The Helicobacter pylori UvrC Nuclease Is Essential for Chromosomal Microimports after Natural Transformation.

Helicobacter pylori is a Gram-negative bacterial carcinogenic pathogen that infects the stomachs of half of the human population. It is a natural mutator due to a deficient DNA mismatch repair pathway and is naturally competent for transformation. As a result, it is one of the most genetically diverse human bacterial pathogens. The length of chromosomal imports in H. pylori follows an unusual bimodal distribution consisting of macroimports with a mean length of 1,645 bp and microimports with a mean length of 28 bp. The mechanisms responsible for this import pattern were unknown. Here, we used a high-throughput whole-genome transformation assay to elucidate the role of nucleotide excision repair pathway (NER) components on import length distribution. The data show that the integration of microimports depended on the activity of the UvrC endonuclease, while none of the other components of the NER pathway was required. Using H. pylori site-directed mutants, we showed that the widely conserved UvrC nuclease active sites, while essential for protection from UV light, one of the canonical NER functions, are not required for generation of microimports. A quantitative analysis of recombination patterns based on over 1,000 imports from over 200 sequenced recombinant genomes showed that microimports occur frequently within clusters of multiple imports, strongly suggesting they derive from a single strand invasion event. We propose a hypothetical model of homologous recombination in H. pylori, involving a novel function of UvrC, that reconciles the available experimental data about recombination patterns in H. pylori. IMPORTANCE Helicobacter pylori is one of the most common and genetically diverse human bacterial pathogens. It is responsible for chronic gastritis and represents the main risk factor for gastric cancer. In H. pylori, DNA fragments can be imported by recombination during natural transformation. The length of those fragments determines how many potentially beneficial or deleterious alleles are acquired and thus influences adaptation to the gastric niche. Here, we used a transformation assay to examine imported fragments across the chromosome. We show that UvrC, an endonuclease involved in DNA repair, is responsible for the specific integration of short DNA fragments. This suggests that short and long fragments are imported through distinct recombination pathways. We also show that short fragments are frequently clustered with longer fragments, suggesting that both pathways may be mechanistically linked. These findings provide a novel basis to explain how H. pylori can fine-tune the genetic diversity acquired by transformation.

Evolved to vary: genome and epigenome variation in the human pathogen Helicobacter pylori.

Helicobacter pylori is a Gram-negative, spiral shaped bacterium that selectively and chronically infects the gastric mucosa of humans. The clinical course of this infection can range from lifelong asymptomatic infection to severe disease, including peptic ulcers or gastric cancer. The high mutation rate and natural competence typical of this species are responsible for massive inter-strain genetic variation exceeding that observed in all other bacterial human pathogens. The adaptive value of such a plastic genome is thought to derive from a rapid exploration of the fitness landscape resulting in fast adaptation to the changing conditions of the gastric environment. Nevertheless, diversity is also lost through recurrent bottlenecks and H. pylori's lifestyle is thus a perpetual race to maintain an appropriate pool of standing genetic variation able to withstand selection events. Another aspect of H. pylori's diversity is a large and variable repertoire of restriction-modification systems. While not yet completely understood, methylome evolution could generate enough transcriptomic variation to provide another intricate layer of adaptive potential. This review provides an up to date synopsis of this rapidly emerging area of H. pylori research that has been enabled by the ever-increasing throughput of Omics technologies and a multitude of other technological advances.

In Vivo Genome and Methylome Adaptation of cag-Negative Helicobacter pylori during Experimental Human Infection.

Multiple studies have demonstrated rapid bacterial genome evolution during chronic infection with Helicobacter pylori In contrast, little was known about genetic changes during the first stages of infection, when selective pressure is likely to be highest. Using single-molecule, real-time (SMRT) and Illumina sequencing technologies, we analyzed genome and methylome evolution during the first 10 weeks of infection by comparing the cag pathogenicity island (cagPAI)-negative H. pylori challenge strain BCS 100 with pairs of H. pylori reisolates from gastric antrum and corpus biopsy specimens of 10 human volunteers who had been infected with this strain as part of a vaccine trial. Most genetic changes detected in the reisolates affected genes with a surface-related role or a predicted function in peptide uptake. Apart from phenotypic changes of the bacterial envelope, a duplication of the catalase gene was observed in one reisolate, which resulted in higher catalase activity and improved survival under oxidative stress conditions. The methylomes also varied in some of the reisolates, mostly by activity switching of phase-variable methyltransferase (MTase) genes. The observed in vivo mutation spectrum was remarkable for a very high proportion of nonsynonymous mutations. Although the data showed substantial within-strain genome diversity in the challenge strain, most antrum and corpus reisolates from the same volunteers were highly similar to each other, indicating that the challenge infection represents a major selective bottleneck shaping the transmitted population. Our findings suggest rapid in vivo selection of H. pylori during early-phase infection providing adaptation to different individuals by common mechanisms of genetic and epigenetic alterations.IMPORTANCE Exceptional genetic diversity and variability are hallmarks of Helicobacter pylori, but the biological role of this plasticity remains incompletely understood. Here, we had the rare opportunity to investigate the molecular evolution during the first weeks of H. pylori infection by comparing the genomes and epigenomes of H. pylori strain BCS 100 used to challenge human volunteers in a vaccine trial with those of bacteria reisolated from the volunteers 10 weeks after the challenge. The data provide molecular insights into the process of establishment of this highly versatile pathogen in 10 different human individual hosts, showing, for example, selection for changes in host-interaction molecules as well as changes in epigenetic methylation patterns. The data provide important clues to the early adaptation of H. pylori to new host niches after transmission, which we believe is vital to understand its success as a chronic pathogen and develop more efficient treatments and vaccines.

The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori.

Helicobacter pylori encodes a large number of restriction-modification (R-M) systems despite its small genome. R-M systems have been described as 'primitive immune systems' in bacteria, but the role of methylation in bacterial gene regulation and other processes is increasingly accepted. Every H. pylori strain harbours a unique set of R-M systems resulting in a highly diverse methylome. We identified a highly conserved GCGC-specific m5C MTase (JHP1050) that was predicted to be active in all of 459 H. pylori genome sequences analyzed. Transcriptome analysis of two H. pylori strains and their respective MTase mutants showed that inactivation of the MTase led to changes in the expression of 225 genes in strain J99, and 29 genes in strain BCM-300. Ten genes were differentially expressed in both mutated strains. Combining bioinformatic analysis and site-directed mutagenesis, we demonstrated that motifs overlapping the promoter influence the expression of genes directly, while methylation of other motifs might cause secondary effects. Thus, m5C methylation modifies the transcription of multiple genes, affecting important phenotypic traits that include adherence to host cells, natural competence for DNA uptake, bacterial cell shape, and susceptibility to copper.

Genome and Methylome Variation in Helicobacter pylori With a cag Pathogenicity Island During Early Stages of Human Infection.

BACKGROUND & AIMS: Helicobacter pylori is remarkable for its genetic variation; yet, little is known about its genetic changes during early stages of human infection, as the bacteria adapt to their new environment. We analyzed genome and methylome variations in a fully virulent strain of H pylori during experimental infection. METHODS: We performed a randomized Phase I/II, observer-blind, placebo-controlled study of 12 healthy, H pylori-negative adults in Germany from October 2008 through March 2010. The volunteers were given a prophylactic vaccine candidate (n = 7) or placebo (n = 5) and then challenged with H pylori strain BCM-300. Biopsy samples were collected and H pylori were isolated. Genomes of the challenge strain and 12 reisolates, obtained 12 weeks after (or in 1 case, 62 weeks after) infection were sequenced by single-molecule, real-time technology, which, in parallel, permitted determination of genome-wide methylation patterns for all strains. Functional effects of genetic changes observed in H pylori strains during human infection were assessed by measuring release of interleukin 8 from AGS cells (to detect cag pathogenicity island function), neutral red uptake (to detect vacuolating cytotoxin activity), and adhesion assays. RESULTS: The observed mutation rate was in agreement with rates previously determined from patients with chronic H pylori infections, without evidence of a mutation burst. A loss of cag pathogenicity island function was observed in 3 reisolates. In addition, 3 reisolates from the vaccine group acquired mutations in the vacuolating cytotoxin gene vacA, resulting in loss of vacuolization activity. We observed interstrain variation in methylomes due to phase variation in genes encoding methyltransferases. CONCLUSIONS: We analyzed adaptation of a fully virulent strain of H pylori to 12 different volunteers to obtain a robust estimate of the frequency of genetic and epigenetic changes in the absence of interstrain recombination. Our findings indicate that the large amount of genetic variation in H pylori poses a challenge to vaccine development. ClinicalTrials.gov no: NCT00736476.

Two different rpf clusters distributed among a population of Stenotrophomonas maltophilia clinical strains display differential diffusible signal factor production and virulence regulation.

The quorum-sensing (QS) system present in the emerging nosocomial pathogen Stenotrophomonas maltophilia is based on the signaling molecule diffusible signal factor (DSF). Production and detection of DSF are governed by the rpf cluster, which encodes the synthase RpfF and the sensor RpfC, among other components. Despite a well-studied system, little is known about its implication in virulence regulation in S. maltophilia. Here, we have analyzed the rpfF gene from 82 S. maltophilia clinical isolates. Although rpfF was found to be present in all of the strains, it showed substantial variation, with two populations (rpfF-1 and rpfF-2) clearly distinguishable by the N-terminal region of the protein. Analysis of rpfC in seven complete genome sequences revealed a corresponding variability in the N-terminal transmembrane domain of its product, suggesting that each RpfF variant has an associated RpfC variant. We show that only RpfC-RpfF-1 variant strains display detectable DSF production. Heterologous rpfF complementation of ΔrpfF mutants of a representative strain of each variant suggests that RpfF-2 is, however, functional and that the observed DSF-deficient phenotype of RpfC-RpfF-2 variant strains is due to permanent repression of RpfF-2 by RpfC-2. This is corroborated by the ΔrpfC mutant of the RpfC-RpfF-2 representative strain. In line with this observations, deletion of rpfF from the RpfC-RpfF-1 strain leads to an increase in biofilm formation, a decrease in swarming motility, and relative attenuation in the Caenorhabditis elegans and zebrafish infection models, whereas deletion of the same gene from the representative RpfC-RpfF-2 strain has no significant effect on these virulence-related phenotypes.