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).