bovisBCG (black bar). in both Mtb andM. bovisBCG, but broadly affected cAIG regulation only inM. bovisBCG. These studies identify Cmr as a transcription factor that regulates cAIGs within macrophages, and suggest that multiple factors affect cAMP-associated gene regulation in TB-complex mycobacteria. cAMP signaling and Cmr-mediated gene regulation during Mtb contamination of macrophages may have implications for TB pathogenesis. Keywords:Mycobacterium tuberculosis,M. bovisBCG, cyclic AMP (cAMP), Rv1675c (cmr), gene regulation, macrophages == Introduction == Tuberculosis (TB) is usually a serious global health problem, with one third of the worlds Sibutramine hydrochloride populace currently infected withMycobacterium tuberculosis(Mtb), and a new case rate of Sibutramine hydrochloride approximately 8 million per year (WHO Report 2007,http://www.who.int/tb/en/). Contamination is further complicated by increasing levels of drug resistance and a deadly synergy with human immunodeficiency computer virus (HIV) (Nunnet al., 2005). A better understanding of the mechanisms by which Mtb senses and responds to its environment during contamination may contribute to the identification of improved antitubercular drug candidates. cAMP has recently been shown to be a global regulator of gene expression in Mtb, and it is likely to play a role in Mtb-host interactions, including virulence (Agarwalet al., 2006;Gazdik and McDonough, 2005;Lowrieet al., 1975;Lowrieet al., 1979;Rickmanet al., 2005). A wide range of cellular responses are controlled by cAMP levels in both prokaryotic and eukaryotic cells (Botsford and Harman, 1992;Peterkofskyet al., 1993;Tang and Hurley, 1998). cAMP signaling is also critical for virulence in a number of protozoan, fungal and bacterial pathogens (Alspaughet al., 1997;Alspaughet al., 2002;Grosset al., 2003;Liebmannet al., 2003;Petersen and Young, 2002). In particular, the recent identification of cAMP as a regulator of the virulence-associated plasminogen activator gene,pla, inY. pestis(Kimet al., 2007), and type III secretion systems in bothPseudomonus aeruginosaandYersinia enterocolitica(Petersen and Small, 2002;Smithet al., 2004), suggests that the importance of cAMP signaling to bacterial pathogenesis is usually greater than previously acknowledged. The classical model for cAMP regulation in prokaryotes is based on the well characterized cAMP response inEscherichia coli(Botsford and Harman, 1992).E. colicontains a single class I adenylate cyclase (AC) which catalyzes the conversion of ATP to cAMP (Barzu and Danchin, 1994;Botsford and Harman, 1992). The cAMP signal is usually transduced inE. coliby the cAMP receptor protein (CRP). CRP undergoes a conformational change upon binding cAMP, which activates it as a transcription factor (Botsford and Harman, 1992). By comparison, the Mtb genome contains 15 putative class III ACs (McCueet al., 2000), 10 of which have confirmed biochemical activity (Abdel Motaalet al., 2006;Castroet al., 2005;Guoet al., 2001;Linderet al., 2002;Reddyet al., 2001;Shenoy and Visweswariah, 2006;Sinhaet al., 2005;Tewset al., 2005).M. bovishas a similar number of cyclase genes, but the genomes of other Actinobacteria, includingCorynebacteriumandStreptomyceseach contain only a single adenylyl cyclase gene (Shenoyet al., 2004). The surprisingly high number of ACs suggests a large role for cAMP signal transduction in Mtbs gene regulation. The cAMP signaling paradigm in Mtb is also likely to be far more complex than the classicalE. colimodel, due to this large number and diversity of AC proteins. However, very little is currently known about cAMP signaling pathways in Mtb. Two of Mtbs ten predicted nucleotide binding proteins, CRPMt(also called Rabbit Polyclonal to GPR175 Rv3676) and Rv1675c (referred to hereafter as Cmr, forcAMP andmacrophageregulator), were initally classified as members of the CRP/FNR transcriptional regulatory factor family, based on in silico analyses (McCueet al., 2000). Deletion ofcrpattenuates Mtb virulence in a murine model, although in vitro growth of these mutants is also reduced (Rickmanet al., 2005). CRPMts DNA and cAMP binding properties have recently been experimentally confirmed, and we identified a potential CRPMtregulon of 114 members (Baiet al., 2005;Baiet al., 2007). We also separately identified a set of five cAMP-induced genes (cAIGs) in Mtb using an exogenous cAMP supplementation model (Gazdik and McDonough, 2005). However, none of these cAIGs belongs to the predicted CRPMtregulon. In this study we show that four cAIGs are regulated by Mtbs second predicted CRP-like protein, Cmr. Sibutramine hydrochloride Elevated cAMP levels and intramacrophage conditions were identified as key cAIG regulatory signals in both virulent Mtb and attenuatedM. bovisBCG. == Results == == cmris required for regulation of cAMP-induced protein expression == We reasoned that a.
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