Supplementary File 1
Article Title:
A lacZ reporter based strategy for rapid expression analysis and target validation of Mycobacterium tuberculosis latent
infection genes
Journal Name:
Current Microbiology
Author Names:
Shivani Sood1 • Satinder Kaur2 • Rahul Shrivastava1*
*
Corresponding author
Affiliations:
1
Department of Biotechnology & Bioinformatics, Jaypee University of Information Technology, Waknaghat,
Solan, Himachal Pradesh - 173234, India
2
Division of Microbiology, Central Drug Research Institute, Lucknow, Uttar Pradesh - 226001, India
E-mail of the corresponding author:
juit.rahul@gmail.com
1
Fig. 1 M. smegmatis cells show anaerobiosis induced non-replicating persistent (NRP) state.
Log10 CFU/mL as a function of time is shown in the graph. Cells were grown to an O.D. (600nm) of 0.4-0.7 and
incubated in sealed tubes. CFU was monitored for a period of 3 weeks post anaerobiosis, during which, the cells
showed constant CFU. ‘f’ represents fading and ‘d’ represents complete discoloration of methylene blue, indicating
hypoxic condition. [Data shows a mean of CFU counts done in triplicate from 3 independent experiments with S.D.
bars]
10
9
f
d
8
Log 10 CFU/mL
Constant CFU
7
6
5
4
3
2
1
Time period (in number of days after sealing of vials)
2
Fig. 2 ZN staining of M. smegmatis cultures under actively replicating and NRP state [1000X].
M. smegmatis cells were stained using Ziehl-Neelsen method for acid fast bacteria. (A) Actively replicating cells
show normal morphology and staining characteristics (bacilli stained dark pink in color and longer in size) in
comparison to (B) Non-replicating persistent state cells exhibiting altered staining characteristics (bacilli stained
light pink in color and shorter size).
(A) Normal stained M. smegmatis cells under actively replicating state
(B) M. smegmatis cells under NRP state showing altered staining characteristics
3
Fig. 3 TEM images of M. smegmatis cells under actively replicating and NRP state.
(A) Actively replicating cells showing normal size (1431.58nm), and bacillary shape in comparison to (B) NRP state
cells, which appear shorter in size (524.48nm) and exhibit an altered coccus or coccobacillary morphology.
(A) Normal bacillary morphology of actively replicating M. smegmatis cells
(B) Altered cocco-bacillary morphology of NRP state M. smegmatis cells
4
Table 1 List of genes and corresponding primers used for amplification of their upstream sequence
Gene name/
Primer Sequence 5’- 3’
Amplicon size
Rv number
Rv3417c (hsp60)
F – GGGGTACCCTAGAGGTCACCACAACG
435bp
R – GCTCTAGACATTGCGAAGTGATTCCT
Rv0467 (icl)
F – GGGGTACCGATCGAGTTCGGCGACAT
842bp
R - GCTCTAGAAGACAACTCCTTAACGGTCT
Rv2031c (hspX)
F - GGGGTACCCACCCGAAAGTGGTCGAGTA
412bp
R - GCTCTAGATTGATGCCTCCTAATCGATGG
Rv0227c
F – GGGGTACCGTCAGCGGCACATAGAT
960bp*
R – GCTCTAGAACATGACTGCCCGGTTCA
Rv0347
F - GGGGTACCAAGACCGTACCGACGATCAG
948bp*
R – GCTCTAGACATCGACGCCGAAATAGAG
Rv0430
F – GGGGTACCCGCAATCGTAGACGAAGA
551bp
R – GCTCTAGACGCTGTCCATATCGCTTTC
Rv0628
F - GGGGTACCGATTTGGTCGATGGCTTCT
817bp*
R – GCTCTAGAGACTCCGATCCGCACACAT
Rv0883c
F- GGGGTACCCCCGTCAGTCGATGTGAT
925bp
R - GCTCTAGAACCCTGGCAGATGATGTTTTT
KpnI and XbaI sites were added at 5’ ends of forward and reverse primers respectively, to facilitate directional
cloning into pMV206::lacZ vector. * KpnI site present in the genomic DNA sequence was used for cloning; hence
these sequences had modified amplicon size of 673, 817 and 514 bases respectively.
5
Table 2 Drug susceptibility and percentage survival of M. smegmatis under actively replicating and hypoxia
induced NRP states
Antibiotic
Actively Replicating State
NRP State
Ethambutol (20.0µg/ml)
S
R
0.06%
77%
S
R
28.85%
92%
S
R
0.07%
84.6%
R
S
~100%
1.5%
Isoniazid (0.4µg/ml)
Rifampicin (0.5 µg/ml)
Metronidazole (120.0µg/ml)
S = Sensitive, R = Resistant (indicate comparative susceptibility of M. smegmatis under both states)
MIC for different drugs was determined for actively replicating M. smegmatis and the same concentration was used
for NRP state cells. Percentage of M. smegmatis cells surviving under both conditions was determined to record any
deviation in the susceptibility pattern. For NRP stage cultures, antibiotics were added 10 days post anaerobiosis
through rubber septum and CFU were determined. Bacilli under hypoxia exhibited tolerance to common
antimycobacterial drugs and were sensitive to metronidazole, indicating prevalence of NRP.
6