The seven Mycobacterium tuberculosis whiB-like genes encode small proteins postulated to be transcriptional regulators. A systematic real-time reverse transcription-PCR analysis following exposure to antibiotics and a variety of growth and in vitro stress conditions indicates differential, and in some cases dramatic, transcription modulations for the different M. tuberculosis whiB family members. This information together with biochemical analyses of the whiB1 to whiB7 gene products will be important for understanding the biology of this novel family of proteins in mycobacteria and related actinomycetes.Upon infection, Mycobacterium tuberculosis has the ability to adapt to many different environments within the host organism. Tubercle bacilli are able to avoid immune system detection and persist inside the host for decades. Many of the conditions to which the bacteria are exposed, such as the acidic environment within the phagolysosomal compartment, are harsh (14). In the lung, there is a recruitment of activated macrophages to the infection site, and these, along with other immune cells, contain the infection by forming a tuberculous granuloma (49). M. tuberculosis is adept at surviving within the hypoxic and fatty acid-rich granulomatous environment (8), a facet critical to its ability to persist despite immune pressure (25,28). Moreover, tubercle bacilli can survive for long periods of exposure to low-nutrient conditions, such as may occur within a granuloma (4, 34), as well as temperature and oxidative stress which may act to disrupt the M. tuberculosis cell membrane. The bacterium also has a remarkable tolerance to a wide range of antibiotics (12,26). To survive, the organism must sense and respond to exogenous stress conditions. Hence, differential expression of transcriptional regulators, which control sets of genes that respond to environmental stimuli, is an important mechanism of stress survival.A family of genes which may be key transcriptional regulators in M. tuberculosis is the whiB gene family. The whiB-like genes are exclusive to the actinomycetes, such as Mycobacterium and Streptomyces spp., and are absent from all other organisms studied thus far (9, 44). whiB was identified in Streptomyces coelicolor as an essential gene for sporulation of aerial hyphae (9), and early sporulation genes were identified by morphological studies of a collection of mutants unable to form normal gray spore pigment (17). Because their aerial mycelium remained white upon prolonged incubation, these mutants were designated whi mutants. S. coelicolor whiB encodes an 87-amino-acid polypeptide with attributes suggesting that it may be a DNA binding protein (9). S. coelicolor WhiB and its related homologues, including S. coelicolor WhiD, each contain four cysteine residues, a feature common in metalcoordinating DNA binding proteins (33). Indeed, a recent study has found that Sc WhiD binds a [4Fe-4S] cluster under anaerobic conditions similar to the situation with Escherichia coli DNA binding regulators SoxR and Fnr (6,10,...

The unique PE/PPE multigene family of proteins occupies almost 10% of the coding sequence of Mycobacterium tuberculosis (M.tb), the causative agent of human tuberculosis. Although some members of this family have been shown to be involved in pathways essential to M.tb pathogenesis, their precise physiological functions remain largely undefined. Here, we investigate the roles of the conserved members of the ‘PE only’ subfamily Rv0285 (PE5) and Rv1386 (PE15) in mediating host-pathogen interactions. Recombinant Mycobacterium smegmatis strains expressing PE5 and PE15 showed enhanced survival vs controls in J774.1 and THP-1 macrophages - this increase in viable counts was correlated with a reduction in transcript levels of inducible nitric oxide synthase. An up-regulation of anti- and down-regulation of pro-inflammatory cytokine levels was also observed in infected macrophages implying an immuno-modulatory function for these proteins. Induction of IL-10 production upon infection of THP-1 macrophages was associated with increased phosphorylation of the MAP Kinases p38 and ERK1/2, which was abolished in the presence of the pharmacological inhibitors SB203580 and PD98059. The PE5-PPE4 and PE15-PPE20 gene pairs were observed to be co-operonic in M.tb, hinting at an additional level of complexity in the functioning of these proteins. We conclude that M.tb exploits the PE proteins to evade the host immune response by altering the Th1 and Th2 type balance thereby favouring in vivo bacillary survival.

The wbl (whiB-like) genes encode putative transcription factors unique to actinomycetes. This study characterized the promoter element of one of the seven wbl genes of Mycobacterium tuberculosis, whiB1 (Rv3219c). The results reveal that whiB1 is transcribed by a class I-type cAMP receptor protein (CRP)-dependent promoter, harbouring a CRP-binding site positioned at "58?5 with respect to its transcription start point. In vivo promoter activity analysis and electrophoretic mobility shift assays suggest that the expression of whiB1 is indeed regulated by cAMP-dependent binding of CRP M (encoded by the M. tuberculosis gene Rv3676) to the whiB1 59 untranslated region (59UTR). b-Galactosidase gene fusion analysis revealed induction of the whiB1 promoter in M. tuberculosis on addition of exogenous dibutyric cAMP (a diffusible cAMP analogue) only when an intact CRP-binding site was present. These results indicate that M. tuberculosis whiB1 transcription is regulated in part by cAMP levels via direct binding of cAMP-activated CRP M to a consensus CRP-binding site in the whiB1 59UTR. INTRODUCTIONMycobacterium tuberculosis, the aetiological agent of tuberculosis, accounts for nearly 2 million human deaths every year. Approximately one-third of the world's population is latently infected with M. tuberculosis, and 7 to 8 million new TB cases occur annually (Dye et al., 1999). M. tuberculosis can survive in diverse surroundings ranging from the human host to droplet nuclei in the atmosphere. Adaptation to such diverse conditions requires controlled regulation of the expression of key genes that allow the bacillus to alter its physiology in response to changes in environmental stimuli. Sequencing of the M. tuberculosis genome has revealed the presence of more than 100 regulatory proteins, 13 sigma factors and 11 two-component systems (Cole et al., 1998), which provide the bacterium with a high degree of adaptability.The Wbl (WhiB-like) family of proteins is present throughout the actinomycetes but absent from all other organisms evaluated so far (Molle et al., 2000;Soliveri et al., 2000). Due to the presence of a conserved helix-turn-helix motif, these proteins are believed to function as DNA-binding transcription regulators. The first of these proteins, WhiB, was identified in Streptomyces coelicolor, a Gram-positive sporulating bacterium closely related to M. tuberculosis. 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 inv...

The rabbit model of tuberculosis is attractive because of its pathophysiologic resemblance to the disease in humans. Rabbits are naturally resistant to infection but may manifest cavitary lung lesions. We describe here a novel approach that utilizes presensitization and bronchoscopic inoculation to reliably produce cavities in the rabbit model. With a fixed inoculum of bacilli, we were able to reproducibly generate cavities by using Mycobacterium bovis Ravenel, M. bovis AF2122, M. bovis BCG, M. tuberculosis H37Rv, M. tuberculosis CDC1551, and the M. tuberculosis CDC1551 ⌬sigC mutant. M. bovis infections generated cavitary CFU counts of 10 6 to 10 9 bacilli, while non-M. bovis species and BCG yielded CFU counts that ranged from 10 4 to 10 8 bacilli. Extrapulmonary dissemination was almost exclusively noted among rabbits infected with M. bovis Ravenel and AF2122. Though all of the species yielded secondary lesions at intrapulmonary sites, M. bovis infections led to the most apparent gross pathology. Using the M. tuberculosis icl and dosR gene expression patterns as molecular sentinels, we demonstrated that both the cavity wall and cavity lumen are microenvironments associated with hypoxia, upregulation of the bacterial dormancy program, and the use of host lipids for bacterial catabolism. This unique cavitary model provides a reliable animal model to study cavity pathogenesis and extrapulmonary dissemination.Tuberculosis (TB) continues to be a global public health problem, with approximately one-third of the world's population latently infected (14). Nine million active infections are diagnosed annually, and approximately two million people died of the disease last year (33). Cavitary pulmonary lesions are key means of disease transmission (1, 21). The lesions present an ideal environment for tremendous bacillary growth, with up to 10 7 to 10 9 organisms being able to be routinely cultured from a single cavity (4). High titers of bacilli also lead to an increased likelihood of antimicrobial resistance due to spontaneous mutations (11).For these reasons, animal models of cavity formation and TB disease transmission have been sought. The murine model is limited because mice form cellular granuloma-like lesions which usually lack necrosis, caseation, and cavitation (16). Guinea pigs form necrotic granulomas with caseation but rarely produce cavitary lesions after aerosol Mycobacterium tuberculosis exposure (27). However, rabbits form caseating, necrotic granulomas which, under the correct conditions, may liquefy and cavitate.Early investigations by Wells and Lurie yielded cavity formation in rabbits presensitized with heat-killed M. bovis and challenged with aerosolized low-dose M. bovis (32). The importance of presensitization was further elucidated by Ratcliffe and Wells in their demonstration that more numerous cavities develop after reinfection with high-dose M. bovis following an initial primary low-dose aerosolized infection (23). Yamamura et al. reliably produced lung cavities in rabbits 30 to 60 days after in...

Methionine aminopeptidase (MetAP) is a metalloprotease that removes the N-terminal methionine during protein synthesis. To assess the importance of the two MetAPs in M. tuberculosis, we overexpressed and purified each of the MetAPs to near homogeneity and showed that both were active as MetAP enzymes in vitro. We screened a library of 175,000 compounds against MtMetAP1c and identified 2, 3-dichloro-1, 4-napthoquinone class of compounds as inhibitors of both MtMetAPs. It was found that the MtMetAP inhibitors were active against replicating and aged non-growing M. tuberculosis. Overexpression of either MtMetAP1a or MtMetAP1c in M. tuberculosis conferred resistance of bacterial cells to the inhibitors. Moreover, knockdown of MtMetAP1a, but not MtMetAP1c, resulted in decreased viability of M. tuberculosis. These results suggest that MtMetAP1a is a promising target for developing anti-tuberculosis agents.

The pathogenesis of Mycobacterium tuberculosis involves the coordinate action of multiple bacillary components that modulate host immune responses to ensure its survival. One such group of factors is the multigenic PE_PPE protein family, several members of which have been implicated in host immune evasion. Here we investigate the function of the PE-PPE gene pair PE35 (Rv3872)-PPE68 (Rv3873), located in the region of difference 1, encoding a specialized mycobacterial secretion system that is deleted in all vaccine strains of Mycobacterium bovis BCG. We report that this gene pair is co-operonic in M. tuberculosis, and demonstrate that its gene products interact with each other. Stimulation of THP-1 macrophages with recombinant PE35 and PPE68, singly or in combination, led to a dose-dependent increase in levels of the anti-inflammatory cytokine interleukin (IL)-10 and the chemokine monocyte chemoattractant protein-1, and caused a reciprocal decrease in levels of the proinflammatory cytokine IL-12. PE35/PPE68-stimulated production of IL-10 and monocyte chemoattractant protein-1 was observed to be dependent on toll-like receptor 2, as receptor blockade caused a significant reduction in their levels. Pharmacological inhibition indicated that this induction involved activation of the mitogen-activated protein kinase signalling axis. In a transwell migration assay, culture supernatants from PE35/PPE68-treated THP-1 cells were observed to stimulate the migration of monocytes. Our findings suggest that the PE35-PPE68 gene pair plays an important immunomodulatory role in regulating the pathophysiology of M. tuberculosis. Structured digital abstractTLR2 physically interacts with PPE68 by anti bait coimmunoprecipitation (View interaction) PE35 binds to PPE68 by pull down (View interaction) PE35 physically interacts with PPE68 by anti tag coimmunoprecipitation (View interaction) TLR2 physically interacts with PE35 by anti bait coimmunoprecipitation (View interaction) PPE68 and PE35 physically interact by dihydrofolate reductase reconstruction (View interaction)

A growing body of evidence suggests that the WhiB-like proteins exclusive to the GC-rich actinomycete genera play significant roles in pathogenesis and cell division. Each of these proteins contains four invariant cysteine residues and a conserved helix-turn-helix motif. whmD, the Mycobacterium smegmatis homologue of Streptomyces coelicolor whiB, is essential in M. smegmatis, and the conditionally complemented mutant M. smegmatis 628-53 undergoes filamentation under nonpermissive conditions. To identify residues critical to WhmD function, we developed a cotransformation-based assay to screen for alleles that complement the filamentation phenotype of M. smegmatis 628-53 following inducer withdrawal. Mycobacterium tuberculosis whiB2 and S. coelicolor whiB complemented the defect in M. smegmatis 628-53, indicating that these genes are true functional orthologues of whmD. Deletion analysis suggested that the N-terminal 67 and C-terminal 12 amino acid residues are dispensable for activity. Site-directed mutagenesis indicated that three of the four conserved cysteine residues (C 90 , C 93 , and C 99 ) and a conserved aspartate (D 71 ) are essential. Mutations in a predicted loop glycine (G 111 ) and an unstructured leucine (L 116 ) were poorly tolerated. The region essential for WhmD activity encompasses 6 of the 10 residues conserved in all seven M. tuberculosis WhiBs, as well as in most members of the WhiB family identified thus far. WhmD structure was found to be sensitive to the presence of a reducing agent, suggesting that the cysteine residues are involved in coordinating a metal ion. Iron-specific staining strongly suggested that WhmD contains a bound iron atom. With this information, we have now begun to comprehend the functional significance of the conserved sequence and structural elements in this novel family of proteins.Streptomyces coelicolor, a gram-positive, sporulating bacterium, is phylogenetically a close relative of Mycobacterium tuberculosis, with a similarly high GC content (65 to 70%). This organism follows a differentiation cycle in which young colonies send hyphal extensions into the agar and later-generation cells form white aerial hyphae which become pigmented and produce spores (4). Mutants which show an arrest in the development of mature spores and pigment formation remain white, and mutations leading to this phenotype have been described to map to eight independent loci (5, 12). The firstcharacterized nonsporulating mutant was shown to be defective in whiB, a gene encoding a basic polypeptide 87 amino acids long with a putative helix-turn-helix (HTH) motif (8). whiB mutants are viable but incapable of sporulating. Secondary-structure prediction and mutational analysis suggest that WhiB is a DNA binding protein (8). In addition, whiB mutants show reduced expression of the whiE cluster encoding spore pigment, indicating that this gene is likely to be a transcription factor.The large database of DNA sequences of members of the order Actinomycetales has revealed a family of genes encoding pro...

Toll-like receptor (TLR)-mediated interactions of Mycobacterium tuberculosis (M. tb) with macrophages are major determinant in the outcome of innate immune defence and subsequent adaptive immune responses. Here we report a novel interaction of the M. tb protein pair PE9 (Rv1088)-PE10 (Rv1089) with the macrophage TLR4 leading to apoptosis and modulation of cytokine levels. We demonstrate that the two proteins physically interact, and that PE9 is required for the cell wall localization of PE10 in Mycobacterium smegmatis. Interaction of the PE9-PE10 complex with TLR4 in THP-1 macrophages was associated with increased levels of phospho-IRF-3, which correlated with an increase in transcript levels of its target gene interferon-β. THP-1 macrophages treated with PE9-PE10 complex showed multiple hallmarks of apoptosis and modulation of interleukin (IL)-1b and IL-10 levels. All of these effects were abrogated when cells were treated either with an antibody to PE10 or an anti-TLR4 antibody, indicating that the complex specifically interacts with TLR4 through PE10, establishing this protein pair as a TLR4 ligand. This novel observation of two proline-glutamate (PE) proteins forming functional heterodimers represents a considerable expansion of the PE_PPE repertoire in the context of receptor engagement and the concomitant modulation of host responses by this unique class of proteins.