Cell Lines
A549 cells were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Sigma), penicillin-streptomycin (100 U/mL, Gibco), and L-glutamine (2mM, Gibco), at 37°C in 5% CO2. AllSaccharomyces cerevisiae strains used were derivatives of BY4742 (MAT \(\alpha\) his3 1 leu2 0 lys2 0 ura3 0 ), some of which were modified to induce allGAL1 promoters via addition of \(\beta\)-estradiol due to the incorporation of the gene for Gal4-estrogen receptor-VP16 protein within the leu2 0 locus (Louvion et al. , 1993). Yeast strains either lacking lipid droplets (VCY2; BY4742 are1::KanMX are2::KanMX trp1::URA3 lro1::TRP1 dga1::lox-HIS-lox ) or containing the galactose-inducible DGA1 gene (VCY3; BY4742 are1::KanMX are2::KanMX trp1::URA3 lro1::TRP1 GAL-DGA1::HIS3 ) were kind gifts from Dr. William Prinz (NIDDK).
Plasmids and Transfections
Plasmid pMRLB.7 containing gene Rv3875 (Protein ESAT-6) from Mycobacterium tuberculosis, NR-50170 and genomic DNA from Mycobacterium tuberculosis, Strain H37Rv, NR-48669 were obtained through BEI Resources (National Institutes of Health, NIAID). The expression vector mEmerald-N1 used as a control in the ESAT-6 studies, was a gift from Michael Davidson (Addgene plasmid # 53976; http://n2t.net/addgene:53976; RRID:Addgene_53976).
Gene Rv3875 containing the coding region for Protein ESAT-6 was amplified from plasmid pMRLB.7 using primers Esat6XbaF (5’-CGAGTCTAGATGCCACCATGACAGAGCAG-3’) and Esat6ApaR (5’-GTTCGAAGGGCCCGAGTGCGAACATCCCAGTGAC-3’). The fragment was then cloned in-frame with the myc tag in pcDNA3.1(+)Myc-His A (Thermofisher) using the XbaI and ApaI restriction sites to create the plasmid pcDNA3.1myc-hisA-ESAT6.
The Mtb genomic region containing Rv1646 (PE17) was amplified from
The Mtb genomic region containing Rv1646 (PE17) was amplified from Mycobacterium tuberculosis H37Rv genomic DNA and inserted into pcDNA3.1(+) using the following primers: PE17HindIIF (5’-GGATTGCTAAGCTTTAGACTTTATTTC-3’) and PE17XbaR (5’-GTCTCTAGACGGTTCGAAACCG-3’). PE17-Myc-His and pcDNA3.1-PE171-233-Myc-His were generated by cloning into the Hind III and Xba I (New England BioLabs) restriction sites of pcDNA3.1(+)Myc-His A (Thermofisher). Identical forward primers were used in generation of both constructs (5’-GGATTGCTAAGCTTTAGACTTTATTTC). Reverse primers are as follows: PE17, 5’-GTCTCTAGACGGTTCGAAACCG; PE171-223, 5’-GCCGGTCACCGCTCTAGAGGGAGCTTGCAG. All constructs were confirmed by DNA sequencing. The plasmids were amplified in RapidTrans TAM1 competentE.coli (Active Motif) and purified using a genElute plasmid miniprep kit or HiSpeed plasmid maxi kit prep kit (Sigma, Qiagen, respectively). DNA concentrations were quantified using a NanoDrop 2000 Spectrophotometer (Thermofisher).
pYES2-PE17 was constructed by amplifying PE17 with primers (forward: 5’- CGGGATCTGTACGACGATGACGATAAGGTAATGTCGTTTCTCACCGTGGC; reverse: 5’-GAGACCGAGGAGAGGGTTAGGGATAGGCTTTTAGAAACCGTTGAGTA
GGGCGGGAAG) containing 30 bp of homology upstream and downstream of the pYES2/NT A (Thermo Scientific) multiple cloning site, thereby fusing the N-terminal Xpress epitope to the PE17 open reading frame. pYES2/NT A was linearized via Bam HI digestion, and linear plasmid was co-transformed into yeast with the above PCR amplicon to form the pYES2-PE17 construct via gap repair. Successful incorporation of PE17 was verified by sequencing.
pYES2-PE17-mRuby2 was constructed by amplifying the mRuby2 open reading frame from pFA6a-link-yomRuby2-SpHis5 (Lee et al. , 2013) using the primers 5’- GGCCTTCTTCCCGCCCTACTCAACGGTTTCGGTGGTGGTGCTTCT ATGGTGTCCAAAGGAGAGGAGTTAATC and 5’- ATAACTAATTACATGATGCGGCCCTCTAGGGATCTATATTACCCTGTTATCCCTAGCG, which contain 30-bp homology to both the C-terminus of PE17 and the pYES2-PE17 vector, as well as introducing a [G4A] linker sequence (underlined) between the PE17 and mRuby2 open reading frames. The resultant amplicon was co-transformed into yeast with the pYES2-PE17 plasmid that had been previously linearized with PmeI, and selected for growth on CSM plates lacking uracil to construct pYES2-PE17-mRuby2 via gap repair. When used in the 3∆ dga1∆ or 3∆GAL-DGA1 backgrounds, pYES2-PE17-mRuby2 was converted from aURA3 selection to LYS2 by transforming a BY4742 strain harboring pYES2-PE17-mRuby2 with HindIII-digested pM2660 (ATCC plasmid #87559 (Cross, 1997), and selected for growth on CSM plates lacking lysine. Resultant colonies were then screened for no growth on CSM plates lacking uracil, creating the pYES2-PE17-mRuby2 (ura3::LYS2 ) plasmid.
To construct a plasmid expressing GFP-Erg6, the ERG6 open reading frame was amplified from the BY4742 yeast genome using the primer pair 5’- ATGGATGAACTA TACAAGTCCGGACTCAGATCTATGAGTGAAACAGAATTGAGAAAAAG and 5’- GTC GACTGCAGAATTCGAAGCTTGAGCTCGAGATCTTTATTGAGTTGC TTCTTGGGAAG. The resultant amplicon was co-transformed into yeast with plasmid pGO36 (Odorizzi et al. , 1998) which had been previously linearized with BglII. Resultant gap-repaired plasmids were selected on CSM plates lacking uracil, thus constructing the pGO36-Erg6 (GFP-Erg6) plasmid. In order to convert the pGO36-Erg6 plasmid from uracil to leucine selection, BY4742 harboring pGO36-Erg6 was transformed with SmaI-digested pUL9 (ATCC plasmid #87552 (Cross, 1997)) and transformants were selected on CSM plates lacking leucine. Resultant colonies were then screened for no growth on plates lacking uracil, creating the pGO36-Erg6 (ura3::LEU2 ) plasmid.
Mammalian cells were grown on sterilized coverlips for 24 hours prior to transient transfection using jetPRIME (Polyplus) according to the manufacturers protocol with the following exception: for each well of a six well plate, 1 µg DNA was used per transfection. Transient transfections were allowed to incubate for 48 hours before processing.
Infection of A549 cells with Mtb and post-fixation staining with Mitotracker was performed as described previously (Fine-Coulson et al. , 2015).