The Mycobacterium tuberculosis methyltransferase Rv2067c manipulates host epigenetic programming to promote its own survival.

Mycobacterium tuberculosis has evolved several mechanisms to counter host defense arsenals for its proliferation. Here we report that M. tuberculosis employs a multi-pronged approach to modify host epigenetic machinery for its survival. It secretes methyltransferase (MTase) Rv2067c into macrophages, trimethylating histone H3K79 in a non-nucleosomal context. Rv2067c downregulates host MTase DOT1L, decreasing DOT1L-mediated nucleosomally added H3K79me3 mark on pro-inflammatory response genes. Consequent inhibition of caspase-8-dependent apoptosis and enhancement of RIPK3-mediated necrosis results in increased pathogenesis. In parallel, Rv2067c enhances the expression of SESTRIN3, NLRC3, and TMTC1, enabling the pathogen to overcome host inflammatory and oxidative responses. We provide the structural basis for differential methylation of H3K79 by Rv2067c and DOT1L. The structures of Rv2067c and DOT1L explain how their action on H3K79 is spatially and temporally separated, enabling Rv2067c to effectively intercept the host epigenetic circuit and downstream signaling.

Rv0802c is an acyltransferase that succinylates and acetylates Mycobacterium tuberculosis nucleoid-associated protein HU.

Among the nucleoid-associated proteins (NAPs), HU is the most conserved in eubacteria, engaged in overall chromosome organization and regulation of gene expression. Unlike other bacteria, HU from Mycobacterium tuberculosis (MtHU), has a long carboxyl terminal domain enriched in basic amino acids, resembling eukaryotic histone N-terminal tails. As with histones, MtHU undergoes post-translational modifications and we have previously identified interacting kinases, methyltransferases, an acetyltransferase and a deacetylase. Here we show that Rv0802c interacts and succinylates MtHU. Although categorized as a succinyltransferase, we show that this GNAT superfamily member can catalyse both succinylation and acetylation of MtHU with comparable kinetic parameters. Like acetylation of MtHU, succinylation of MtHU caused reduced interaction of the NAP with DNA, determined by electrophoretic mobility shift assay and surface plasmon resonance. However, in vivo expression of Rv0802c did not significantly alter the nucleoid architecture. Although such succinylation of NAPs is rare, these modifications of the archetypal NAP may provide avenues to the organism to compensate for the underrepresentation of NAPs in its genome to control the dynamics of nucleoid architecture and cellular functions.

DNA flowerstructure co-localizes with human pathogens in infected macrophages.

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.

Conditional down-regulation of GreA impacts expression of rRNA and transcription factors, affecting Mycobacterium smegmatis survival.

Gre, one of the conserved transcription factors in bacteria, modulates RNA polymerase (RNAP) activity to ensure processivity and fidelity of RNA synthesis. Gre factors regulate transcription by inducing the intrinsic-endonucleolytic activity of RNAP, allowing the enzyme to resume transcription from the paused and arrested sites. While Escherichia coli and a number of eubacteria harbor GreA and GreB, genus mycobacteria has a single Gre (GreA). To address the importance of the GreA in growth, physiology and gene expression of Mycobacterium smegmatis, we have constructed a conditional knock-down strain of GreA. The GreA depleted strain exhibited slow growth, drastic changes in cell surface phenotype, cell death, and increased susceptibility to front-line anti-tubercular drugs. Transcripts and 2D-gel electrophoresis (2D-PAGE) analysis of the GreA conditional knock-down strain showed altered expression of the genes involved in transcription regulation. Among the genes analysed, expression of RNAP subunits (ß, ß' and ω), carD, hupB, lsr2, and nusA were affected to a large extent. Severe reduction in the expression of genes of rRNA operon in the knock-down strain reveal a role for GreA in regulating the core components of the translation process.

Folate gene polymorphisms MTR A2756G, MTRR A66G, and BHMT G742A and risk for coronary artery disease: a meta-analysis.

UNLABELLED: Folate pathway gene polymorphisms may be a risk factor for coronary artery disease (CAD). However, studies of the association between these polymorphisms and CAD have reported conflicting results. Therefore, we performed a meta-analysis to better assess the association. OBJECTIVE: To investigate the association between 3 major polymorphisms in genes encoding enzymes involved in remethylation of homocysteine to methionine--methionine synthase (MTR) A2756G, methionine synthase reductase (MTRR) A66G, and betaine homocysteine methyltransferase (BHMT) G742A--and CAD, with assessment of small-study bias and differences between studies. METHODS: Case-control studies were identified by searching electronic literature databases for relevant reports published before February 2011. Data on genotype frequency were extracted, and 4 genetic models were applied. Heterogeneity was explored with stratification by ethnicity of the study sample. RESULTS: We found weak evidence of a recessive effect of the G allele in MTR A2756G (odds ratio, 1.61 [95% confidence interval, 0.98-2.66]; p=0.06). No effect of MTRR A66G and BHMT G742A in dominant, recessive, homozygous, and contrast allele genetic models was observed. CONCLUSION: Known common single-nucleotide polymorphisms in MTRR and BHMT genes may not be significant risk factors for CAD. The functional impact of these polymorphisms on enzyme activity is still unknown. Before additional epidemiologic studies are done, the functional impact of these polymorphisms, if any, should be established.

Gene polymorphisms and low dietary intake of micronutrients in coronary artery disease.

BACKGROUND/AIMS: Coronary artery disease (CAD) is a complex disorder involving genetic and non-genetic factors. Food is an important component of the latter. We examined if DNA polymorphisms in genes encoding enzymes of one-carbon metabolism coupled with low consumption of micronutrients such as folate, vitamins B(6) and B(12) might increase the risk of CAD. METHODS: A case-control study consisting of 252 CAD patients and 252 controls were included. Three single nucleotide polymorphisms (SNP), 2 insertion/deletion and 1 repeat polymorphism were typed. The micronutrient intake was estimated from a standard 24-hour dietary recall coupled to a food frequency questionnaire. RESULTS: The results suggest an association of 'early-onset CAD' with betaine homocysteine S-methyl transferase (BHMT) 742G→A SNP (odds ratio = 1.52; 95% confidence interval, 0.96-2.41; p = 0.04). No association was observed for all age of onset, but more patients than controls whose micronutrient intake was in the lowest quintile also carried the minor allele (50% patients vs. 37% controls; p = 0.042). Furthermore, dietary intake of folate micronutrients below the recommended daily allowance was observed in a larger percent of patients than controls with the minor BHMT allele (51% patients vs. 44% controls; p = 0.021). CONCLUSIONS: In the presence of the minor BHMT allele, a decreased consumption of folate micronutrients might increase the risk of CAD.