Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guérin attenuation

Abstract

The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) is a live attenuated organism, but the mutation responsible for its attenuation has never been defined. Recent genetic studies identified a single DNA region of difference, RD1, which is absent in all BCG strains and present in all Mycobacterium tuberculosis (MTB) strains. The 9 open-reading frames predicted within this 9.5-kb region are of unknown function, although they include the TB-specific immunodominant antigens ESAT-6 and CFP-10. In this study, RD1 was deleted from MTB strain H37Rv, and virulence of H37Rv:DeltaRD1 was assessed after infections of the human macrophage-like cell line THP-1, human peripheral blood monocyte-derived macrophages, and C57BL/6 mice. In each of these systems, the H37Rv:DeltaRD1 strain was strikingly less virulent than MTB and was very similar to BCG controls. Therefore, it was concluded that genes within or controlled by RD1 are essential for MTB virulence and that loss of RD1 was important in BCG attenuation.

Figure 1

Bacterial growth and cytotoxicity in a human macrophage THP-1 cell line (A and B) and growth in human peripheral blood mononuclear cell (PBMC)–derived macrophages (C) after infection by H37Rv ([unk]), H37Rv:ΔRD1 (■), or BCG-Russia (▲). Data for each time point are the mean and SD of 4 infections in panels A and B and of a representative infection (of 4) in panel C. Bacteria were quantitated by luciferase assay (A) or by plating for colony-forming units (C). Cytotoxicity is indicated by the decline in macrophage metabolism over the course of infection (B).

Figure 2

Bacterial growth in lungs (A and B) and spleens (C and D) after low-dose aerosol infection of 6–8-week-old C57BL/6 mice with wild-type H37Rv ([unk]), H37Rv:ΔRD1 (■), or BCG-Russia (▲). A and C, Early-phase infection and dissemination. B and D, Progression of infection through 21 weeks. Data at each time point are the mean and SD of 15 mice per strain from 3 separate infections.

Figure 3

Lung histopathology after low-dose aerosol infection by H37Rv (A, D, and G), H37Rv:ΔRD1 (B, E, and H), or BCG-Russia (C, F, and I). At each time point, the left lung was removed, inflated, fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylineosin. Photographs shown are representative of at least 15 mice/strain. A, B, and C, Differences in overall lung involvement (original magnification, ×10) at week 6 after infection. D, E, and F, Differences in granuloma formation (original magnification, ×50; at week 6 after infection). D, Well-organized granuloma with tight rim of lymphocytes surrounding central core of histiocytes in a mouse infected with wild-type MTB. E and F, Areas of greatest inflammation in the H37:ΔRD1-infected (E) or BCG-Russia–infected (F) lung, consisting only of mild perivascular lymphocyte cuffs. G, H, and I, Differences in lung inflammation at week 21 after infection (original magnification, ×10).

Figure 4

Survival of C57BL/6 mice after high-dose aerosol infection with H37Rv or H37Rv:ΔRD1. Results are from 15 mice infected with an average dose of 316 H37Rv bacilli and from 18 mice infected with an average of 730 H37Rv:ΔRD1 bacilli.