MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm), knockout strain of M. smegmatis (Delta rel(Msm)) is expected to show a (p)ppGpp null (p)ppGpp(0)] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA(.)DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel(Msm) in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present RHII, RNase HII domain, originally identified as (d) under bar omain of (u) under bar nknown (f) under bar unction 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.

Distinct and Contrasting Transcription Initiation Patterns at Mycobacterium tuberculosis Promoters

Although sequencing of Mycobacterium tuberculosis genome lead to better understanding of transcription units and gene functions, interactions occurring during transcription initiation between RNA polymerase and promoters is yet to be elucidated. Different stages of transcription initiation include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance. We have now analyzed these events with four promoters of M. tuberculosis viz. P-gyrB1, P-gyrR, P-rrnPCL1 and P-metU. The promoters differed from each other in their rates of open complex formation, decay, promoter clearance and abortive transcription. The equilibrium binding and kinetic studies of various steps revealed distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. Surprisingly, the transcription at gyr promoter was enhanced in the presence of initiating nucleotides and decreased in the presence of alarmone, pppGpp, a pattern typically seen with rRNA promoters studied so far. The gyr promoter of M. smegmatis, on the other hand, was not subjected to pppGpp mediated regulation. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. Moreover, the distinct rate limiting steps during transcription initiation of each one of the promoters studied point at variations in their intracellular regulation.

Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from Mycobacterium smegmatis

An analysis of the Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1), has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 angstrom resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six beta-strands forming an anti-parallel beta-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function.

Crystallization and preliminary X-ray studies of MutT1 (MSMEG_2390) from Mycobacterium smegmatis

MutT1 (MSMEG_2390) from Mycobacterium smegmatis has been crystallized and the crystals have been characterized using X-ray diffraction. The crystals belonged to space group P2(1)2(1)2(1). The Matthews coefficient suggested the possibility of one protein molecule in the asymmetric unit of the orthorhombic unit cell. Solution of the structure using the known three-dimensional structure of a bacterial MutT1 is anticipated.

Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis

The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, remains unknown. In this regard, M. tuberculosis fic gene (Mtfic) was cloned, overexpressed, and purified to homogeneity for its biochemical characterisation. It has the characteristic FIC motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Neither the His-tagged nor the GST-tagged MtFic protein, overexpressed in Escherichia coil, nor expression of Mtfic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtFic (MBP-MtFic) could be obtained partly in the soluble fraction. The cloned, overexpressed, and purified recombinant MBP-MtFic showed conversion of ATP, GTP, CTP, and UTP into AMP. GMP, CMP, and UMP, respectively. Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The design of NMP formation assay using the recombinant, soluble MtFic would enable identification of its target substrate for NMPylation. (C) 2012 Elsevier Inc. All rights reserved.

Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg2+ co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg2+ for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

Biochemical Properties of MutT2 Proteins from Mycobacterium tuberculosis and M. smegmatis and Their Contrasting Antimutator Roles in Escherichia coli

Mycobacterium tuberculosis, the causative agent of tuberculosis, is at increased risk of accumulating damaged guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP because of its residency in the oxidative environment of the host macrophages. By hydrolyzing the oxidized guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role in allowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we purified recombinant M. tuberculosis MutT2 (MtuMutT2) and M. smegmatis MutT2 (MsmMutT2) proteins from M. tuberculosis (a slow grower) and M. smegmatis (fast growing model mycobacteria), respectively, for their biochemical characterization. Distinct from the Escherichia coli MutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (K-m and V-max) revealed that while MtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP, MsmMutT2 hydrolyzes them nearly equally. Also, MsmMutT2 is about 14 times more efficient than MtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP. Consistent with these observations, MsmMutT2 but not MtuMutT2 rescues E. coli for MutT deficiency by decreasing both the mutation frequency and A-to-C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

Regulation of lipid biosynthesis, sliding motility, and biofilm formation by a membrane-anchored nucleoid-associated protein of mycobacterium tuberculosis

Bacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria.

Mycobacterium tuberculosis mutT1 (Rv2985) and ADPRase (Rv1700) proteins constitute a two-stage mechanism of 8-Oxo-dGTP and 8-Oxo-GTP detoxification and adenosine to cytidine mutation avoidance

Approximately one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. A better understanding of the pathogen biology is crucial to develop new tools/strategies to tackle its spread and treatment. In the host macrophages, the pathogen is exposed to reactive oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP, respectively. Incorporation of the damaged nucleotides in nucleic acids is detrimental to organisms. MutT proteins, belonging to a class of Nudix hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. Mycobacteria possess several MutT proteins. However, a functional homolog of Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1 and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then converts them to the corresponding nucleoside monophosphates. This observation suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with a K-m of similar to 50 mu M and V-max of similar to 0.9 pmol/min per ng of protein, and Rv1700 converts 8-oxo-dGDP to 8-oxo-dGMP with a K-m of similar to 9.5 mu M and V-max of similar to 0.04 pmol/min per ng of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for its MutT deficiency by decreasing A to C mutations (a hallmark of MutT deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a crucial role in survival of bacteria against oxidative stress.

Mycobacterium tuberculosis RsdA provides a conformational rationale for selective regulation of sigma-factor activity by proteolysis

The relative levels of different sigma factors dictate the expression profile of a bacterium. Extracytoplasmic function sigma factors synchronize the transcriptional profile with environmental conditions. The cellular concentration of free extracytoplasmic function sigma factors is regulated by the localization of this protein in a sigma/anti-sigma complex. Anti-sigma factors are multi-domain proteins with a receptor to sense environmental stimuli and a conserved anti-sigma domain (ASD) that binds a sigma factor. Here we describe the structure of Mycobacterium tuberculosis anti-sigma(D) (RsdA) in complex with the -35 promoter binding domain of sigma(D) (sigma(D)(4)). We note distinct conformational features that enable the release of sigma(D) by the selective proteolysis of the ASD in RsdA. The structural and biochemical features of the sigma(D)/RsdA complex provide a basis to reconcile diverse regulatory mechanisms that govern sigma/anti-sigma interactions despite high overall structural similarity. Multiple regulatory mechanisms embedded in an ASD scaffold thus provide an elegant route to rapidly re-engineer the expression profile of a bacterium in response to an environmental stimulus.

Heat and SDS insensitive NDK dimers are largely stabilised by hydrophobic interaction to form functional hexamer in Mycobacterium smegmatis

The primary structure and function of nucleoside diphosphate kinase (NDK), a substrate non-specific enzyme involved in the maintenance of nucleotide pools is also implicated to play pivotal roles in many other cellular processes. NDK is conserved from bacteria to human and forms a homotetramer or hexamer to exhibit its biological activity. However, the nature of the functional oligomeric form of the enzyme differs among different organisms. The functional form of NDKs from many bacterial systems, including that of the human pathogen, Mycobacterium tuberculosis (MtuNDK), is a hexamer, although some bacterial NDKs are tetrameric in nature. The present study addresses the oligomeric property of MsmNDK and how a dimer, the basic subunit of a functional hexamer, is stabilized by hydrogen bonds and hydrophobic interactions. Homology modeling was generated using the three-dimensional structure of MtuNDK as a template; the residues interacting at the monomer-monomer interface of MsmNDK were mapped. Using recombinant enzymes of wild type, catalytically inactive mutant, and monomer-monomer interactive mutants of MsmNDK, the stability of the dimer was verified under heat, SDS, low pH, and methanol. The predicted residues (Gln17, Ser24 and Glu27) were engaged in dimer formation, however the mutated proteins retained the ATPase and GTPase activity even after introducing single (MsmNDK- Q17A, MsmNDK-E27A, and MsmNDK-E27Q) and double (MsmNDK-E27A/Q17A) mutation. However, the monomer monomer interaction could be abolished using methanol, indicating the stabilization of the monomer-monomer interaction by hydrophobic interaction.

Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress Major histocompatibility complex II expression during Mycobacterium bovis Bacillus Calmette-Guerin infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guerin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-gamma-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Mining large-scale response networks reveals `topmost activities' in Mycobacterium tuberculosis infection

Mycobacterium tuberculosis owes its high pathogenic potential to its ability to evade host immune responses and thrive inside the macrophage. The outcome of infection is largely determined by the cellular response comprising a multitude of molecular events. The complexity and inter-relatedness in the processes makes it essential to adopt systems approaches to study them. In this work, we construct a comprehensive network of infection-related processes in a human macrophage comprising 1888 proteins and 14,016 interactions. We then compute response networks based on available gene expression profiles corresponding to states of health, disease and drug treatment. We use a novel formulation for mining response networks that has led to identifying highest activities in the cell. Highest activity paths provide mechanistic insights into pathogenesis and response to treatment. The approach used here serves as a generic framework for mining dynamic changes in genome-scale protein interaction networks.