The Mycobacterium tuberculosis genome contains four copies of an operon called mce (mce1-4). Previously we reported that M. tuberculosis disrupted in the mce1 operon is more virulent than wild-type M. tuberculosis in mice. We generated single deletion mutants in mce3 (Dmce3) and mce4 (Dmce4) operons and a double deletion mutant (Dmce3/4). Similar doubling times and growth characteristics were observed for all mutants and the wild-type (parent) M. tuberculosis H37Rv strain in culture and in macrophages. In addition, similar bacterial burdens were detected in organs from mice infected with Dmce3 and the parent strain. However, the bacterial burdens of mice infected with Dmce4 and Dmce 3/4 were less than those of mice infected with the parent strain. The median survival times of mice infected with wild-type M. tuberculosis, Dmce3, Dmce4 and Dmce3/4 were 40.5, 46, 58 and 62 weeks, respectively. Histopathological examination of lungs at 15 weeks post-infection showed that the extent of the lung lesions was less prominent in mice infected with Dmce4 and Dmce 3/4 mutants than in mice infected with the other two strains. These observations suggest that the mce3 and mce4 operons have a role distinct from that of mce1 for in vivo survival of M. tuberculosis.

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-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 wild-type, and the mutant never attained the plateau phase of infection in the mouse lungs. The steady-state infection phase was maintained for up to 168 days at a level one to two logs less than that shown by wild-type. These observations raise a rather intriguing possibility that FadD5 may serve to recycle mycolic acids for the long-term survival of the tubercle bacilli.

Binding of the complement inhibitor factor H (fH) to the surface of Neisseria meningitidis is critical for evasion of innate host defenses. The meningococcal vaccine candidate factor H-binding protein (fHbp) serves as an fH ligand. We prepared 16 recombinant fHbp natural sequence variants. By enzyme-linked immunosorbent assay (ELISA), the variants from a New Zealand epidemic strain (fHbp ID 14) and from an endemic United Kingdom strain (ID 15) showed 10-fold lower fH binding than a reference fHbp from an epidemic Norwegian strain (ID 1). By surface plasmon resonance, association rate constants (k a ) for fHbp ID 14 and 15 were similar to those for ID 1, but dissociation rate constants (k d ) were 4-to 10-fold higher than those for ID 1. To determine the effect of fH affinity on fHbp fitness, we prepared isogenic mutants of strain H44/76 that expressed fHbp ID 1, 14, or 15. By flow cytometry, mutants expressing fHbp ID 14 or 15 had lower fH binding than ID 1. When incubated in plasma or blood of nonimmune donors, all three mutants showed similar increases in CFU/ml. In contrast, an isogenic fHbp knockout mutant, which grew well in broth, was rapidly killed in plasma or blood. Thus, although fHbp expression was required for survival of strain H44/76 in blood or plasma, expression of two natural fHbp sequence variants with lower fH affinity had minimal or no effect on nonimmune clearance. One reason may be the high fH concentrations in normal serum, which favor saturation of fH binding to fHbp, even when dissociation rates varied over 10-fold.