The mycobacterial desaturase DesA2 is associated with mycolic acid biosynthesis

Abstract

Mycolic acids are critical for the survival and virulence of Mycobacterium tuberculosis, the causative agent of tuberculosis. Double bond formation in the merochain of mycolic acids remains poorly understood, though we have previously shown desA1, encoding an aerobic desaturase, is involved in mycolic acid desaturation. Here we show that a second desaturase encoded by desA2 is also involved in mycolate biosynthesis. DesA2 is essential for growth of the fast-growing Mycobacterium smegmatis in laboratory media. Conditional depletion of DesA2 led to a decrease in mycolic acid biosynthesis and loss of mycobacterial viability. Additionally, DesA2-depleted cells also accumulated fatty acids of chain lengths C₁₉-C₂₄. The complete loss of mycolate biosynthesis following DesA2 depletion, and the absence of any monoenoic derivatives (found to accumulate on depletion of DesA1) suggests an early role for DesA2 in the mycolic acid biosynthesis machinery, highlighting its potential as a drug target.

Figure 1

Structures of subclasses of mycolic acids found in M. tuberculosis and M. smegmatis.

Figure 2

DesA2 is present in mycolic acid producing species that encode a FAS-II (A) Multiple sequence alignment of DesA2 orthologs (B) Genomic maps of regions containing desA2.

Figure 3

Growth of the ∆desA2 mutant and the parental merodiploid strain on TSB agar plate (A) and 7H9 broth (B) in the presence or absence of acetamide. Serial dilutions spotted onto the TSB agar plates are indicated as a guide in the plate on the left (C) Growth curve of the ∆desA2 mutant in 7H9 broth with or without acetamide.

Figure 4

(A) Autoradiographs of two-dimension silver TLC plates used to separate [¹⁴C]-labelled fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) extracted from the ∆desA2 mutant. Ac; acetamide (B) Densitometric quantification of mycolic acid subspecies shown in (A). Comparative values shown for each subspecies, with those for − Ac cultures expressed as a percentage of those from + Ac cultures. (C) ESI–MS of MAMES extracted from the ∆desA2 mutant.

Figure 5

Autoradiographs of 2D-TLC analysis of apolar (Systems C, D) and polar lipids (System E) extracted from WT and ∆desA2 mutant. Solvent systems C, D and E are as described by Dobson et al.. Ac acetamide, TMM trehalose monomycolate, TDM trehalose dimycolate, PIM phosphatidyl inositol mannoside.

Figure 6

Autoradiograph of reverse phase TLC of [¹⁴C]-labelled fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) extracted from the ∆desA2 mutant. Ac acetamide.

Figure 7

Molecular species compositions of extractable fatty acid methyl esters (FAMEs) from ∆desA2 mutant as determined by GC. (A) GC analysis of the extracts from the ∆desA2 mutant. (B) Relative quantities of FAME peaks comparing cultures grown in the presence or absence of acetamide.