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. 2002 Mar 5;99(5):3147-52.
doi: 10.1073/pnas.052705399.

Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth

Adrie J C Steyn  1 , Desmond M Collins, Mary K Hondalus, William R Jacobs Jr, R Pamela Kawakami, Barry R Bloom
Affiliations

Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth

Adrie J C Steyn et al. Proc Natl Acad Sci U S A. .

Abstract

Previous work established that the principal sigma factor (RpoV) of virulent Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, restores virulence to an attenuated strain containing a point mutation (Arg-515-->His) in the 4.2 domain of RpoV. We used the 4.2 domain of RpoV as bait in a yeast two-hybrid screen of an M. tuberculosis H37Rv library and identified a putative transcription factor, WhiB3, which selectively interacts with the 4.2 domain of RpoV in virulent strains but not with the mutated (Arg-515-->His) allele. Infection of mice and guinea pigs with a M. tuberculosis H37Rv whiB3 deletion mutant strain showed that whiB3 is not necessary for in vivo bacterial replication in either animal model. In contrast, an M. bovis whiB3 deletion mutant was completely attenuated for growth in guinea pigs. However, we found that immunocompetent mice infected with the M. tuberculosis H37Rv whiB3 mutant strain had significantly longer mean survival times as compared with mice challenged with wild-type M. tuberculosis. Remarkably, the bacterial organ burdens of both mutant and wild-type infected mice were identical during the acute and persistent phases of infection. Our results imply that M. tuberculosis replication per se is not a sufficient condition for virulence in vivo. They also indicate a different role for M. bovis and M. tuberculosis whiB3 genes in pathogenesis generated in different animal models. We propose that M. tuberculosis WhiB3 functions as a transcription factor regulating genes that influence the immune response of the host.

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Figures

Figure 1
Figure 1
(A) General diagram and alignment illustrating the C-terminal amino acids of RpoV (SigA) used as bait in the yeast two-hybrid system. Indicated are amino acids (vertical arrows) that affect activation by the corresponding transcription factors. The single point mutation (R515→H) causing attenuation of an MTB complex strain is circled. Mtb, M. tuberculosis; Mlep, M. leprae; Msm, M. smegmatis; Ren, Renibacterium salmoninarum; Sgris, Streptomyces griseus; Coryn, Corynebacterium ammoniagenes; Ecoli, E. coli. (B) Alignment of the most conserved regions of WhiB3-related proteins. Indicated are the four Cys residues (filled dots). A single amino acid substitution that abolishes sporulation in S. coelicolor, (Leu→Pro) is also indicated. S. coelicolor [ScoelA (T35596), ScoelB (CAC36616.1)], M. leprae [MlepA (CAC30314.1), MlepB (CAC31823.1)], M. tuberculosis CDC1551 (MtbCDC). Msm, M. smegmatis; Rhod, Rhodococcus opacus; TM 4 mycobacteriophage TM 4, gp49; +, positively charged residues; −, negatively charged residues. (C) The effect of the R515→H mutation on the interaction between RpoV and WhiB3 using the yeast two-hybrid system. S. cerevisiae PJ69–4α and S. cerevisiae PJ69–4A were transformed separately with pWB1 and the corresponding bait plasmids and mated. A heavy suspension of diploid cells were streaked out on SC lacking Ade and His and containing 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside, incubated at 30°C, and photographed 4 days later. (1) S. cerevisiae [pWB1/pRpoV54]. (2) S. cerevisiae [pWB1/pRpoVR515H]. (3) S. cerevisiae [pLAM5′/pRpoV54]. (4) S. cerevisiae [pLAM5′/pRpoVR515H]. The control plasmid pLAM5′ contains the unrelated human lamin C protein fused to the DNA-binding domain.
Figure 2
Figure 2
In vitro interaction of immobilized WhiB3(his) with GST-RpoV using SELDI-TOF. Proteins were analyzed on H4 or IMAC-3 chips. (A) Purified GST-RpoV analyzed on a hydrophobic H4 chip. (B) The eluate analyzed on an H4 chip. The hydrophobic surface of the H4 chip preferentially captured GST-RpoV but not WhiB3(his) (laser intensity 270; sensitivity 9). Because the matrix solution disrupts noncovalent complexes, each protein is represented by a separate peak. Intriguingly, the apparent m/z of GST-RpoV in the eluate was altered slightly but consistently. (C) Analysis of the eluate on an IMAC-3 chip. The eluate was dialyzed against PBS to remove the imidazole, incubated on the IMAC-3 surface for 30 min to allow binding of WhiB3(his) to the chelated Ni2+, and washed with sterile water before analysis. Under these conditions, GST-RpoV did not bind to the IMAC-3 surface and was not observed in the spectra. Peaks corresponding to WhiB3(his) were observed by using a laser intensity of 240 and a sensitivity of 9. Mass identification was made by 100 average shots with the Ciphergen PBS II ProteinChip reader. Note the double-charged ions (+2H) in A and B. External calibration was performed as suggested by the manufacturer.
Figure 3
Figure 3
Disruption of the H37Rv and M. bovis ATCC35723 whiB3 loci. (A) Southern blot analysis of H37Rv parental (lanes 4 and 8) and two hygromycin resistant clones, WH1 (lanes 2 and 6) and WH2 (lanes 3 and 7). A 974-bp NcoI/StuI fragment adjacent to whiB3 was used to probe NcoI- (lanes 2–4) and BsmBI- (lanes 6–8) digested chromosomal DNA separated on a 0.8% agarose gel. Lanes 1 and 5 contain a 1-kb DNA ladder (New England Biolabs). (B) Southern blot analysis of wt M. bovis ATCC35723 (lane 1), an M. bovis hygromycin-resistant clone (lane 2), and marker DNA (lane 3). Chromosomal DNA in lanes 1 and 2 were digested with BsmB1. The marker DNA corresponds to a DNA fragment size of 1,650 bp.
Figure 4
Figure 4
Disruption of H37Rv whiB3 does not affect organ burden during the acute and persistent phases of infection in mice and guinea pigs, whereas M. bovis whiB3 is required for in vivo growth in guinea pigs. (A) Bacterial burden in the lung (1), liver (2), and spleen (3) of C57BL/6 mice intravenously infected with 106 cfu of H37Rv and MTwb3H. Bacterial burdens in the spleens of guinea pigs (six per group) infected with H37Rv, MTwb3H, M. bovis ATCC35723, and MBwb3H (4). The results for each time point are the means and SDs of four mice per experimental group. Guinea pigs were killed 3 weeks after infection. (B) Disruption of H37Rv whiB3 significantly prolongs host survival. C57BL/6 mice (9–10 per group) were infected intravenously with 106 cfu of the indicated strain.
Figure 5
Figure 5
Cellular infiltration in the lungs of H37Rv- (A) and MTwb3H- (B) infected C57BL/6 mice. Mice were infected with 106 cfu H37Rv or MTwb3H cells, and organs were harvested at day 126, fixed in formalin, embedded and stained with hematoxylin and eosin. Magnification 100×. Spleens from guinea pigs infected with MBwb3H (C) and M. bovis ATCC35723 (D). Note the presence of multiple lesions (arrows) in the M. bovis ATCC35723-infected spleen.
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