Comparison of in vitro Susceptibility of Mycobacteria Against PA-824 to Identify Key Residues of Ddn, the Deazoflavin-Dependent Nitroreductase from Mycobacterium tuberculosis

Figures & data

Table 1 MIC Distributions for Various Mycobacteria Species Against PA-824

Species	Classification		No. of Strains with Different MIC (mg/L)													MIC50 (mg/L)	MIC90 (mg/L)
		≤0.016	0.031	0.062	0.12	0.25	0.5	1	2	4	8	16	32	>32	Total
M. tuberculosis	SGM	18	23	21	8	1	1	0	0	0	0	0	0	0	72	0.031	0.12
M. avium	SGM	0	0	1	0	0	0	0	0	0	0	0	0	21	22	>32	>32
M. intracellulare	SGM	0	0	0	0	0	0	0	0	0	0	0	0	34	34	>32	>32
M. kansasii	SGM	0	0	0	0	0	0	0	1	13	16	0	0	3	33	8	8
M. abscessus	RGM	0	0	0	0	0	0	0	0	0	1	0	1	34	36	>32	>32
M. fortuitum	RGM	0	0	0	0	0	0	0	0	0	1	2	15	12	30	32	>32

Abbreviations: SGM, slowly growing mycobacteria; RGM, rapidly growing mycobacteria. MIC₅₀, the concentration required to inhibit the growth of 50% of the strains; MIC₉₀, the concentration required to inhibit the growth of 90% of the strains.

Figure 1 Multiple sequence alignments and phylogenetic analysis of Ddn sequences in MTB and its homologues of NTMs. (A) Multiple sequence alignments of Ddn of MTB and its homologues of NTMs. Shared amino acids are highlighted in red and stars mark non-shared amino acids selected for testing in this study. (B) Phylogenetic tree of MTB Ddn and NTMs Ddn homologous sequences constructed based on protein sequence similarity.

Figure 2 Protein expression analysis and growth curve analysis of M. smegmatis transformed with plasmids encoding MTB Ddn and its mutants. (A) Western blot analysis of MTB Ddn and Ddn mutants recombinantly expressed by M. smegmatis. (B) Growth curve analysis of M. smegmatis transformed with plasmids encoding MTB Ddn and Ddn mutants.

Table 2 MIC of Ddn Mutants in M. smegmatis Against PA-824

Strains	Mutation Site	MIC (mg/L)
WT	—	384
pMV261	—	384
Ddn	—	24
Ddn-Y65L	194/195tAC→tTG	384
Ddn-V75L	223gTC→cTC	12
Ddn-A76V	227gCg→gTg	192
Ddn-Q125K	373Cag→Aag	12
Ddn-Y133F	398tAt→tTt	192
Ddn-V148I	442Gtt→Att	48