The nature of the mutation underlying attenuation of BCG has been a mystery since Calmette's first observations of his attenuated strain [26
]. During the last decade, associations have been made regarding the origin of BCG, mycobacterial virulence, and the RD1 region or gene(s) within it [6
]. Our data demonstrate that loss of RD1 affects MTB growth and survival in cultured macrophages and greatly alters mycobacterial growth and dissemination, as well as the resultant histopathology and survival of the mouse. In every analysis, the phenotype of H37Rv:ΔRD1 was distinctly different from that of virulent H37Rv and remarkably similar to that of BCG. These studies indicate that sequences within or controlled by the RD1 region are needed for MTB virulence and argue for the hypothesis that loss of RD1 was an important event in the creation of BCG.
The role of RD1 in virulence of MTB and attenuation of BCG is substantiated by the fact that completely independent H37Rv:ΔRD1 strains yielded the same results in cultured macrophage and mouse infection assays (data not shown). We also have deleted the RD1 region from MTB strain Erdman (ATCC 35801). In cultured macrophages, this strain is as attenuated as H37Rv:ΔRD1 (data not shown). However, thus far we have been unable to complement deletion of RD1 in the traditional fashion. For reasons that are unclear, the genes in the RD1 region expressed only very poorly after we restored the entire region into the phage L5 attachment site [28
] of H37Rv:ΔRD1 (data not shown). Complementation in trans
of a 9-gene deletion with a 12-kb DNA fragment is not straightforward. Efforts to reinstall the RD1 DNA in situ and to complement the ΔRD1 phenotypes with smaller portions of the RD1 region are underway.
Although clearly attenuated, BCG and H37Rv:ΔRD1 were able to both grow and persist in C57BL/6 mice. Although these mice are relatively resistant to MTB, they carry the Nramp1
allele, which confers sensitivity to BCG [20
]. Previously, BCG has been shown to grow in an Nramp
-susceptible mouse strain after aerosol infection [29
]. Furthermore, although the BCG-Russia strain that we used may be a more aggressive BCG [30
], we saw essentially identical growth in vivo after aerosol infection with BCG Pasteur (ATCC 35734; data not shown). As noted elsewhere [33
], growth and persistence in vivo and the ability to induce pathology are separate manifestations of mycobacterial virulence that can be dissociated under the proper conditions.
The mechanism by which the RD1 region exerts its effects remains to be elucidated. At present, our data highlight 2 broad areas for further study. First, impaired replication in cultured macrophages and in the first weeks after aerosol challenge of mice implies that H37Rv:ΔRD1 and BCG are defective in some aspect of metabolism or response to innate host defenses that is revealed by these environments. At the same time, continued replication of these strains in mice beyond the time when H37Rv is contained and the lack of histopathology in vivo suggest that H37Rv:ΔRD1 and BCG elicit a very different adaptive immune response than virulent MTB. Perhaps expression of immunogens encoded by RD1 [34
] at early times in vivo is necessary for induction of a powerful adaptive immune response.
It has been noted recently that ESAT-6 and CFP-10 form a tight 1:1 complex [38
] and that homologues are widespread among gram-positive bacteria [39
]. It was suggested that these proteins may contribute to a novel bacterial secretion system, with the energy for protein export supplied by a conserved, membrane-bound ATPase encoded nearby (Rv3870
in the RD1 region). This idea is attractive for 2 reasons. First, it provides a mechanism by which ESAT-6 and CFP-10, which lack signal sequences, may be exported from bacilli. Second, various secretion systems are essential for virulence of many other bacterial pathogens [41
]. Among other options, we are currently testing the hypothesis that RD1 deletion has interrupted a novel secretion system that is necessary for MTB virulence.
Regardless of its mechanism of action, analysis of RD1-deficient MTB could contribute to development of a better TB vaccine. The ability to multiply and persist in vivo may be necessary for a vaccine to stimulate lasting protective immunity against MTB [32
]. Because it derives from MTB and not M. bovis,
H37Rv:ΔRD1 should share more antigenic determinants with MTB, may persist in vivo longer than BCG, and may provoke a more protective immune response. Thus, continued focus on the RD1 region should provide novel insight into mechanisms of MTB pathogenesis, help elucidate the nature of a productive immune response, and may help stimulate much needed improvements in the TB vaccine.