cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.     

Identification of cyclic AMP-regulated genes in Mycobacterium tuberculosis complex bacteria under low-oxygen conditions

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which kills approximately 2 million people a year despite current treatment options. A greater understanding of the biology of this bacterium is needed to better combat TB disease. The M. tuberculosis genome encodes as many as 15 adenylate cyclases, suggesting that cyclic AMP (cAMP) has an important, yet overlooked, role in mycobacteria. This study examined the effect of exogenous cAMP on protein expression in Mycobacterium bovis BCG grown under hypoxic versus ambient conditions. Both shaking and shallow standing cultures were examined for each atmospheric condition. Different cAMP-dependent changes in protein expression were observed in each condition by two-dimensional gel electrophoresis. Shaking low-oxygen cultures produced the most changes (12), while standing ambient conditions showed the fewest (2). Five upregulated proteins, Rv1265, Rv2971, GroEL2, PE_PGRS6a, and malate dehydrogenase, were identified from BCG by mass spectrometry and were shown to also be regulated by cAMP at the mRNA level in both M. tuberculosis H37Rv and BCG. To our knowledge, these data provide the first direct evidence for cAMP-mediated gene regulation in TB complex mycobacteria.     

Comparative analysis of iron regulated genes in mycobacteria

Iron dependent regulator, IdeR, regulates the expression of genes in response to intracellular iron levels in M. tuberculosis. Orthologs of IdeR are present in all the sequenced genomes of mycobacteria. We have used a computational approach to identify conserved IdeR regulated genes across the mycobacteria and the genes that are specific to each of the mycobacteria. Novel iron regulated genes that code for a predicted 4-hydroxy benzoyl coA hydrolase (Rv1847) and a protease dependent antibiotic regulatory system (Rv1846c, Rv0185c) are conserved across the mycobacteria. Although Mycobacterium natural-resistance-associated macrophage protein (Mramp) is present in all mycobacteria, it is, as predicted, an iron-regulated gene in only one species, M. avium subsp. paratuberculosis. We also observed an additional iron-regulated exochelin biosynthetic operon, which is present only in non-pathogenic Mycobacterium, M. smegmatis.     

New messages from old messengers: cAMP and mycobacteria

Cyclic nucleotides are ancient second messengers, and the enzymes that synthesize cAMP and cGMP [cyclic nucleotide monophosphates (cNMPs)] are encoded in the genomes of several bacteria. We focus here on recent biochemical and structural information on the proteins that make and break cyclic nucleotides in mycobacteria, namely the nucleotide cyclases and phosphodiesterases, respectively. The presence of these enzymes along with putative cNMP-binding proteins suggests an intricate regulation of cAMP metabolism and utilization by these organisms. It is anticipated that future research will be directed towards identifying cellular processes that are regulated by cAMP in mycobacteria and deciphering the cross-talk between mycobacterial pathogens and their eukaryotic host.     

Comparison of cAMP receptor protein (CRP) and a cAMP-independent form of CRP by Raman spectroscopy and DNA binding.

The secondary structures of the cAMP receptor protein (CRP), a complex of CRP and cAMP, and a cAMP-independent receptor protein mutant (CRP*141 gln) were examined by using Raman spectroscopy. Spectra were obtained from CRP and CRP*141 gln dissolved in 0.3 M NaCl and 30 mM sodium phosphate at protein concentrations of 30-40 mg/mL. CRP and CRP.cAMP1 were compared at lower protein concentrations (10-12 mg/mL) in a solvent of 0.35 M NaCl and 20 mM sodium phosphate. Raman analysis indicates that CRP structural changes induced by one bound cAMP or by the Gly to Gln mutation at residue 141 are small. Spectra of the three CRP samples are essentially identical from 400 to 1900 cm-1. This result differs from the Raman spectroscopy study of CRP and CRP.cAMP2 cocrystals [DeGrazia et al. (1990) Biochemistry 29, 3557]. The latter work showed spectral differences between CRP and CRP.cAMP2 consistent with alterations in the protein conformation. These studies indicate that CRP and CRP.cAMP1 in solution are similar in structure and differ from CRP.cAMP2 cocrystals. Protease digestion and a DNA binding assay were also employed to characterize the wild-type and mutant proteins. CRP*141 gln exhibited the same conformational characteristics of previously reported cAMP-independent mutant proteins. It was sensitive to proteolytic attack in the absence of cAMP, or upon addition of cGMP. In the absence of cAMP, both wild-type and mutant CRPs bound noncooperatively to a 62 bp lac promoter DNA. The equilibrium constants were approximately 10(6) M-1 in 0.1 M Na+. CRP*141 gln had a 2-4-fold higher affinity for the 62 bp DNA than CRP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the synthesis of adenylate cyclase in Escherichia coli by the cAMP -- cAMP receptor protein complex

Summary The synthesis of the adenylate cyclase [ATP pyrophosphatelyase-(cyclizing), E.C. 4.6.1.1.] of Escherichia coli, appears to be regulated negatively by the cAMP receptor protein CRP. This conclusion is based on a comparison of adenylate cyclase activities measured in vitro with the rates of cAMP synthesis by intact bacteria. The activity of adenylate cyclase, depending on conditions of growth, is also regulated by CRP; this effect, however, is indirect insofar as it is mediated by a protein or proteins under CRP control.     

Ligand-modulated binding of a gene regulatory protein to DNA. Quantitative analysis of cyclic-AMP induced binding of CRP from Escherichia coli to non-specific and specific DNA targets

This paper describes a generally applicable method for quantitative investigation of ligand-dependent binding of a regulatory protein to its target DNA at equilibrium. It is used here to analyse the coupled binding equilibria of cAMP receptor protein from Escherichia coli K12 (CRP) with DNA and the physiological effector cAMP. In principle, the DNA binding parameters of CRP dimers with either one or two ligands bound are determinable in such an approach. The change of protein fluorescence was used to measure CRP binding to its recognition sequence in the lac control region and to non-specific DNA. Furthermore, the binding of cAMP to preformed CRP-DNA complexes was independently studied by equilibrium dialysis. The data were analysed using a simple interactive model for two intrinsically identical sites and site-site interactions. The intrinsic binding constant K and the co-operativity factor alpha for binding of cAMP to free CRP depend only slightly on salt concentration between 0.01 M and 0.2 M. In contrast, the affinity of cAMP for CRP pre-bound to non-specific DNA increases with the salt concentration and the co-operativity changes from positive to negative. This results from cation rebinding to the DNA lattice upon forming the cAMP-CRP-DNA complex from cAMP and the pre-formed CRP-DNA complex. The CRP-cAMP1 complex shows almost the same affinity for specific and non-specific DNA as the CRP-cAMP2 complex, and both displace the same number of cations. It is concluded that the allosteric activation of CRP is induced upon binding of the first cAMP. These results are used to estimate the occupation of the CRP site in the lac control region in relation to the cAMP concentration in vivo. Under physiological conditions the lac promoter is activated by the CRP dimer complexed with only one cAMP. Furthermore, a model for the differential activation of various genes expressed under catabolite repression is presented and discussed.     

Cyclic AMP-induced conformational changes in mycobacterial protein acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.     

Effects of Mycobacterium tuberculosis ESAT-6/CFP-10 fusion protein on the autophagy function of mouse macrophages

Autophagy plays specific roles in host innate and adaptive immune responses to numerous intracellular pathogens, including Mycobacterium tuberculosis. The ESAT-6 and CFP-10 proteins are secreted by M. tuberculosis and play important roles in pathogenesis. We hypothesized that these two proteins may affect the autophagy function of host macrophages during infection with M. tuberculosis, thereby shaping the immune reaction toward the pathogen. Interestingly, we found that rapamycin-induced autophagy of macrophages infected with M. tuberculosis H37Rv enhanced localization of mycobacteria with autophagosomes and lysosomes. Ectopic expression of the ESAT-6/CFP-10 fusion in macrophages dramatically inhibited autophagosome formation, and M. tuberculosis survival inside infected macrophages was significantly affected as well. Further, M. tuberculosis viability was increased by the fusion protein. Expression levels of autophagy-related genes (ATG), especially atg8, also decreased (p<0.05). These results suggested that ESAT-6 and CFP-10 proteins play significant roles in autophagy formation in M. tuberculosis infection and that autophagosome formation is regulated through the expression of ATG.