Exploring the Roles of Phase Variable HpyAII Restriction-modification System in the Human Pathogen Helicobacter pylori

Abstract

Helicobacter pylori is a Gram-negative microaerophilic bacteria known to infect as much as 80% of some populations with an average morbidity range of around 50% of the world population. It has been recognized as a definitive carcinogen (Type I). H. pylori shows extraordinary genetic diversity and this property is critical to its success as a human pathogen. High genetic diversity and interstrain variations seen in H. pylori is attributed to its remarkable ability to take foreign DNA by natural transformation. Natural transformation in H. pylori is governed to some extent by the presence of Restriction-Modification systems (R-M systems). Three types of DNA methylation are associated with R-M systems in bacteria, N6-adenine (m6A), C5-cytosine (m5C) and N4-cytosine (m4C). Recent studies in pathogenic bacteria have shown the epigenetic roles of m6A in virulence, gene regulation and genetic evolution of the organism. This is in contrast to eukaryotes where m5C is known to be the epigenetic signal. In mammals and plants DNA cytosine methyltransferases epigenetically regulate the gene expression through the precise epigenetic modification of certain cytosine residues with a methyl group. Moreover, aberrant methylation patterns are embryonic lethal in mammals, and can also lead to diseases including cancer. In plant it can result in pleiotropic morphological defects. The role of cytosine methylation in bacteria is not very well known. A recent study has shown that the loss of m5C in H. pylori strains alters the expression of genes involved in motility, adhesion, and virulence. Another study in E. coli has shown the role of m5C in stationary phase stress regulation. However, no physiological role of the other form of cytosine methylation (m4C), aside restriction protection is known in bacteria. Genome sequences of various strains of H. pylori reveal an abundance of R-M systems. Typically 25-34 R-M systems are present in different H. pylori strains. Methylome analysis of H. pylori 26695 strain has revealed the presence of numerous m6A and m5C methyltransferases. H. pylori 26695 strain harbors a phase variable type IIS HpyAII R-M system. This R-M system is composed of two exocyclic methyltransferases, M1.HpyAII (m6A) and M2.HpyAII (m4C) and one type IIS phase variable endonuclease (HpyAII). HpyAII recognizes the sequence 5' GAAGA 3' / 3' CTTCT 5' and cleaves eight bp downstream on the top strand and seven bp downstream on the bottom strand. HpyAII is a novel phase-variable restriction endonuclease containing multiple repetitive stretches of adenine residues in the ORF. M1.HpyAII methylates the final adenine residue of GAAGA sequence, whereas M2.HpyAII methylates the first cytosine of the complementary TCTTC sequence. M2.HpyAII is the only N4-cytosine (m4C) MTase present in H. pylori strain 26695. The aim of the present study is to understand the potential epigenetic role of m4C modification by understanding the roles of HpyAII R-M system in virulence, gene expression and natural transformation of H. pylori. Understanding the biochemical properties of the novel phase variable HpyAII endonuclease can reveal critical information about the regulation of natural transformation in H. pylori. Bioinformatics analysis shows that HpyAII is an HNH catalytic motif containing endonuclease. The biochemical study on HpyAII indicates that the enzyme prefers two-site substrate over a one-site substrate for maximal activity. A strong preference for two-sites was observed with supercoiled plasmid and oligonucleotide duplex DNA. Cofactor analysis revealed the preference of R.HpyAII for transition metals (Ni2+, Cd2+, and Co2+) over alkaline earth metals (Mg2+, Ca2+) for maximal cleavage activity. Mutational analysis of the conserved residues of the HNH motif in HpyAII confirmed the presence of functional HNH motif. Interestingly, mutation of first His residue (general acid) of the HNH motif to Ala does not abolish the enzymatic activity but instead causes loss of fidelity compared to wild type HpyAII. The H328A mutant displayed promiscuous DNA cleavage activity on different DNA substrates. The novelty of this observation lies in the fact that mutation of first His residue (general acid) of the HNH motif in other known HNH motif containing enzymes has always abolished enzymatic activity. Mutation at a single amino acid residue leading to the loss of fidelity provides insights into the regulation of fidelity and evolution of restriction enzymes by point mutation.