[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis (Mtb) Cmr (Rv1675c) is a CRP/FNR family transcription factor known to be responsive to cAMP levels and during macrophage
infections. However, Cmr's DNA binding properties, cellular targets and overall role in tuberculosis (TB) complex bacteria
have not been characterized. In this study, we used experimental and computational approaches to characterize Cmr's DNA binding
properties and identify a putative regulon. Cmr binds a 16-bp palindromic site that includes four highly conserved nucleotides
that are required for DNA binding. A total of 368 binding sites, distributed in clusters among ∼200 binding regions throughout
the Mycobacterium bovis BCG genome, were identified using ChIP-seq. One of the most enriched Cmr binding sites was located upstream of the cmr promoter, and we demonstrated that expression of cmr is autoregulated. cAMP affected Cmr binding at a subset of DNA loci in vivo and in vitro, including multiple sites adjacent to members of the DosR (DevR) dormancy regulon. Our findings of cooperative binding of
Cmr to these DNA regions and the regulation by Cmr of the DosR-regulated virulence gene Rv2623 demonstrate the complexity
of Cmr-mediated gene regulation and suggest a role for Cmr in the biology of persistent TB infection.
Preview · Article · Sep 2015 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Cyclic di-AMP (c-di-AMP) is a signaling molecule that has been shown to play important roles in bacterial physiology and infections. Currently, c-di-AMP detection and quantification relies mostly on the use of high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). In this study, a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of c-di-AMP was developed, which utilizes a novel pneumococcal c-di-AMP binding protein (CabP) and a newly commercialized c-di-AMP derivative. With this new method, c-di-AMP concentrations in biological samples can be quickly and accurately quantified. Furthermore, this assay is much more efficient than current methods as it requires less overall cost and training while processing many samples at once. Therefore, this assay can be extensively used in research into c-di-AMP signaling.
No preview · Article · Sep 2014 · Journal of Microbiological Methods
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. The pathogenesis by the causative agent, Mycobacterium tuberculosis, is still not fully understood. We have previously reported that M. tuberculosis Rv3586 (disA) encodes a diadenylate cyclase, which converts ATP to cyclic di-AMP (c-di-AMP). In this study, we demonstrated that a protein encoded by Rv2837c (cnpB) possesses c-di-AMP phosphodiesterase activity and cleaves c-di-AMP exclusively to AMP. Our results showed that in M. tuberculosis, deletion of disA abolished bacterial c-di-AMP production, whereas deletion of cnpB significantly enhanced the bacterial c-di-AMP accumulation and secretion. The c-di-AMP levels in both mutants could be corrected by expressing the respective gene. We also found that macrophages infected with ΔcnpB secreted much higher levels of IFN-β than those infected with the wildtype (WT) or the complemented mutant. Interestingly, mice infected with M. tuberculosis ΔcnpB displayed significantly reduced inflammation, less bacterial burden in the lungs and spleens, and extended survival compared to those infected with the WT or the complemented mutant. These results indicate that deletion of cnpB results in attenuated virulence, which is correlated with elevated c-di-AMP levels
Full-text · Article · May 2014 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Cyclic di-AMP (c-di-AMP) has been shown to play important roles as a second messenger in bacterial physiology and infections.
However, understanding of how the signal is transduced is still limited. Previously, we have characterized a diadenylate cyclase
and two c-di-AMP phosphodiesterases in Streptococcus pneumoniae, a Gram-positive pathogen. In this study, we identified a c-di-AMP binding protein (CabP) in S. pneumoniae using c-di-AMP affinity chromatography. We demonstrated that CabP specifically bound c-di-AMP and that this interaction could
not be interrupted by competition with other nucleotides, including ATP, cAMP, AMP, phosphoadenylyl adenosine (pApA), and
cyclic di-GMP (c-di-GMP). By using a bacterial two-hybrid system and genetic mutagenesis, we showed that CabP directly interacted
with a potassium transporter (SPD_0076) and that both proteins were required for pneumococcal growth in media with low concentrations
of potassium. Interestingly, the interaction between CabP and SPD_0076 and the efficiency of potassium uptake were impaired
by elevated c-di-AMP in pneumococci. These results establish a direct c-di-AMP-mediated signaling pathway that regulates pneumococcal
Full-text · Article · Nov 2013 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology
and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and
SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved
c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but
not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth,
resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is
essential for pneumococcal biology and disease.
Full-text · Article · Sep 2013 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: The arginine deiminase system (ADS) is associated with arginine catabolism and plays a role in virulence of several pathogenic
bacteria. In Streptococcus pneumoniae, the ADS genes exist as a locus consisting of arcABCDT. A recent genome-wide mutagenesis approach revealed that both arcD and arcT are potentially essential in a chinchilla otitis media (OM) model. In the present study, we generated ΔarcD, ΔarcT, and ΔarcDT mutants by homologous recombination and evaluated their infectivity. Our results showed that only arcD, and not arcT, of an OM isolate is required during chinchilla middle ear infection. Additionally, D39 ΔarcD exhibited enhanced nasopharyngeal colonization and was attenuated in both mouse pneumonia and bacteremia models. In vitro, D39 ΔarcD displayed enhanced adherence to A549 epithelial cells and increased phagocytosis by J774A.1 macrophages compared to those
with the parental strain. This mutant also exhibited an impaired capsule, as detected using electron microscopy, immunofluorescence,
and a capsule assay. We demonstrated that the capsule defect in the D39 ΔarcD mutant may not be associated with a deficiency in arginine but rather is likely caused by a loss of interaction between the
capsule and the transmembrane protein ArcD.
Full-text · Article · Aug 2013 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: Vitamin B6 is an essential cofactor for a large number of enzymes in both prokaryotes and eukaryotes. In this study, we characterized
the pyridoxal 5′-phosphate (PLP) biosynthesis pathway in Streptococcus pneumoniae. Our results revealed that S. pneumoniae possesses a de novo vitamin B6 biosynthesis pathway encoded by the pdxST genes. Purified PdxS functionally displayed as PLP synthase, whereas PdxT exhibited glutaminase activity in vitro. Deletion of pdxS, but not pdxT, resulted in a vitamin B6 auxotrophic mutant. The defective growth of the ΔpdxS mutant in a vitamin B6-depleted medium could be chemically restored in the presence of the B6 vitamers at optimal concentrations. By analyzing PdxS expression levels, we demonstrated that the expression of pdxS was repressed by PLP and activated by a transcription factor, PdxR. A pneumococcal ΔpdxR mutant also exhibited as a vitamin B6 auxotroph. In addition, we found that disruption of the vitamin B6 biosynthesis pathway in S. pneumoniae caused a significant attenuation in a chinchilla middle ear infection model and a minor attenuation in a mouse pneumonia
model, indicating that the impact of vitamin B6 synthesis on virulence depends upon the bacterial infection niche.
Full-text · Article · Mar 2013 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg(2+), Mn(2+) or Co(2+). DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis CRP(Mt), encoded by Rv3676 (crp), is a CRP-like transcription factor that binds with the serC-Rv0885 intergenic region. In the present study, we evaluated CRP(Mt) 's regulation of serC and Rv0885 in M. tuberculosis and M. bovis BCG, using site-specific mutagenesis, promoter fusions and reverse-transcriptase PCR (RT-PCR). The CRP(Mt) 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 CRP(Mt) 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 CRP(Mt) 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 CRP(Mt)-regulated factors required by M. tuberculosis during macrophage and host infection.
[Show abstract][Hide abstract] ABSTRACT: cAMP is an ancient second messenger, and is used by many organisms to regulate a wide range of cellular functions. Mycobacterium tuberculosis complex bacteria are exceptional in that they have genes for at least 15 biochemically distinct adenylyl cyclases, the enzymes that generate cAMP. cAMP-associated gene regulation within tubercle bacilli is required for their virulence, and secretion of cAMP produced by M. tuberculosis bacteria into host macrophages disrupts the host's immune response to infection. In this review, we discuss recent advances in our understanding of the means by which cAMP levels are controlled within mycobacteria, the importance of cAMP to M. tuberculosis during host infection, and the role of cAMP in mycobacterial gene regulation. Understanding the myriad aspects of cAMP signalling in tubercle bacilli will establish new paradigms for cAMP signalling, and may contribute to new approaches for prevention and/or treatment of tuberculosis disease.
Preview · Article · Mar 2011 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Yersinia pestis, the etiologic agent of plague, has only recently evolved from Yersinia pseudotuberculosis. hfq deletion caused severe growth restriction at 37°C in Y. pestis but not in Y. pseudotuberculosis. Strains from all epidemic plague biovars were similarly affected, implicating Hfq, and likely small RNAs (sRNAs), in the
unique biology of the plague bacillus.
Preview · Article · Aug 2010 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c. 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 tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.
Preview · Article · Jan 2009 · FEMS Immunology & Medical Microbiology
[Show abstract][Hide abstract] ABSTRACT: Cyclic AMP (cAMP) has recently been shown to be a global regulator of gene expression in Mycobacterium tuberculosis (Mtb). In this study we identified a new cAMP-associated regulon in Mtb and Mycobacterium bovis BCG, which is distinct from the previously described CRP(Mt) regulon. Proteomic comparison of wild-type M. bovis BCG with a Rv1675c (cmr) knockout strain showed dysregulated expression of four previously identified proteins encoded by the cAMP-induced genes (cAIGs) mdh, groEL2, Rv1265 and PE_PGRS6a. Regulated expression of these four cAIGs also occurred during macrophage infection, and this regulation required cmr in both Mtb and M. bovis BCG. Purified His-Cmr bound to the DNA sequences upstream of three cAIGs (mdh, groEL2, Rv1265) in electrophoretic mobility shift assays, suggesting direct regulation of these genes by Cmr. We also found that low pH stimulated cAMP production in both Mtb and M. bovis BCG, but broadly affected cAIG regulation only in M. bovis BCG. These studies identify Cmr as a transcription factor that regulates cAIGs within macrophages, and suggest that multiple factors affect cAMP-associated gene regulation in tuberculosis-complex mycobacteria. cAMP signalling and Cmr-mediated gene regulation during Mtb infection of macrophages may have implications for TB pathogenesis.
Full-text · Article · Dec 2008 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis Rv3676 encodes a cyclic AMP (cAMP) receptor-like protein (CRPMt) that has been implicated in global gene regulation and may play an important role during tuberculosis infection. The CRPMt ortholog in Mycobacterium bovis BCG, CRPBCG, is dysfunctional in an Escherichia coli CRP competition assay and has been proposed as a potential source of M. bovis BCG's attenuation. We compared CRPBCG and CRPMt in vitro and in vivo, in M. bovis BCG and M. tuberculosis, to evaluate CRPBCG's potential function in a mycobacterial system. Both proteins formed dimers in mycobacterial lysates, bound to the same target
DNA sequences, and were similarly affected by the presence of cAMP in DNA binding assays. However, CRPMt and CRPBCG differed in their relative affinities for specific DNA target sequences and in their susceptibilities to protease digestion.
Surprisingly, CRPBCG DNA binding activity was stronger than that of CRPMt both in vitro and in vivo, as measured by electrophoretic mobility shift and chromatin immunoprecipitation assays. Nutrient
starvation-associated regulation of several CRPMt regulon members also differed between M. bovis BCG and M. tuberculosis. We conclude that CRPBCG is a functional cAMP-responsive DNA binding protein with an in vivo DNA binding profile in M. bovis BCG similar to that of CRPMt in M. tuberculosis. However, biologically significant functional differences may exist between CRPBCG and CRPMt with respect to gene regulation, and this issue warrants further study.
Full-text · Article · Dec 2007 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: The molecular basis of the biological differences between Yersinia pestis and Yersinia pseudotuberculosis remains largely unknown, and relatively little is known about environmental regulation of gene expression in these bacteria. We used a proteomic approach to explore the regulatory response of each bacterium to carbon dioxide-supplemented hypoxic conditions. Both organisms responded similarly and the magnitude of their responses was similar to what was observed in low iron conditions. We also identified proteins that were expressed at different levels in Y. pestis and Y. pseudotuberculosis, and found that SodB is expressed more strongly at both the protein and RNA levels in Y. pseudotuberculosis than in Y. pestis. Enzyme activity did not directly correlate with levels of protein expression, and we propose that an amino acid change difference between these orthologous proteins has the potential to affect catalytic activity. In addition, the upstream regulatory regions of several chromosomal genes were found to exhibit specific binding with a putative transcription factor, CDS4, from the Y. pestis-specific pPCPI plasmid. The potential role of this protein in modulating Y. pestis- specific gene regulation warrants further investigation.
No preview · Article · Feb 2007 · Advances in Experimental Medicine and Biology
[Show abstract][Hide abstract] ABSTRACT: Little is known about cyclic AMP (cAMP) function in Mycobacterium tuberculosis, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins. M. tuberculosis Rv3676, which we have designated CRPMt, is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRPMt's interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRPMt DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for Escherichia coli. CRPMt binding sites were identified in a total of 73 promoter regions regulating 114 genes in the M. tuberculosis genome, which are being explored as a regulon. Specific CRPMt binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete
loss of binding to CRPMt. cAMP enhanced CRPMt's ability to bind DNA and caused allosteric alterations in CRPMt conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in M. tuberculosis, suggesting a role for cAMP signal transduction in M. tuberculosis and implicating CRPMt as a cAMP-responsive global regulator.
Full-text · Article · Dec 2005 · Journal of Bacteriology