The Regulation of ManLAM-Related Gene Expression in Mycobacterium tuberculosis with Different Drug Resistance Profiles Following Isoniazid Treatment

Figures & data

Table 1 Primer Sequences for Multiplex Real-Time PCR Target Stress-Related Genes

Target	Forward (F) and Reverse (R) primers 5’→ 3’	Amplicon Length (bp)	Source or Reference
hspX	F: CGACAAGGACGTCGACATTA R: CCTTGTCGTAGGTGGCCTTA	173 bp	[39]
sigE	F: GCAGTGCAAATTCGGAGGAT R: ATTGGTCAGACGGCTCCA	114 bp	This work
tgs1	F: TAGCTGGGCTCTCCGATGAA R: ATTGACACGGAATCCACCCC	107 bp	This work
sigA	F: GTGATTTCGTCTGGGATGAAGA R: TACCTTGCCGATCTGTTTGAG	116 bp	This work

Table 2 Probes Sequences Used in Multiplex Real-Time PCR Target Stress-Related Genes

Name	Probe Sequence 5’→ 3’	Reporter dye 5’	Quencher 3’
hspX	TGCCGGTAGGTGCTGACGAG	Texas Red	BHQ-2
sigE	ACCTTGAGTCCGACCAGCATGTCT	Cy5	BHQ-2
tgs1	ACCATGCAGTCGCAATCCGCA	HEX	BHQ-1
sigA	ACGCAAGGACGCCGAACTCA	FAM	BHQ-1

Table 3 The Condition for Multiplex Real-Time PCR Assay Target Stress-Related Genes

Steps	Temperature (°C)	Time	Cycles
Pre-denaturation	95	1 min	1
Denaturation	95	30 sec	40
Extension	56	30 sec	40

Table 4 Primer Sequences for ManLAM-Related Genes and Genes Associated with Drug Resistance

Target	Forward (F) and Reverse (R) Primers 5’→ 3’	Amplicon Length (bp)	Source or Reference
sigA	F: GTCGGAGGCCCTGCGTCAAG R: AGGCCAGCCTCGATCCGCTT	147 bp	[31]
pimB	F: GCGGTAGGTATTCCAACGAAG R: TATGCACCGCAGTGGAAAG	394 bp	[44]
mptA	F: CGGTTGATTTGGCTACAGCG R: CGTAAGGATCCAGACCGTCG	435 bp	This work*
mptC	F: ATAGCCCTCAAACTCACCCC R: TCATCGCCAATCGTCAACC	245 bp	[13]
embC	F: GCTGCACATCCTGGACAC R: ACGACATTGCCACCGATAC	914 bp	[45]
dprE1	F: TATCCACTCCATTGACGCCG R: ATGATGATGCCGGTGAGACC	318 bp	This work*
dprE2	F: ATGGTTCTTGATGCCGTAGG R: TCAGATGGGCAGCTTGCGGAAG	765 bp	[46]
InhA promoter region	F: TGCCCAGAAAGGGATCCGTCATG R: ATGAGGAATGCGTCCGCGGA	455 bp	[47]
katG	F: AACGACGTCGAAACAGCGGC R: GCGAACTCGTCGGCCAATTC	455 bp	[47]
rpoB	F: GAGGGTCAGACCACGATGAC R: GAGCCGATCAGACCGATG	449 bp	[48]

Note: *Primers were designed in this study using NCBI primer blast.

Table 5 PCR Conditions for ManLAM-Related Genes Expression Analysis

Steps	Temperature (°C)	Time	Cycles
Pre-denaturation	95	5 min	1
Denaturation	95	30 sec	40
Annealing	60	30 sec	40
Extension	72	30 sec	40
Final extension	72	5 min	1

Table 6 PCR Conditions for DNA Sequencing

Steps	Temperature (°C)	Time	Cycles
Pre-denaturation	95	5 min	1
Denaturation	95	30 sec	35
Annealing	68 (inhA and katG), 65 (ManLAM-related genes), 56 (rpoB)	15 sec	35
Extension	72	10 sec	35
Final extension	72	5 min	1

Figure 1 The relative expression of hspX (A), tgs1 (B), and sigE (C) of MTB in response to isoniazid treatment. *Significant at ρ < 0.05; **significant at ρ < 0.01 were determined by one-way analysis of variance with Tukey’s multiple comparison.

Figure 2 The relative expression of pimB (A), mptA (B), mptC (C), dprE1 (D), dprE2 (E) and embC (F) of MTB fours strain in response to isoniazid treatment. *Significant at ρ < 0.05; **significant at ρ < 0.01; ***significant at ρ < 0.001 were determined by one-way analysis of variance with Tukey’s multiple comparison.

Figure 3 The relative expression of ManLAM-related genes that were up-regulated in INH-R. The relative expression was compared among ManLAM-related genes that were up-regulated in INH-R, including pimB, mptA and mptC. *Significant at ρ < 0.05 was determined by one-way analysis of variance with Tukey’s multiple comparison.

Figure 4 The Venn diagram represents a distinct expression pattern of ManLAM-related genes of drug resistance MTB response to isoniazid treatment. ManLAM-related genes that were up-regulated are listed in each circle that represents each MTB strain. *Represents a gene which is up-regulated in H37Rv (a drug-sensitive strain).

Figure 5 Sequence alignment of inhA for MTB with different drug resistance profiles. *Represents the position of the nucleotide mutation in the inhA regulatory region of INH-R.

Figure 6 Sequence alignment of rpoB for MTB with different drug resistance profiles. *In the upper part indicates the location of the nucleotide mutation and *in the lower part indicates amino acid substitutions of MDR and RIF-R.

Figure 7 Sequence alignment of katG for MTB with different drug resistance profiles. *In the upper part indicates the location of the nucleotide mutation and *in the lower part indicates amino acid substitutions of MDR.

Figure 8 Sequence alignment of dprE1 for MTB with different drug resistance profiles. *Indicates the location of the nucleotide mutation in dprE1 gene of MDR and RIF-R.

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