Helicobacter pylori enhances HLA-C expression in the human gastric adenocarcinoma cells AGS and can protect them from the cytotoxicity of natural killer cells.

Helicobacter pylori (H. pylori) seems to play causative roles in gastric cancers. H. pylori has also been detected in established gastric cancers. How the presence of H. pylori modulates immune response to the cancer is unclear. The cytotoxicity of natural killer (NK) cells, toward infected or malignant cells, is controlled by the repertoire of activating and inhibitory receptors expressed on their surface. Here, we studied H. pylori-induced changes in the expression of ligands, of activating and inhibitory receptors of NK cells, in the gastric adenocarcinoma AGS cells, and their impacts on NK cell responses. AGS cells lacked or had low surface expression of the class I major histocompatibility complex (MHC-I) molecules HLA-E and HLA-C-ligands of the major NK cell inhibitory receptors NKG2A and killer-cell Ig-like receptor (KIR), respectively. However, AGS cells had high surface expression of ligands of activating receptors DNAM-1 and CD2, and of the adhesion molecules LFA-1. Consistently, AGS cells were sensitive to killing by NK cells despite the expression of inhibitory KIR on NK cells. Furthermore, H. pylori enhanced HLA-C surface expression on AGS cells. H. pylori infection enhanced HLA-C protein synthesis, which could explain H. pylori-induced HLA-C surface expression. H. pylori infection enhanced HLA-C surface expression also in the hepatoma Huh7 and HepG2 cells. Furthermore, H. pylori-induced HLA-C surface expression on AGS cells promoted inhibition of NK cells by KIR, and thereby protected AGS cells from NK cell cytotoxicity. These results suggest that H. pylori enhances HLA-C expression in host cells and protects them from the cytotoxic attack of NK cells expressing HLA-C-specific inhibitory receptors.

CRISPR Correction of the GBA Mutation in Human-Induced Pluripotent Stem Cells Restores Normal Function to Gaucher Macrophages and Increases Their Susceptibility to Mycobacterium tuberculosis.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the ß-glucocerebrosidase (GCase) GBA gene, which result in macrophage dysfunction. CRISPR (clustered regularly interspaced short palindromic repeats) editing of the homozygous L444P (1448T→C) GBA mutation in type 2 GD (GBA-/-) human-induced pluripotent stem cells (hiPSCs) yielded both heterozygous (GBA+/-) and homozygous (GBA+/+) isogenic lines. Macrophages derived from GBA-/-, GBA+/- and GBA+/+ hiPSCs showed that GBA mutation correction restores normal macrophage functions: GCase activity, motility, and phagocytosis. Furthermore, infection of GBA-/-, GBA+/- and GBA+/+ macrophages with the Mycobacterium tuberculosis H37Rv strain showed that impaired mobility and phagocytic activity were correlated with reduced levels of bacterial engulfment and replication suggesting that GD may be protective against tuberculosis.

Kinetic and catalytic properties of M.HpyAXVII, a phase-variable DNA methyltransferase from Helicobacter pylori.

The bacterium Helicobacter pylori is one of the most common infectious agents found in the human stomach. H. pylori has an unusually large number of DNA methyltransferases (MTases), prompting speculation that they may be involved in the cancerization of epithelial cells. The mod-4a/4b locus, consisting of the hp1369 and hp1370 ORFs, encodes for a truncated and inactive MTase in H. pylori strain 26695. However, slipped-strand synthesis within the phase-variable polyguanine tract in hp1369 results in expression of an active HP1369-1370 fusion N6-adenine methyltransferase, designated M.HpyAXVII. Sequence analysis of the mod-4a/4b locus across 74 H. pylori strain genomes has provided insights into the regulation of M.HpyAXVII expression. To better understand the role of M.HpyAXVII in the H. pylori biology, here we cloned and overexpressed the hp1369-70 fusion construct in Escherichia coli BL21(DE3) cells. Results from size-exclusion chromatography and multi-angle light scattering (MALS) analyses suggested that M.HpyAXVII exists as a dimer in solution. Kinetic studies, including product and substrate inhibition analyses, initial velocity dependence between substrates, and isotope partitioning, suggested that M.HpyAXVII catalyzes DNA methylation in an ordered Bi Bi mechanism in which the AdoMet binding precedes DNA binding and AdoMet's methyl group is then transferred to an adenine within the DNA recognition sequence. Altering the highly conserved catalytic motif (DPP(Y/F)) as well as the AdoMet-binding motif (FXGXG) by site-directed mutagenesis abolished the catalytic activity of M.HpyAXVII. These results provide insights into the enzyme kinetic mechanism of M.HpyAXVII. We propose that AdoMet binding conformationally "primes" the enzyme for DNA binding.

N4-cytosine DNA methylation regulates transcription and pathogenesis in Helicobacter pylori.

Many bacterial genomes exclusively display an N4-methyl cytosine base (m4C), whose physiological significance is not yet clear. Helicobacter pylori is a carcinogenic bacterium and the leading cause of gastric cancer in humans. Helicobacter pylori strain 26695 harbors a single m4C cytosine methyltransferase, M2.HpyAII which recognizes 5' TCTTC 3' sequence and methylates the first cytosine residue. To understand the role of m4C modification, M2.hpyAII deletion strain was constructed. Deletion strain displayed lower adherence to host AGS cells and reduced potential to induce inflammation and apoptosis. M2.hpyAII gene deletion strain exhibited reduced capacity for natural transformation, which was rescued in the complemented strain carrying an active copy of M2.hpyAII gene in the genome. Genome-wide gene expression and proteomic analysis were carried out to discern the possible reasons behind the altered phenotype of the M2.hpyAII gene deletion strain. Upon the loss of m4C modification a total of 102 genes belonging to virulence, ribosome assembly and cellular components were differentially expressed. The present study adds a functional role for the presence of m4C modification in H. pylori and provides the first evidence that m4C signal acts as a global epigenetic regulator in H. pylori.

Mechanism of formation of a toroid around DNA by the mismatch sensor protein.

The DNA mismatch repair (MMR) pathway removes errors that appear during genome replication. MutS is the primary mismatch sensor and forms an asymmetric dimer that encircles DNA to bend it to scan for mismatches. The mechanism utilized to load DNA into the central tunnel was unknown and the origin of the force required to bend DNA was unclear. We show that, in absence of DNA, MutS forms a symmetric dimer wherein a gap exists between the monomers through which DNA can enter the central tunnel. The comparison with structures of MutS-DNA complexes suggests that the mismatch scanning monomer (Bm) will move by nearly 50 Å to associate with the other monomer (Am). Consequently, the N-terminal domains of both monomers will press onto DNA to bend it. The proposed mechanism of toroid formation evinces that the force required to bend DNA arises primarily due to the movement of Bm and hence, the MutS dimer acts like a pair of pliers to bend DNA. We also shed light on the allosteric mechanism that influences the expulsion of adenosine triphosphate from Am on DNA binding. Overall, this study provides mechanistic insight regarding the primary event in MMR i.e. the assembly of the MutS-DNA complex.

Correction to: Mutations in the nucleotide binding and hydrolysis domains of helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function.

Following publication of the original article [1], the authors notified us of an error in the presentation of Fig. 6G.

Generation of Catalytic Antibodies Is an Intrinsic Property of an Individual's Immune System: A Study on a Large Cohort of Renal Transplant Patients.

Renal transplant is the treatment of choice for patients with terminal end-stage renal disease. We have previously identified low levels of catalytic IgG as a potential prognosis marker for chronic allograft rejection. The origin and physiopathological relevance of catalytic Abs is not well understood, owing to the fact that catalytic Abs have been studied in relatively small cohorts of patients with rare diseases and/or without systematic follow-up. In the current study, we have followed the evolution of the levels of catalytic IgG in a large cohort of renal transplant patients over a 2-y period. Our results demonstrate that, prior to transplant, patients with renal failure present with heterogeneous levels of IgG hydrolyzing the generic proline-phenylalanine-arginine-methylcoumarinamide (PFR-MCA) substrate. PFR-MCA hydrolysis was greater for patients' IgG than for a therapeutic preparation of pooled IgG from healthy donors. Renal transplant was marked by a drastic decrease in levels of catalytic IgG over 3 mo followed by a steady increase during the next 21 mo. Patients who displayed high levels of catalytic IgG pretransplant recovered high levels of catalytic Abs 2 y posttransplant. Interestingly, IgG-mediated hydrolysis of a model protein substrate, procoagulant factor VIII, did not correlate with that of PFR-MCA prior transplantation, whereas it did 12 mo posttransplant. Taken together, our results suggest that the level of circulating catalytic IgG under pathological conditions is an intrinsic property of each individual's immune system and that recovery of pretransplant levels of catalytic IgG is accompanied by changes in the repertoire of target Ags.

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N(6)-adenine DNA methyltransferases of Neisseria meningitidis.

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N(6)-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N(6)-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY M6A: G-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC M6A: CC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC M6A: GC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY M6A: G-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC M6A: GG-3' sites, to 100% at 5'-ACGT M6A: GG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.

The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient anti-recombination function of Helicobacter pylori†MutS2.

Helicobacter pylori, a human pathogen, is a naturally and constitutively competent bacteria, displaying a high rate of intergenomic recombination. While recombination events are essential for evolution and adaptation of H. pylori to dynamic gastric niches and new hosts, such events should be regulated tightly to maintain genomic integrity. Here, we analyze the role of the nuclease activity of MutS2, a protein that limits recombination during transformation in H. pylori. In previously studied MutS2 proteins, the C-terminal Smr domain was mapped as the region responsible for its nuclease activity. We report here that deletion of Smr domain does not completely abolish the nuclease activity of HpMutS2. Using bioinformatics analysis and mutagenesis, we identified an additional and novel nuclease motif (LDLK) at the N-terminus of HpMutS2 unique to Helicobacter and related ε-proteobacterial species. A single point mutation (D30A) in the LDLK motif and the deletion of Smr domain resulted in ∼ 5-10-fold loss of DNA cleavage ability of HpMutS2. Interestingly, the mutant forms of HpMutS2 wherein the LDLK motif was mutated or the Smr domain was deleted were unable to complement the hyper-recombination phenotype of a mutS2(-) strain, suggesting that both nuclease sites are indispensable for an efficient anti-recombinase activity of HpMutS2.

Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function.

BACKGROUND: Helicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive. RESULTS: To explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited ~2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by ~3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by ~10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2. CONCLUSIONS: These data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.

Type III restriction-modification enzymes: a historical perspective.

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

Mycobacterium tuberculosis DinG is a structure-specific helicase that unwinds G4 DNA: implications for targeting G4 DNA as a novel therapeutic approach.

The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' → 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' → 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.

Helicobacter pylori DprA alleviates restriction barrier for incoming DNA.

Helicobacter pylori is a Gram-negative bacterium that colonizes human stomach and causes gastric inflammation. The species is naturally competent and displays remarkable diversity. The presence of a large number of restriction-modification (R-M) systems in this bacterium creates a barrier against natural transformation by foreign DNA. Yet, mechanisms that protect incoming double-stranded DNA (dsDNA) from restriction enzymes are not well understood. A DNA-binding protein, DNA Processing Protein A (DprA) has been shown to facilitate natural transformation of several Gram-positive and Gram-negative bacteria by protecting incoming single-stranded DNA (ssDNA) and promoting RecA loading on it. However, in this study, we report that H. pylori DprA (HpDprA) binds not only ssDNA but also dsDNA thereby conferring protection to both from various exonucleases and Type II restriction enzymes. Here, we observed a stimulatory role of HpDprA in DNA methylation through physical interaction with methyltransferases. Thus, HpDprA displayed dual functional interaction with H. pylori R-M systems by not only inhibiting the restriction enzymes but also stimulating methyltransferases. These results indicate that HpDprA could be one of the factors that modulate the R-M barrier during inter-strain natural transformation in H. pylori.

Role of DNA methyltransferases in epigenetic regulation in bacteria.

In prokaryotes, alteration in gene expression was observed with the modification of DNA, especially DNA methylation. Such changes are inherited from generation to generation with no alterations in the DNA sequence and represent the epigenetic signal in prokaryotes. DNA methyltransferases are enzymes involved in DNA modification and thus in epigenetic regulation of gene expression. DNA methylation not only affects the thermodynamic stability of DNA, but also changes its curvature. Methylation of specific residues on DNA can affect the protein-DNA interactions. DNA methylation in prokaryotes regulates a number of physiological processes in the bacterial cell including transcription, DNA mismatch repair and replication initiation. Significantly, many reports have suggested a role of DNA methylation in regulating the expression of a number of genes in virulence and pathogenesis thus, making DNA methlytransferases novel targets for the designing of therapeutics. Here, we summarize the current knowledge about the influence of DNA methylation on gene regulation in different bacteria, and on bacterial virulence.

Diversity of DNA methyltransferases that recognize asymmetric target sequences.

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.

Structural similarities between SAM and ATP recognition motifs and detection of ATP binding in a SAM binding DNA methyltransferase.

S-adenosylmethionine (SAM) is a ubiquitous co-factor that serves as a donor for methylation reactions and additionally serves as a donor of other functional groups such as amino and ribosyl moieties in a variety of other biochemical reactions. Such versatility in function is enabled by the ability of SAM to be recognized by a wide variety of protein molecules that vary in their sequences and structural folds. To understand what gives rise to specific SAM binding in diverse proteins, we set out to study if there are any structural patterns at their binding sites. A comprehensive analysis of structures of the binding sites of SAM by all-pair comparison and clustering, indicated the presence of 4 different site-types, only one among them being well studied. For each site-type we decipher the common minimum principle involved in SAM recognition by diverse proteins and derive structural motifs that are characteristic of SAM binding. The presence of the structural motifs with precise three-dimensional arrangement of amino acids in SAM sites that appear to have evolved independently, indicates that these are winning arrangements of residues to bring about SAM recognition. Further, we find high similarity between one of the SAM site types and a well known ATP binding site type. We demonstrate using in vitro experiments that a known SAM binding protein, HpyAII.M1, a type 2 methyltransferase can bind and hydrolyse ATP. We find common structural motifs that explain this, further supported through site-directed mutagenesis. Observation of similar motifs for binding two of the most ubiquitous ligands in multiple protein families with diverse sequences and structural folds presents compelling evidence at the molecular level in favour of convergent evolution.

A nucleotide insertion between two adjacent methyltransferases in Helicobacter pylori results in a bifunctional DNA methyltransferase.

Helicobacter pylori has a dynamic R-M (restriction-modification) system. It is capable of acquiring new R-M systems from the environment in the form of DNA released from other bacteria or other H. pylori strains. Random mutations in R-M genes can result in non-functional R-M systems or R-M systems with new properties. hpyAVIAM and hpyAVIBM are two solitary DNA MTase (methyltransferase) genes adjacent to each other and lacking a cognate restriction enzyme gene in H. pylori strain 26695. Interestingly, in an Indian strain D27, hpyAVIAM-hpyAVIBM encodes a single bifunctional polypeptide due to insertion of a nucleotide just before the stop codon of hpyAVIBM and, when a similar mutation was made in hpyAVIAM-hpyAVIBM from strain 26695, a functional MTase with an N-terminal C5-cytosine MTase domain and a C-terminal N6-adenine MTase domain was constructed. Mutations in the AdoMet (S-adenosylmethionine)-binding motif or in the catalytic motif of M.HpyAVIA or M.HpyAVIB selectively abrogated the C5-cytosine or N6-adenine methylation activity of M.HpyAVIA-M.HpyAVIB fusion protein. The present study highlights the ability of H. pylori to evolve genes with unique functions and thus generate variability. For organisms such as H. pylori, which have a small genome, these adaptations could be important for their survival in the hostile host environment.

Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris.

Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E. coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOX1 promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.