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

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-5153 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-5153 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.T he increased susceptibility of HIV-infected individuals and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) results in the death of 2-3 million people each year (1) and underscores the urgency of deciphering the molecular mechanisms of virulence of this pathogen. The highly variable protective efficacy of Mycobacterium bovis bacillus Calmette-Guérin in adults (0-80%; ref. 2) emphasizes the urgency for developing second-generation antituberculosis antimicrobial agents and vaccines. With these aims in mind, research stimulated by the advances in mycobacterial genetics (3, 4) has led to the identification of several genes that have been implicated in virulence (5-12).MTB requires sophisticated genetic mechanisms to recognize appropriate environmental signals and to convey this information to the transcriptional apparatus of the organism. The activation of bacterial sigma factors to regulate gene expression is an effective response mechanism that enables pathogens to respond instantly to a multitude of environmental signals. Bacterial 70 -type sigma factors are composed of four major regions, called regions 1, 2, 3 and 4 (13). Region 4 is subdivided further into sub regions 4.1 and 4.2; the latter is known to interact with the Ϫ35 region of promoters (13) and other transcription factors. Mutations in or close to the helix-turn-helix (HTH) motif in region 4.2 can result in either positive or negative effects on activation by transcri...

MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

“…The mycobacterial growth rate in guinea pigs is not the only indicator of virulence, and studies have shown that strains with no differences in in vivo growth rates can produce infections that differ significantly by histopathological analysis 46,47 . Despite the reduced bacterial loads, histopathological assessments revealed that lung sections from guinea pigs infected with DmazF3D9D6 exhibited higher degrees of granulomatous inflammation than guinea pigs infected with the parental strain (Fig.…”

Regulation of the expression of whiB1 in Mycobacterium tuberculosis: role of cAMP receptor protein

“…S. coelicolor whiB mutants produce abnormally long, tightly coiled aerial hyphae that are completely blocked in their ability to form sporulation septa (Chater, 1972;Davis & Chater, 1992;Flardh et al, 1999). Studies of whiB orthologues in mycobacteria have shown that the M. smegmatis whiB2 gene (also called whmD) is essential ; M. tuberculosis whiB3 plays a role in virulence and its gene product may interact with a sigma factor of RNA polymerase (Steyn et al, 2002); whiB7 of M. tuberculosis is involved in multi-drug resistance (Morris et al, 2005). Each of the Wbl family of proteins contains four invariant cysteine residues, which are believed to be involved in binding a [4Fe-4S] cluster (Jakimowicz et al, 2005).…”

Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR

“…It is worthy to note that the results of these studies revealed that the end of the in vivo exponential growth phase of M. tuberculosis in the lungs of infected mice coincides with the bacillary load-independent downregulation of all the host genes studied. Recently, characterization of the M. tuberculosis WhiB3 mutant has provided evidence that the tubercle bacillus can profoundly affect the host immune response independent of tissue bacillary load (Steyn et al, 2002). The significance and specificity of the downregulation of host gene expression observed at 25 days post infection in the present study can be better assessed using the microarray technology.…”