Helicobacter Pylori Restriction-Modification Systems : Possible Roles Beyond Genome Protection

Abstract

Helicobacter pylori is one of the most potential and successful human pathogen which colonizes atleast 50% of world population. One of the important characteristics of this pathogen is the degree of allelic diversity and genetic variability which helps it to adapt and colonize. Phase variation is one of the mechanisms used by Helicobacter pylori to generate variation, where presence of homopolymeric nucleotide or dinucleotide repeats in an ORF make it prone to frequent length changes as a consequence of slipped strand mispairing mediated mutagenesis. An important feature of H. pylori biology is the presence of a large number of Restriction-Modification (R-M) systems in its genome. Till date, seven strains have been completely sequenced and each have more than 20 R-M systems. The presence of homopolymeric nucleotide or dinucleotide repeats in many of R-M systems make them an interesting subject for investigation. Here, we show that hp0051 which codes for a C5 cytosine methyltransferase from H. pylori is a hypermutable gene, which undergoes random mutations. In addition it exhibits phase variation due to presence of a dinucleotide (AG) repeat which results in a truncated protein. hp0051 homologs were amplified and sequenced from different clinical isolates of H. pylori. Sequence analysis showed that hp0051 homologs from 23 clinical isolates are different from each other suggesting a hypervariable nature of the sequence. It was observed that when over expressed in E. coli hp0051 undergoes random mutations. These mutations give rise to different variants of HP0051 with different biochemical properties. Different variants of HP0051 were biochemically characterized. All variants recognize 5´-CCTC-3´ and methylate the first cytosine. A few of the isoforms exhibit degeneracy in the recognition site as they recognize 5´- CNCC-3´ (N being any nucleotide) and methylates third cytosine. Molecular modelling studies suggest that HP0051 has two domains, one large domain having catalytic and AdoMet binding motifs and small domain having target recognition domain. DNA sequencing, peptide finger mapping, MALDI MS-MS and CD have been used to establish the differences between the different isoforms of HP0051. Interestingly when a mutant protein which lacks methylation activity was expressed in E.coli random mutations were not observed. To understand the role of methylation in the occurrence of random mutations, microarray analysis was done. Microarray analysis have shown that the overexpression of HP0051 results in the down regulation of deoxyadenine methyltransferase (dam) in E.coli. Microarray data were further authenticated by RT PCR analysis. dam plays a vital role in mismatch repair pathway and down regulation of dam results in enhanced mutation rates. A large number of clinical isolates were analysed for the presence of hp0051 and hp0051 was found to be present in 83% of strains obtained from patients compared to 25 % of strains from healthy volunteers. Single colonies obtained from the same patient were analysed and it was found that variation in hp0051 exists within a patient also. Deletion of an orphan C5 cytosine methyltransferase, hp0051 in H. pylori strains 26695, SS1 and 98.4 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. 98.4∆hp0051 mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. H. pylori strain 26695∆hp0051 is more motile than the wild- type. hp0051 from strain 26695 is able to complement mutant SS1 and 98.4 strains. This study highlights the possible significance of cytosine methylation in the physiology of H. pylori. hp0050 is a N6 DNA adenine methyltransferase which overlaps with the hp0051 ORF .hp0050 was cloned, over expressed and purified to near homogeneity. It recognizes the sequence 5´GRRG 3´ (where R is A or G) and most intriguingly methylates both adenines when R is A (5´GAAG 3´). Kinetic analysis suggest a non processive (repeated hit) mechanism of methylation in which HP0050 methyltransferase methylates one adenine at a time in sequence 5´GAAG 3´. Interestingly, HP0050 homologs from two clinical strains PG227 and 128 methylate only 5´GAGG 3´ compared to 5´GRRG 3´ in strain 26695. HP0050 MTase is highly conserved as it is present in more than 90% of strains. Inactivation of hp0050 in strain PG227 resulted in poor growth suggesting its important role in the physiology of Helicobacter pylori. Collectively, these findings provide impetus for exploring the role(s) of this conserved DNA methyltransferase in the cellular processes of Helicobacter pylori. In one of the clinical isolate it was found that hp0051 and hp0050 can code for a single polypeptide due to an insertion mutation. This mutant ( hp0050 and hp0051 fusion ) was cloned, overexpressed and purified. It was found that fusion protein is able to methylate both adenine and cytosine in the cognate sequence. Similarly, hp1369 - hp1370 is a phase variable type III MTase and it belongs to ɛ group of MTases based on the arrangement of motifs. It has a poly G repeat in its ORF and any change in the number of repeats can result in a functional (full length) or non functional (truncated) protein. Within strain 26695, it has 10 G repeat which results in a truncated protein. Addition of a single nucleotide by site directed mutagenesis in the repeat results in a full length functional protein. HP1369_HP1370 fusion protein recognizes and methylates 5´ TCAGC 3´. DNA methyltransferases are known to play a critical role in gene regulation, cell cycle regulation and pathogenesity in a number of pathogens. H. pylori genome is rich in DNA methyltransferases and this study shows that these methyltransferases exhibit unique features like phase - variation and polymorphism .We propose that high degree of variation that exists in these methyltransferases could play a vital role in enhancing the ability of H. pylori to adapt its host.