Attenuation of Mycobacterium tuberculosis functionally disrupted in a fatty acyl-coenzyme A synthetase gene fadD5

Abstract

One key adaptation that Mycobacterium tuberculosis established to survive long term in vivo is a reliance on lipids as an energy source. M. tuberculosis H37Rv has 36 fadD genes annotated as putative fatty acyl-coenzyme A (CoA) synthetase genes, which encode enzymes that activate fatty acids for metabolism. One such gene, fadD5 (Rv0166), is located within the mce1 operon, a cluster of genes associated with M. tuberculosis persistence. We disrupted the putative fatty acid-binding site of fadD5 in H37Rv M. tuberculosis. No significant differences were found in the growth of the mutant and wild-type strains in vitro in nutrient-rich broth or in activated RAW264.7 cells. However, the fadD5 mutant was diminished in growth in minimal medium containing mycolic acid but not other long-chain fatty acids. C57BL/6 mice infected with the fadD5 mutant survived significantly longer than those infected with the wild type, and the mutant never attained the plateau phase of infection in mouse lungs. Infection in the steady-state phase was maintained for up to 168 days at a level that was 1-2 logs less than that noted in the wild type. These observations raise the rather intriguing possibility that FadD5 may serve to recycle mycolic acids for the long-term survival of the tubercle bacilli.

Fig. 1. The fadD5 gene in the mce1 operon and its comparison to FACS consensus sequence

(A) Position of the Rv0166 fadD5 gene within the mce1 operon of the M. tuberculosis H37Rv genome. (B) Alignment of the FACS signature motif (as defined by Black, P.N., et al., 1997) of E. coli and M. tuberculosis FadD enzymes. Among the 25 amino acid residues characterizing the signature motif, 13 are highly conserved and 8 are invariant (underlined). Dashes indicate the absence of a homologous residue, and residues in bold reflect the in-frame substitutions created in the M. tuberculosis fadD5 mutant strain.

Fig. 2. Southern blot analysis of the fadD5 mutant strain and growth kinetics in standard medium

(A) Restriction digestion sites within and surrounding fadD5 genomic region. The 5’ and 3’ probes (black boxes) represent the regions recognized in the Southern blot analysis of H37Rv WT (top) and fadD5 mutant (bottom) genomic DNA. (B) Southern blot analysis of the WT (lane 2) and fadD5 mutant genomic DNA (lane 3) cleaved by the restriction enzyme, KpnI. The lanes are flanked by the digoxigenin-labeled molecular mass standards, II (lane 1) and VII (lane 4) (Roche Diagnostics). (C) Growth kinetics of the WT, fadD5 mutant, and fadD5 complemented strains in 7H9 medium. Cultures were inoculated in triplicate at O.D.₅₈₀ 0.004 and optical densities were measured at 1,4,7,10, and 14 days post-infection.

Fig. 3. Growth of M. tuberculosis strains in a single carbon source medium

Growth kinetics of WT, fadD5 mutant, and fadD5 complemented strains in (A) complete Sauton’s medium (with glycerol), incomplete Sauton’s medium (without glycerol), and with (B) oleate, (C) palmitate, (D) caprylate, or (E) mycolic acid. Growth kinetics comparing WT and mce1 operon mutant in incomplete Sauton’s medium with (F) mycolic acid. The average of three O.D.₅₈₀ measurements per strain was plotted for each time point.

Fig. 4. Growth and cytokine induction of macrophage-like RAW264.7 cells infected with M. tuberculosis strains

(A) Recovery of WT, fadD5 mutant, and fadD5 complemented strains from macrophage-like RAW264.7 cells (n=3 per strain; m.o.i. 10:1) over a 72 hr period. Uninfected RAW cells served as a negative control, while LPS-infected RAW cells served as a positive control. At each time point, supernatants were filtered and collected for ELISA analysis. The production and release into the supernatant of (B) TNFα, (C) IL-6, and (D) MCP-1 were measured.

Fig. 5. Recovery of M. tuberculosis strains from infected C57BL/6 mouse lungs

Mice were infected with a dose of 95–128 bacilli per lung with WT, fadD5 mutant, and fadD5 complemented strains. Bacilli were recovered (n=4 per strain) at the indicated time points and enumerated on 7H11 agar plates.

Fig. 6. Survival kinetics of C57/BL6 mice infected with M. tuberculosis strains

Percent survival of mice after aerosol infection with the WT and complemented strains compared against fadD5-mutant infected mice (5 mice per group). Kaplan-Meier curve was generated by GraphPad Prism software (p<0.05 for comparison of mice infected with WT or complemented strain vs fadD5-infected mice, Mantel-Cox log-rank test).

Fig. 7. M. tuberculosis-infected gross mouse lungs and histology at 168 days p.i

Gross lung pathology of mice infected with WT (A), fadD5 mutant (B), or fadD5 complemented (C) strains. Each black bar represents 1.5mm. Histological sections of H&E-stained lungs of mice infected with (A) WT, (B) fadD5 mutant, or (C) fadD5 complemented strains. Magnified at x25 (middle figures) and x100 (bottom figures).