Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3

Mycobacteria are successful pathogens that modulate the host immune response through unclear mechanisms. Here we show that Rv1988, a secreted mycobacterial protein, is a functional methyltransferase that localizes to the host nucleus and interacts with chromatin. Rv1988 methylates histone H3 at H3R42 and represses the genes involved in the first line of defence against mycobacteria. H3R42me2, a non-tail histone modification, is present at the entry and exit point of DNA in the nucleosome and not within the regulatory sites in the N-terminal tail. Rv1988 deletion in Mycobacterium tuberculosis reduces bacterial survival in the host and experimental expression of M. tuberculosis Rv1988 in non-pathogenic Mycobacterium smegmatis negatively affects the health of infected mice. Thus, Rv1988 is an important mycobacterial virulence factor, which uses a non-canonical epigenetic mechanism to control host cell transcription

ALLIUM SATIVUM LINN. CONTAINS LINEAR ALKYLBENZENE SULFONATES THAT ALTER MEMBRANE FLUIDITY FOR THE INHIBITION OF MYCOBACTERIUM TUBERCULOSIS H37RA

  Objectives: The purpose of the study is to characterize antimycobacterial phytoconstituent from ethyl acetate extract of dried bulbs of Allium sativum Linn. (Alliaceae) and elucidating the probable mode of action of the bioactive molecule.Methods: Serial extraction, Mycobacterium tuberculosis assay by agar well diffusion method, minimal inhibitory concentration by microplate alamar blue assay, Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS)/MS, cell leakage assay, scanning electron microscopy (SEM), inhibition property of linear alkylbenzene sulfonate (LAS) in the presence of rifampicin on M. tuberculosis were performed.Results: Ethyl acetate extract displayed significant inhibition properties against M. tuberculosis H37Ra (MTCC 300). Subsequently, the bioactivity-guided fractionation was employed to purify the phytochemical. Analysis of FT-IR, LC-MS (ESI), 1H, and13C-NMR spectrum revealed that the bioactive phytochemicals are the variants of LAS, with C12-alkyl being predominant, and the minimum inhibitory concentration was found to be 5.56 μg/ml. Morphological examination by SEM and cell leakage assay indicated that these molecules change the membrane fluidity.Conclusion: The results thus suggest the possibility of using low concentrations of LAS to effect changes in membrane fluidity, thereby enhancing the efficacy of antibiotic treatment

The transpeptidase PbpA and non-canonical transglycosylase RodA of Mycobacterium tuberculosis play important roles in regulating bacterial cell lengths

The cell wall of Mycobacterium tuberculosis (Mtb) is a complex structure that protects the pathogen in hostile environments. Peptidoglycan (PG), which helps determine the morphology of the cell envelope, undergoes substantial remodeling under stress. This meshwork of linear chains of sugars, crosslinked through attached peptides, is generated through the sequential action of enzymes termed transglycosylases and transpeptidases. The Mtb genome encodes two classical transglycosylases and four transpeptidases, the functions of which are not fully elucidated. Here, we present work on the yet uncharacterized transpeptidase PbpA and a non-classical transglycosylase RodA. We elucidate their roles in regulating in vitro growth and in vivo survival of pathogenic mycobacteria. We find that RodA and PbpA are required for regulating cell length, but do not affect mycobacterial growth. Biochemical analyses show PbpA to be a classical transpeptidase, while RodA is identified to be a member of an emerging class of non-canonical transglycosylases. Phosphorylation of RodA at T463 modulates its biological function. In a guinea pig infection model, RodA and PbpA are found to be required for both, bacterial survival as well as formation of granuloma structures, thus underscoring the importance of these proteins in mediating mycobacterial virulence in the host. Our results emphasize the fact that while redundant enzymes likely compensate for the absence of RodA or PbpA during in vitro growth, the two proteins play critical roles for the survival of the pathogen inside its host

A charge reversal differentiates (p)ppGpp synthesis by monofunctional and bifunctional Rel proteins

A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p)ppGpp (the stringent response molecule), synthesized and hydrolyzed by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. Although these proteins require Mg2+ for (p)ppGpp synthesis, high Mg2+ was shown to inhibit this reaction in bifunctional Rel proteins from Mycobacterium tuberculosis and Streptococcus equisimilis. This is not a characteristic feature in enzymes that use a dual metal ion mechanism, such as DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polβ and RelSeq structures that share a common fold led to the proposal that the latter would follow a single metal ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of guanosine 5′-triphosphate, 3′-diphosphate (pppGpp) synthesis at higher Mg2+ in the monofunctional RelA from Escherichia coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RXKD motif in the bifunctional proteins is reversed to an EXDD motif. The differential response of these proteins to Mg2+ could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg2+ is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by EXDD. This work, for the first time, brings out differences in (p)ppGpp synthesis by the two classes of Rel proteins

Allium sativum linn. contains linear alkylbenzene sulfonates that alter membrane fluidity for the inhibition of Mycobacterium tuberculosis H37RA

Objectives: The purpose of the study is to characterize antimycobacterial phytoconstituent from ethyl acetate extract of dried bulbs of Allium sativum Linn. (Alliaceae) and elucidating the probable mode of action of the bioactive molecule. Methods: Serial extraction, Mycobacterium tuberculosis assay by agar well diffusion method, minimal inhibitory concentration by microplate alamar blue assay, Fourier Transform Infrared (FT-IR), Nuclear Magnetic Resonance (NMR) spectroscopy, Liquid Chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS)/MS, cell leakage assay, Scanning Electron Microscopy (SEM), inhibition property of Linear Alkylbenzene Sulfonate (LAS) in the presence of rifampicin on M. tuberculosis were performed. Results: Ethyl acetate extract displayed significant inhibition properties against M. tuberculosis H37Ra (MTCC 300). Subsequently, the bioactivity-guided fractionation was employed to purify the phytochemical. Analysis of FT-IR, LC-MS (ESI), 1H and 13C-NMR spectrum revealed that the bioactive phytochemicals are the variants of LAS, with C12-alkyl being predominant, and the minimum inhibitory concentration was found to be 5.56 μg/ml. Morphological examination by SEM and cell leakage assay indicated that these molecules change the membrane fluidity. Conclusion: The results thus suggest the possibility of using low concentrations of LAS to effect changes in membrane fluidity, thereby enhancing the efficacy of antibiotic treatment

Protein kinase B (PknB) of Mycobacterium tuberculosisIs essential for growth of the pathogenin vitroas well as for survival within the host

The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-a-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host

Structure of N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Mycobacterium tuberculosis in a cubic space group

The structure of M. tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) was determined by the molecular-replacement method to 3.4 Å resolution in space group I432 and was refined to a final R work and R free of 0.285 and 0.321, respectively

Phosphorylation of Mycobacterium tuberculosis Ser/Thr Phosphatase by PknA and PknB

Background: The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs. Principal Findings: PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn2+-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr137, Thr141, Thr174 and Thr290 being the target residues of PknB while Thr137 and Thr174 are phosphorylated by PknA. The Mn2+-ion binding residues Asp38 and Asp229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstPcD38G are phosphorylated by PknA and PknB in E. coli and addition of Zn2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent. Conclusions and Significance: This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena

Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function

A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions

Depletion of M. tuberculosis GlmU from infected murine lungs effects the clearance of the pathogen

M. tuberculosis N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmUMtb) is a bi-functional enzyme engaged in the synthesis of two metabolic intermediates N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and UDP-GlcNAc, catalyzed by the C- and N-terminal domains respectively. UDP-GlcNAc is a key metabolite essential for the synthesis of peptidoglycan, disaccharide linker, arabinogalactan and mycothiols. While GlmUMtb was predicted to be an essential gene, till date the role of GlmUMtb in modulating the in vitro growth of Mtb or its role in survival of pathogen ex vivo / in vivo have not been deciphered. Here we present the results of a comprehensive study dissecting the role of GlmUMtb in arbitrating the survival of the pathogen both in vitro and in vivo. We find that absence of GlmUMtb leads to extensive perturbation of bacterial morphology and substantial reduction in cell wall thickness under normoxic as well as hypoxic conditions. Complementation studies show that the acetyl- and uridyl- transferase activities of GlmUMtb are independently essential for bacterial survival in vitro and GlmUMtb is also found to be essential for mycobacterial survival in THP-1 cells as well as in guinea pigs. Depletion of GlmUMtb from infected murine lungs, four weeks post infection, led to significant reduction in the bacillary load. The administration of Oxa33, a novel oxazolidine derivative that specifically inhibits GlmUMtb, to infected mice resulted in significant decrease in the bacillary load. Thus our study establishes GlmUMtb as a strong candidate for intervention measures against established tuberculosis infections