejazi
Abstract
Background: In the leishmaniasis parasitic infection, parasite escapes from the immune system of host or prevents macrophage apoptosis. It seems generating transgenic parasites to express proapoptotic proteins can accelerate the apoptosis of infected macrophage and prevent Leishmania differentiation. Hence, we investigated the efficacy of transgenic L. tropica expressing mLLO-BAX-SMAC in expediting macrophage apoptosis.
Methods: This study was performed at the Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences (2022 to 2023), Isfahan, Iran. mLLO-Bax-Smac coding sequence cloned in the pLexyNeo2 was entered into the L. tropica genome using homologous recombination. PCR, Western blot, and hemolysis tests were used to confirm integration accuracy. In addition, the apoptosis percentage of transgenic L. tropica- infected macrophages was assayed by flow-cytometry.
Results: The proper integration of the mLLO-Bax-Smac fragment into the 18srRNA locus of L. tropica and mLLO -BAX-SMAC fusion protein expression was approved. Furthermore, results of flow cytometry showed the mean percentage of apoptosis among the groups is different and transgenic L. tropica leads to a decrease in the apoptosis time of infected macrophages compared to the wild type of Leishmania and to an increase in the immune system response.
Conclusion: The data suggest the transgenic L. tropica can be used as an experimental model to expedite apoptosis of Leishmania-infected macrophage.
1. Sharma U, Singh S. Immunobiology of leishmaniasis. Indian J Exp Biol. 2009; 47(6):412-23.
2. Zolfaghari A, Beheshti-Maal K, Ahadi AM, et al. Identification of Leishmania species and frequency distribution of lrv1 and lrv2 viruses on cutaneous leishmaniasis patients in isfahan province, Iran. Indian J Med Microbiol. 2023;41:13-18.
3. Faulde MK, Heyl G, Amirih ML. Zoonotic cutaneous leishmaniasis, Afghanistan. Emerg Infect Dis. 2006;12:1623-4.
4. Bogitsh BJ, Carter CE, Oeltmann TN. Human parasitology. Academic Press. 2018 May 28.
5. Mahmoudzadeh-Niknam H, Kiaei SS, Iravani D. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in balb/c mice. Korean J Parasitol. 2007; 45(2):103-9.
6. Mears ER, Modabber F, Don R, et al. A review: The current in vivo models for the discovery and utility of new anti-leishmanial drugs targeting cutaneous leishmaniasis. PLoS Negl Trop Dis. 2015; 9(9):e0003889.
7. Bamorovat M, Sharifi I, Tavakoli Oliaee R, et al. Determinants of unresponsiveness to treatment in cutaneous leishmaniasis: A focus on anthroponotic form due to Leishmania tropica. Front Microbiol. 2021; 12:638957.
8. Field AE, Wagage S, Conrad SM, et al. Reduced pathology following infection with transgenic Leishmania major expressing murine cd40 ligand. Infect Immun. 2007;75:3140-3149.
9. Elmahallawy EK, Alkhaldi AA, Saleh AA. Host immune response against leishmaniasis and parasite persistence strategies: A review and assessment of recent research. Biomed Pharmacother. 2021;139:111671.
10. Awasthi A, Mathur RK, Saha B. Immune response to Leishmania infection. Indian J Med Res. 2004;119:238-258.
11. Seimon T, Tabas I. Mechanisms and consequences of macrophage apoptosis in atherosclerosis. J Lipid Res. 2009; 50 Suppl(Suppl):S382-7.
12. Filardy AA, Costa-da-Silva AC, Koeller CM, et al. Infection with Leishmania major induces a cellular stress response in macrophages. PLoS One. 2014; 9(1):e85715.
13. Grode L, Seiler P, Baumann S, et al. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille calmette-guerin mutants that secrete listeriolysin. J Clin Invest. 2005;115(9):2472-2479.
14. Oliveira SC, Splitter GA. Cd8+ type 1 cd44hi cd45 rblo t lymphocytes control intracellular Brucella abortus infection as demonstrated in major histocompatibility complex class i‐and class ii‐deficient mice. Eur J Immunol. 1995;25(9):2551-2557.
15. Kamir D, Zierow S, Leng L, et al. A Leishmania ortholog of macrophage migration inhibitory factor modulates host macrophage responses. J Immunol. 2008; 180(12):8250-61.
16. Akarid K, Arnoult D, Micic‐Polianski J, et al. Leishmania major‐mediated prevention of programmed cell death induction in infected macrophages is associated with the repression of mitochondrial release of cytochrome c. J Leukoc Biol. 2004;76:95-103.
17. Apte SS, Mattei M-G, Olsen BR. Mapping of the human bax gene to chromosome 19q13. 3–q13. 4 and isolation of a novel alternatively spliced transcript, baxδ. Genomics. 1995;26:592-594.
18. Oltval ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programed cell death. Cell. 1993;74:609-619.
19. Weng C, Li Y, Xu D, et al. Specific cleavage of mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (trail)-induced apoptosis in jurkat leukemia t cells. J Biol Chem. 2005; 280(11):10491-500.
20. McNeish I, Bell S, McKay T, et al. Expression of smac/diablo in ovarian carcinoma cells induces apoptosis via a caspase-9-mediated pathway. Exp Cell Res. 2003;286:186-198.
21. Moshrefi M, Spotin A, Kafil HS, et al. Tumor suppressor p53 induces apoptosis of host lymphocytes experimentally infected by Leishmania major, by activation of bax and caspase-3: A possible survival mechanism for the parasite. Parasitol Res. 2017;116:2159-2166.
22. Cianciulli A, Porro C, Calvello R, et al. Resistance to apoptosis in Leishmania infantum-infected human macrophages: A critical role for anti-apoptotic bcl-2 protein and cellular iap1/2. Clin Exp Med. 2018;18:251-261.
23. Beattie L, Evans K, Kaye P, et al. Transgenic Leishmania and the immune response to infection. Parasite Immunol. 2008;30:255-266.
24. Sambrook J, Russell DW, Sambrook J. The condensed protocols: From molecular cloning: A laboratory manual. CSHL Press, NY; 2006.
25. Maniatis T. Molecular cloning. A laboratory manual. 1982.
26. Donyavi T, Bandehpour M, Kazemi B. Preparation of transgenic Iranian lizard Leishmania coding hil-12. Iran J Microbiol. 2017; 9(5):305-311.
27. Cuervo P, De Jesus JB, Saboia-Vahia L, et al. Proteomic characterization of the released/secreted proteins of Leishmania (viannia) braziliensis promastigotes. J Proteomics. 2009;73:79-92.
28. Datta AR, Wentz BA, Russell J. Cloning of the listeriolysin o gene and development of specific gene probes for Listeria monocytogenes. Appl Environ Microbiol. 1990;56(12):3874-3877.
29. Singh N, Gupta R, Jaiswal AK, et al. Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening. J Antimicrob Chemother. 2009;64:370-374.
30. Rieger AM, Nelson KL, Konowalchuk JD, et al. Modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death. J Vis Exp. 2011; (50):2597.
31. Rabhi I, Rabhi S, Ben-Othman R, et al. Transcriptomic signature of Leishmania infected mice macrophages: A metabolic point of view. PLoS Negl Trop Dis. 2012; 6(8):e1763.
32. Patel AP, Deacon A, Getti G. Development and validation of four Leishmania species constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies. Parasitology. 2014;141(4):501-10.
33. Aghaei M, Khanahmad H, Jalali A, et al. Effect of transgenic Leishmania major expressing mLLO-Bax-Smac fusion gene in the apoptosis of the infected macrophages. Iran J Basic Med Sci. 2021; 24(12):1666-1675.
34. Aghaei M, Khanahmad H, Aghaei S, et al. Evaluation of transgenic Leishmania infantum expressing mLLO-BAX-SMAC in the apoptosis of the infected macrophages in vitro and in vivo. Parasite Immunol. 2020;42(11):e12726.
35. Kianmehr A, Golavar R, Rouintan M, et al. Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR. Protein Expr Purif. 2016;118:120-5.
36. Du C, Fang M, Li Y, et al. Smac, a mitochondrial protein that promotes cytochrome c–dependent caspase activation by eliminating IAP inhibition. Cell. 2000;102(1):33-42.
37. Esseiva AC, Chanez AL, Bochud‐Allemann N, et al. Temporal dissection of Bax‐induced events leading to fission of the single mitochondrion in Trypanosoma brucei. EMBO Rep. 2004;5(3):268-73.
2. Zolfaghari A, Beheshti-Maal K, Ahadi AM, et al. Identification of Leishmania species and frequency distribution of lrv1 and lrv2 viruses on cutaneous leishmaniasis patients in isfahan province, Iran. Indian J Med Microbiol. 2023;41:13-18.
3. Faulde MK, Heyl G, Amirih ML. Zoonotic cutaneous leishmaniasis, Afghanistan. Emerg Infect Dis. 2006;12:1623-4.
4. Bogitsh BJ, Carter CE, Oeltmann TN. Human parasitology. Academic Press. 2018 May 28.
5. Mahmoudzadeh-Niknam H, Kiaei SS, Iravani D. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in balb/c mice. Korean J Parasitol. 2007; 45(2):103-9.
6. Mears ER, Modabber F, Don R, et al. A review: The current in vivo models for the discovery and utility of new anti-leishmanial drugs targeting cutaneous leishmaniasis. PLoS Negl Trop Dis. 2015; 9(9):e0003889.
7. Bamorovat M, Sharifi I, Tavakoli Oliaee R, et al. Determinants of unresponsiveness to treatment in cutaneous leishmaniasis: A focus on anthroponotic form due to Leishmania tropica. Front Microbiol. 2021; 12:638957.
8. Field AE, Wagage S, Conrad SM, et al. Reduced pathology following infection with transgenic Leishmania major expressing murine cd40 ligand. Infect Immun. 2007;75:3140-3149.
9. Elmahallawy EK, Alkhaldi AA, Saleh AA. Host immune response against leishmaniasis and parasite persistence strategies: A review and assessment of recent research. Biomed Pharmacother. 2021;139:111671.
10. Awasthi A, Mathur RK, Saha B. Immune response to Leishmania infection. Indian J Med Res. 2004;119:238-258.
11. Seimon T, Tabas I. Mechanisms and consequences of macrophage apoptosis in atherosclerosis. J Lipid Res. 2009; 50 Suppl(Suppl):S382-7.
12. Filardy AA, Costa-da-Silva AC, Koeller CM, et al. Infection with Leishmania major induces a cellular stress response in macrophages. PLoS One. 2014; 9(1):e85715.
13. Grode L, Seiler P, Baumann S, et al. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille calmette-guerin mutants that secrete listeriolysin. J Clin Invest. 2005;115(9):2472-2479.
14. Oliveira SC, Splitter GA. Cd8+ type 1 cd44hi cd45 rblo t lymphocytes control intracellular Brucella abortus infection as demonstrated in major histocompatibility complex class i‐and class ii‐deficient mice. Eur J Immunol. 1995;25(9):2551-2557.
15. Kamir D, Zierow S, Leng L, et al. A Leishmania ortholog of macrophage migration inhibitory factor modulates host macrophage responses. J Immunol. 2008; 180(12):8250-61.
16. Akarid K, Arnoult D, Micic‐Polianski J, et al. Leishmania major‐mediated prevention of programmed cell death induction in infected macrophages is associated with the repression of mitochondrial release of cytochrome c. J Leukoc Biol. 2004;76:95-103.
17. Apte SS, Mattei M-G, Olsen BR. Mapping of the human bax gene to chromosome 19q13. 3–q13. 4 and isolation of a novel alternatively spliced transcript, baxδ. Genomics. 1995;26:592-594.
18. Oltval ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programed cell death. Cell. 1993;74:609-619.
19. Weng C, Li Y, Xu D, et al. Specific cleavage of mcl-1 by caspase-3 in tumor necrosis factor-related apoptosis-inducing ligand (trail)-induced apoptosis in jurkat leukemia t cells. J Biol Chem. 2005; 280(11):10491-500.
20. McNeish I, Bell S, McKay T, et al. Expression of smac/diablo in ovarian carcinoma cells induces apoptosis via a caspase-9-mediated pathway. Exp Cell Res. 2003;286:186-198.
21. Moshrefi M, Spotin A, Kafil HS, et al. Tumor suppressor p53 induces apoptosis of host lymphocytes experimentally infected by Leishmania major, by activation of bax and caspase-3: A possible survival mechanism for the parasite. Parasitol Res. 2017;116:2159-2166.
22. Cianciulli A, Porro C, Calvello R, et al. Resistance to apoptosis in Leishmania infantum-infected human macrophages: A critical role for anti-apoptotic bcl-2 protein and cellular iap1/2. Clin Exp Med. 2018;18:251-261.
23. Beattie L, Evans K, Kaye P, et al. Transgenic Leishmania and the immune response to infection. Parasite Immunol. 2008;30:255-266.
24. Sambrook J, Russell DW, Sambrook J. The condensed protocols: From molecular cloning: A laboratory manual. CSHL Press, NY; 2006.
25. Maniatis T. Molecular cloning. A laboratory manual. 1982.
26. Donyavi T, Bandehpour M, Kazemi B. Preparation of transgenic Iranian lizard Leishmania coding hil-12. Iran J Microbiol. 2017; 9(5):305-311.
27. Cuervo P, De Jesus JB, Saboia-Vahia L, et al. Proteomic characterization of the released/secreted proteins of Leishmania (viannia) braziliensis promastigotes. J Proteomics. 2009;73:79-92.
28. Datta AR, Wentz BA, Russell J. Cloning of the listeriolysin o gene and development of specific gene probes for Listeria monocytogenes. Appl Environ Microbiol. 1990;56(12):3874-3877.
29. Singh N, Gupta R, Jaiswal AK, et al. Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening. J Antimicrob Chemother. 2009;64:370-374.
30. Rieger AM, Nelson KL, Konowalchuk JD, et al. Modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death. J Vis Exp. 2011; (50):2597.
31. Rabhi I, Rabhi S, Ben-Othman R, et al. Transcriptomic signature of Leishmania infected mice macrophages: A metabolic point of view. PLoS Negl Trop Dis. 2012; 6(8):e1763.
32. Patel AP, Deacon A, Getti G. Development and validation of four Leishmania species constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies. Parasitology. 2014;141(4):501-10.
33. Aghaei M, Khanahmad H, Jalali A, et al. Effect of transgenic Leishmania major expressing mLLO-Bax-Smac fusion gene in the apoptosis of the infected macrophages. Iran J Basic Med Sci. 2021; 24(12):1666-1675.
34. Aghaei M, Khanahmad H, Aghaei S, et al. Evaluation of transgenic Leishmania infantum expressing mLLO-BAX-SMAC in the apoptosis of the infected macrophages in vitro and in vivo. Parasite Immunol. 2020;42(11):e12726.
35. Kianmehr A, Golavar R, Rouintan M, et al. Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR. Protein Expr Purif. 2016;118:120-5.
36. Du C, Fang M, Li Y, et al. Smac, a mitochondrial protein that promotes cytochrome c–dependent caspase activation by eliminating IAP inhibition. Cell. 2000;102(1):33-42.
37. Esseiva AC, Chanez AL, Bochud‐Allemann N, et al. Temporal dissection of Bax‐induced events leading to fission of the single mitochondrion in Trypanosoma brucei. EMBO Rep. 2004;5(3):268-73.
| Files | ||
| Issue | Vol 20 No 3 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v20i3.19611 | |
| Keywords | ||
| Leishmaniasis Transgenic Apoptosis Macrophage Homologous recombination | ||
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How to Cite
1.
Aghaei M, Khanahmad H, Aghaei S, Jalali A, Shahmoradi Z, Hashemi N, Sarmadi A, Aboutalebian S, Mohammadi MA, Hosseini SM, Hejazi SH. Evaluation of Apoptotic Effect of Transgenic Leishmania tropica Expressing MLLO-Bax-Smac Fusion Gene in Infected Macrophages, an In Vitro Study. Iran J Parasitol. 2025;20(3):366-377.
