Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways with their mammalian hosts during infection. In this study, cAMP levels associated with M. tuberculosis and M. bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased approximately 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of TB-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.

Mycobacterium tuberculosisTuberculosis (TB) remains a global epidemic, with one-third of the world's population infected, 9 million active cases, and over 2 million deaths annually (3). Increased drug resistance and a lethal synergism with human immunodeficiency virus exacerbate the morbidity and mortality already associated with Mycobacterium tuberculosis infection (7, 9). The molecular pathogenesis of M. tuberculosis is poorly understood, and better characterization of the mechanisms by which the tubercle bacillus responds to its host environment is needed if we are to control this deadly infection.Cyclic AMP (cAMP), generated by adenylate cyclase, is an important signaling molecule in many bacterial species and eukaryotic cells. cAMP-mediated gene regulation in Escherichia coli, a process which requires interaction with the cAMP receptor protein (CRP), has been well defined (16). Additional roles for cAMP in metabolism have been reported for many pathogens (2,6,8,13,18,19,23,30,36), and recent studies increasingly point to an important role for cAMP signaling in microbial pathogenesis. Vfr, a CRP homolog in Pseudomonas aeruginosa, coordinates the expression of a variety of virulenceassociated factors including type IV pili and the type III secretion system (35). Mutation of a cyclase-encoding gene (cya) in Vibrio vulnificus resulted in attenuation of the bacterium, with a 100-fold increase in the 50% lethal dose for mice (15). Pathogenic fungi also use the highly conserved cAMP signal transduction pathway to regulate cellular differentiation and pathogenesis (20,21,31).The potential significance of cAMP signaling in M. tuberculosis pathogenesis was first explored in an early study that reported elevated levels of cAMP in the cytoplasms of macrophages infected with M. microti (24). Those authors correlated such increased cAMP levels with impaired phagosome-lysosome fusion. However, little more work was done on this topic until an in silico study identified genes for 15 adenylate cyclases and 10 cNMP-binding proteins in the M. tuberculosis genome (27). At least 10 of these adenylate cyclases are functional, as demonstrated by in vitro biochemical assays (33). It was recently shown that cAMP signaling is likely to be an important global regulator of gene regulation in M. tuberculosis and also that Rv3676 (CRP Mt ) is a CRP-like cAMP-responsive transcription factor with a putative regulon of more than 100 genes (4,32,34). Deletion of Rv3676 caused impaired growth in laboratory medium, in bone marrow-derived macrophages, and in a mouse model of TB (32).CRP Mt and its ortholog in M. bovis are identical. However, the Mycobacterium bovis BCG Pasteur ortholog, which we refer to as CRP BCG , contains two amino acid differences, L47P and E178K, relative to the CRP Mt sequence. It was recently reported that the L47P change resulted in loss of cAMP binding and that the E178K substitution caused a DNA binding defect in an E. coli model system (34). The authors proposed a role for CRP BCG in M. bovis BCG's attenuation based...

Summary Mycobacterium tuberculosis CRPMt, encoded by Rv3676 (crp), is a CRP-like transcription factor that binds with the serC – Rv0885 intergenic region. In the present study, we evaluated CRPMt’s regulation of serC and Rv0885 in M. tuberculosis and M. bovis BCG, using site-specific mutagenesis, promoter fusions and RT-PCR. The CRPMt binding site was required for full expression of serC and Rv0885, and expression of both genes was reduced in M. tuberculosis and M. bovis BCG crp mutants. These data show that CRPMt binding directly activates both serC and Rv0885 expression. M. tuberculosis serC restored the ability of an Escherichia coli serC mutant to grow in serine-dropout medium, demonstrating that M. tuberculosis serC encodes a phosphoserine aminotransferase. Serine supplementation, or overexpression of serC, accelerated the growth of M. tuberculosis and M. bovis BCG crp mutants in mycomedium, but not within macrophages. These results establish a role for CRPMt in the regulation of amino acid biosynthesis, and show that reduced serine production contributes to the slow-growth phenotype of M. tuberculosis and M. bovis BCG crp mutants in vitro. Restoration of serine biosynthesis by serC expression will facilitate identification of additional CRPMt-regulated factors required by M. tuberculosis during macrophage and host infection.