Mycobacterium bovis BCG vaccine strains lack narK2 and narX induction and exhibit altered phenotypes during dormancy

Abstract

Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that affects one-third of the world's population. The sole extant vaccine for tuberculosis is the live attenuated Mycobacterium bovis bacillus Calmette-Guerin (BCG). We examined 13 representative BCG strains from around the world to ascertain their ability to express DosR-regulated dormancy antigens. These are known to be recognized by T cells of M. tuberculosis-infected individuals, especially those harboring latent infections. Differences in the expression of these antigens could be valuable for use as diagnostic markers to distinguish BCG vaccination from latent tuberculosis. We determined that all BCG strains were defective for the induction of two dormancy genes: narK2 (Rv1737c) and narX (Rv1736c). NarK2 is known to be necessary for nitrate respiration during anaerobic dormancy. Analysis of the narK2/X promoter region revealed a base substitution mutation in all tested BCG strains and M. bovis in comparison to the M. tuberculosis sequence. We also show that nitrate reduction by BCG strains during dormancy was greatly reduced compared to M. tuberculosis and varied between tested strains. Several dormancy regulon transcriptional differences were also identified among the strains, as well as variation in their growth and survival. These findings demonstrate defects in DosR regulon expression during dormancy and phenotypic variation between commonly used BCG vaccine strains.

FIG. 1.

Induction of narK2 and narX transcription by nitric oxide. Quantitative reverse transcription-PCR was used to quantify transcript levels reported at copy number per genome of narK2, narX, and Rv2626 (as a positive control for general dormancy regulon gene expression). Bars: ▪, M. tuberculosis; ▧, M. bovis; ░⃞, BCG Connaught Jpg; ▩, BCG Pasteur 140.

FIG. 2.

Alignment of narK2 promoter region. The sole variable sequence throughout the 500-bp promoter narK2/narX promoter region is shown boxed, with other features labeled. All BCG strains in the present study and M. bovis were identical in promoter region sequence, while the M. tuberculosis sequence and the BCG sequence reported by Hutter and Dick differed (17).

FIG. 3.

Growth in the anaerobic dormancy model. (A) Sealed tubes were opened at various time points throughout the anaerobic dormancy model and sampled for CFU. (B) The optical density at 600 nm was measured without opening the sealed tubes. M. tuberculosis, M. bovis, and BCG Pasteur 140 increased rapidly in optical density and then stabilized or decreased slightly, while the other strains maintained a slow relatively steady increase in optical density throughout the model. Symbols: •, M. tuberculosis; ○, M. bovis; ▪, Pasteur 140; □, Connaught; ⧫, Canada; ⋄, Vietnam; ▴, Russia; ▵, Brazil.