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

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

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

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

Substrate-bound crystal structures reveal features unique to Mycobacterium tuberculosis N-acetyl-glucosamine 1-phosphate uridyltransferase and a catalytic mechanism for acetyl transfer

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmUMtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmUMtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmUMtb. In this SN2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmUMtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmUMtb, which may be exploited for the development of inhibitors specific to GlmU

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

Protein kinase A (PknA) of Mycobacterium tuberculosis is independently activated and is critical for growth in vitro and survival of the pathogen in the host

The essential mycobacterial protein kinases PknA and PknB play crucial roles in modulating cell shape and division. However, the precise in vivo functional aspects of PknA have not been investigated. This study aims to dissect the role of PknA in mediating cell survival in vitro as well as in vivo. We observed aberrant cell shape and severe growth defects when PknA was depleted. Using the mouse infection model, we observe that PknA is essential for survival of the pathogen in the host. Complementation studies affirm the importance of the kinase, juxtamembrane and transmembrane domains of PknA. Surprisingly, the extracytoplasmic domain is dispensable for cell growth and survival in vitro. We find that phosphorylation of the activation loop at Thr172 of PknA is critical for bacterial growth. PknB has been previously suggested to be the receptor kinase, which activates multiple kinases, including PknA, by trans-phosphorylating their activation loop residues. Using phospho-specific PknA antibodies and conditional pknB mutant, we find that PknA autophosphorylates its activation loop independent of PknB. Fluorescently tagged PknA and PknB show distinctive distribution patterns within the cell, suggesting that although both kinases are known to modulate cell shape and division, their modes of action are likely to be different. This is supported by our findings that expression of kinase-dead PknA versus kinase-dead PknB in mycobacterial cells leads to different cellular phenotypes. Data indicate that although PknA and PknB are expressed as part of the same operon, they appear to be regulating cellular processes through divergent signaling pathways

Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages

Mycobacterium tuberculosis (Mtb) is known to modulate the host immune responses to facilitate its persistence inside the host cells. One of the key mechanisms includes repression of class-II transactivator (CIITA) and MHC-II expression in infected macrophages. However, the precise mechanism of CIITA and MHC-II down-regulation is not well studied. Mtb 6-kDa early secretory antigenic target (ESAT-6) is a known potent virulence and antigenic determinant. Mtb genome encodes 23 such ESAT-6 family proteins. We herein report that Mtb and M. bovis-BCG infection down-regulated the expression of CIITA/MHC-II by inducing hypermethylation in histone H3 Lysine 9 (H3K9me2/3). Further, we show that Mtb ESAT-6 family protein EsxL, encoded by Rv1198, is responsible for the down-regulation of CIITA/MHC-II by inducing H3K9me2/3. We further report that Mtb esxL induced the expression of nitric oxide synthetase (iNOS), NO production and p38-MAPK pathway, which in turn was responsible for the increased H3K9me2/3 in CIITA via up-regulation of euchromatic histone-lysine N-methyltransferase 2 (G9a). In contrast, inhibition of iNOS, p38-MAPK and G9a abrogated H3K9me2/3 resulting in increased CIITA expression. Chromatin immune precipitation assay confirmed that hypermethylation at the promoter IV (pIV) region of CIITA is mainly responsible for the CIITA down regulation and subsequently antigen presentation. We found that co-culture of macrophages infected with esxL expressing M. smegmatis and mouse spleenocytes led to down-regulation of IL-2, a key cytokine involved in T-cell proliferation. In summary, we show that Mtb esxL inhibits antigen presentation by enhancing H3K9me2/3 on CIITA promoter thereby repressing its expression through NO and p38-MAPK activation