Identification of Cyclic AMP-Regulated Genes in
            <i>Mycobacterium tuberculosis</i>
            Complex Bacteria under Low-Oxygen Conditions

Gazdik, Michaela A.; McDonough, Kathleen A.

doi:10.1128/jb.187.8.2681-2692.2005

Cited by 73 publications

(94 citation statements)

References 63 publications

(89 reference statements)

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“…It is postulated that secreted cAMP may prevent phagolysosome formation and thereby protect bacilli from host-mediated destruction (Lowrie et al, 1975(Lowrie et al, , 1979. Recently, it has been observed that exogenous cAMP induced the expression of several biologically significant genes in M. bovis BCG, thus implicating cAMPmediated regulation of gene expression as an important mechanism in the M. tuberculosis complex (Gazdik & McDonough, 2005).…”

Section: Discussionmentioning

confidence: 99%

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

2006

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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...

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Section: Discussionmentioning

confidence: 99%

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

2006

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“…Complex effects of modulating cAMP levels in mycobacterium-infected macrophages have been described in many studies making it difficult to provide a simple model (e.g. Gazdik and McDonough, 2005;Yadav et al, 2004;Roach et al, 2005;Schorey and Cooper, 2003). It is even conceivable that mycobacteria can make and secrete cAMP into the phagosomal lumen, as suggested from earlier studies (Lowrie et al, 1975).…”

Section: Discussionmentioning

confidence: 99%

cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria

Kalamidas

Kuehnel

Peyron

et al. 2006

Journal of Cell Science

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We showed recently that actin assembly by phagosomal membranes facilitates fusion with late endocytic organelles in macrophages. Moreover, lipids that induced phagosomal actin also stimulated this fusion process. In macrophages infected with pathogenic mycobacteria actin-stimulatory lipids led to an increase in pathogen destruction, whereas inhibitors facilitated their growth. A model was proposed whereby phagosomal membrane actin assembly provides tracks for lysosomes to move towards phagosomes, thereby facilitating fusion. Here, we investigated how cAMP affected phagosomal actin assembly in vitro, and phagosomal actin, acidification and late fusion events in J774 macrophages. Latex bead phagosomes are shown to possess adenylyl cyclase activity, which synthesizes cAMP, and phosphodiesterase activity, which degrades cAMP. The system is regulated by protein kinase A (PKA). Increasing cAMP levels inhibited, whereas decreasing cAMP levels stimulated, actin assembly in vitro and within cells. Increasing cAMP levels also inhibited phagosome-lysosome fusion and acidification in cells, whereas reducing cAMP had the opposite effect. High cAMP levels induced an increase in intraphagosomal growth in macrophages of both the non-pathogenic Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis, whereas low cAMP levels or inhibition of PKA correlated with increased bacterial destruction. We argue that the phagosome cAMP-PKA system behaves as a molecular switch that regulates phagosome actin and maturation in macrophages.

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“…In addition to its role as a heat-shock protein, GroEL functions as a chaperonin to assist folding of linear amino acid chains into their respective three-dimensional structure (65). Cyclic-AMP-mediated up-regulation of GroEL2 under low-oxygen/CO 2 -enriched growth conditions was observed by Gazdik and McDonough (2005) (66). A previous study from our laboratory showed that GroEL2 promoter of MTB is highly active in M. smegmatis when subjected to different stress conditions including hypoxia (65).…”

Section: Validation Of Quantitative Proteomic Data By Qpcr-tomentioning

confidence: 99%

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

Gopinath

Raghunandanan

Gomez

et al. 2015

Molecular & Cellular Proteomics

114

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Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free onedimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post reaeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to

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Identification of Cyclic AMP-Regulated Genes in Mycobacterium tuberculosis Complex Bacteria under Low-Oxygen Conditions

Cited by 73 publications

References 63 publications

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

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

cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

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