Subcellular localization of Leishmania donovani dihydrolipoamide dehydrogenase variants*
4.3 MATERIAL AND METHODS .1 Materials
Leishmania donovani strain (BHU 1081) was kindly gifted by Prof, Shyam Sundar, Banaras Hindu University, India. L. donovani culture condition are established in our lab which include M199 medium (Sigma-Aldrich) with 15% fetal bovine serum (HiMedia), and antibiotics (penicillin and streptomycin, HiMedia) (Saudagar 2011). Green fluorescent protein (GFP) coding sequence was amplified from pGL1686 vector was gifted by Prof.
David Mottram (University of Glasgow, Scotland, UK). All the enzymes for Gibson based cloning were purchased from Imperial life sciences (ILS) with high purity grades. All the buffer constituents for electroporation were purchased from HiMedia with high purity.
Protocols for parasite electroporation were also optimized in our lab (Ruchika et al., 2017;
Prakash et al. 2018). Stain (Hoechst 33342, Alexa fluor, and Mitotracker) used for localization studies were from Invitrogen.
4.3.2 Cloning of LdDLDH_Variant1 and LdDLDH_Variant2 gene with GFP in B3324 pXGPhleo (Phleomycin) vector
The coding sequence of LdDLDH_Variant1 and LdDLDH_Variant2 with GFP were cloned in pXGPhleo vector, (Leishmania- specific vector) by Gibson chew back and anneal assembly (Gibson et al, 2009). This method can able to ligate any fragments (4-5 insert + vector) without the use of restriction enzyme. Initially, all the coding sequences were amplified by using overlapping primers (Table 4.1). Whereas, the coding sequence of GFP was amplified from pGL1686 vector. Total 4 overlapped primers were designed, two for variants and remaining two for GFP. Forward primers (FP) of variants were made with the overlapping sequence with pXGPhleo vector at XmaI site and reverse primer (RP) with GFP 5’ overhanging part. Similarly for GFP-FP were prepared with over hanged region of 3’ end of variant and RP with the overlapping region with pXGPhleo vector at BamHI site. Two amplified fragments (coding sequence of variants and GFP) with vector (pXGPhleo) digested with XmaI and BamHI in Gibson master mix (5x Isothermal buffer, Taq Ligase, T5 exonuclease, Phusion polymerase) were incubated at 50 ºC for 60 min in PCR to make complete construct. Subsequently, the ligated construct was transformed in
E.coli DH5α strain which was selected on ampicillin-containing LB plate. Finally, positive clones were confirmed through amplification of both the fragments by gene-specific primer through colony PCR.
Table 4.1: List of primers for cloning of LdDLDH_Variant1 and LdDLDH_Variant2 with GFP in pXGPhleo vector.
S.No Sequence Name Sequence 1. LdDLDH_Variant1_F
P_Localzn
GCCCTCCCCCTGTCCCCGGGATGAAGCGCACTATCTTTGC 2. LdDLDH_Variant1_R
P_ Localzn
AACAGTTCCTCGCCCTTGCTCATTTTATTGTGCGCGTACGCG C
3. LdDLDH_Variant2_F P_ Localzn
GCCCTCCCCCTGTCCCCGGGATGTTCCGCAGGAACATAGC 4. LdDLDH_Variant2_R
P_ Localzn
AACAGTTCCTCGCCCTTGCTCATGAAGTTGATCGTCTGCGC AAA
5. LdDLDH_Variant1_F P_ Localzn
GCGCGTACGCGCACAATAAAATGAGCAAGGGCGAGGAACT GTT
6. LdDLDH_Variant1/V ariant2_RP_ Localzn
GAGGATCTGCTAGTGGATCCTCACTTGTACAGCTCGTCCAT GCC
7. LdDLDH_Variant2_F P_ Localzn
TTTGCGCAGACGATCAACTTCATGAGCAAGGGCGAGGAAC TGTT
4.3.3 Generation of L. donovani expressing GFP tagged LdDLDH variants
Log phage L.donovani promastigote cells were electroporated with pXGPhleo_LdDLDH_Variant1-GFP/pXGPhleo_LdDLDH_Variant2-GFP. Protocol for electroporation was established in our lab (Tiwari and Dubey, 2018; Bhardwaj et al, 2017).
Briefly, one million promastigote cells were centrifuged at 2500 rpm for 5 min to remove dead cell debris. Media above the cell pellet were discarded and cells were washed with two times by 2 ml PBSG buffer (10 mM NaH2PO4, 10 mM Na2HPO4, 145 mM NaCl and 2% glucose). After each washing, the cells were centrifuged at low rpm (2500 rpm, 5 min).
Subsequently, the supernatant was discarded and the pellet was processed for further washing with electroporation buffer (21 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose). Finally, the pellet was suspended in 1 ml electroporation buffer and distributed in three electroporation cuvette (Control, Test 1 and Test 2). In control cells, no plasmid was added whereas, in test 1 and 2, pXGPhleo_LdDLDH_Variant1-GFP and pXGPhleo_LdDLDH_Variant2-GFP were added, respectively. Thereafter, cells were incubated in ice for 15 min followed by electroporation by application of exponential protocol (450 V, 400 μF capacitance, 50 Ω resistance for 2 mm cuvette) in Gene Pulsar Xcell™ Electroporation system (Bio-Rad). After electroporation cells were further incubated at 4 ºC for 15 min which was finally transferred in M199 media. Next day, cells
were transferred in antibiotic (Phleomycin, Sigma-Aldrich) containing M199 media for selection. Positively selected all sets of cells were used to visualize green fluorescence.
4.3.4 Western blot analysis to confirm the expression of LdDLDH_Variant1-GFP and LdDLDH_Variant2-GFP
Initially, GFP tagged LdDLDH variant expressing cells were washed with two times by 1 x PBS and lysed in RIPA buffer (1x Protease Inhibitor cocktail, 1 mM PMSF and 10 mM EDTA). Total cell lysates were utilized for GFP detection by western blot (Towbin et al, 1979). Briefly, Cell lysate of both the variants with the control cell was run on 12% SDS- PAGE followed by protein were transferred on methanol charged polyvinylidene fluoride (PVDF) membrane. Protein containing PVDF membrane was blocked with 5% skimmed milk for 2 hr at room temperature. Further, the membrane was washed with 1 x TBST buffer three times followed membrane was incubated with primary anti-GFP primary antibody (1:1000 dilution) (Sigma-Aldrich, cat no. AB10145) overnight at 4 ºC.
Afterward, the membrane was again washed with 1x TBST buffer (thrice) and further incubated for 60 min at room temperature with secondary anti-rabbit IgG antibody (1:10,000 dilution). The band was detected in chemiluminescent HRP substrate (Bio-Rad) under Chemi-Doc, BioRad. L. donovani anti-α-tubulin (1:1000 dilution) used as endogenous control and anti-rabbit IgG conjugated with HRP (1:10,000) was used as a secondary antibody against tubulin.
4.3.5 Cellular imaging through fluorescence microscope
The phleomycin positive L. donovani promastigotes cells were visualized for GFP tagged protein under a Nikon inverted microscope (Eclipse Ti-U). The cells were washed with 1 x PBS (pH 7.4) (twice) to remove residual media and dead cells followed by cells were fixed by 2 % formaldehyde by keeping at 25 ºC for 30 min. Further, the cells were permeabilized by application of 0.1 % Triton X 100 which was kept for 10 min at 25 ºC.
After, permeabilization, cells were washed two times with PBS and counterstained with different organelle specific stain (Mitotracker, Hoechst, and Alexa fluor). Again, the cells were washed with PBS followed by mounted with anti-fade reagent (n-propyl gallate).
Finally, mounted cells were imaged under 100 x oil immersion of Nikon inverted microscope (Eclipse Ti-U) with an excitation wavelength of 488 nm for GFP. The plasma membrane was stained with Alexa Fluor 594 (excitation wavelength 594 nm), nucleus by
Hoechst 3342 (excitation at 350 nm) and mitochondria by Mito-Tracker (excitation at 579 nm).