Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori.

Bacterial antibiotic resistance is a major threat to human health. A combination of antibiotics with metals is among the proposed alternative treatments. Only one such combination is successfully used in clinics; it associates antibiotics with the metal bismuth to treat infections by Helicobacter pylori. This bacterial pathogen colonizes the human stomach and is associated with gastric cancer, killing 800,000 individuals yearly. The effect of bismuth in H. pylori treatment is not well understood in particular for sublethal doses such as those measured in the plasma of treated patients. We addressed this question and observed that bismuth induces the formation of homogeneously sized membrane vesicles (MVs) with unique protein cargo content enriched in bismuth-binding proteins, as shown by quantitative proteomics. Purified MVs of bismuth-exposed bacteria were strongly enriched in bismuth as measured by inductively coupled plasma optical emission spectrometry (ICP-OES), unlike bacterial cells from which they originate. Thus, our results revealed a novel function of MVs in bismuth detoxification, where secreted MVs act as tool to discard bismuth from the bacteria. Bismuth also induces the formation of intracellular polyphosphate granules that are associated with changes in nucleoid structure. Nucleoid compaction in response to bismuth was established by immunogold electron microscopy and refined by the first chromosome conformation capture (Hi-C) analysis of H. pylori. Our results reveal that even low doses of bismuth induce profound changes in H. pylori physiology and highlight a novel defense mechanism that involves MV-mediated bismuth extrusion from the bacteria and a probable local DNA protective response where polyphosphate granules are associated with nucleoid compaction. IMPORTANCE Bacterial resistance to antibiotics is a major threat to human health. Treatments combining antibiotics with metals were proposed to circumvent this hurdle. Only one such combination is successfully used in clinics associating antibiotics with the metal bismuth to treat infections by the human pathogen Helicobacter pylori. H. pylori causes 800,000 deaths by gastric cancer yearly. How bismuth impacts H. pylori and its response to this toxic metal were ill defined. We discovered that upon bismuth exposure, H. pylori secretes membrane vesicles that are enriched in bismuth. Bismuth also induces the formation of intracellular polyphosphate granules associated with compaction of the chromosome. Upon bismuth exposure, H. pylori displays both defense and protection mechanisms, with bismuth extrusion by vesicles and shielding of the chromosome.

Nickel, an essential virulence determinant of Helicobacter pylori: Transport and trafficking pathways and their targeting by bismuth.

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Nickel is required for the pathogenicity of H. pylori. This bacterial pathogen colonizes the stomach of about half of the human population worldwide and is associated with gastric cancer that is responsible for 800,000 deaths per year. H. pylori possesses two nickel-enzymes that are essential for in vivo colonization, a [NiFe] hydrogenase and an abundant urease responsible for resistance to gastric acidity. Because of these two enzymes, survival of H. pylori relies on an important supply of nickel, implying tight control strategies to avoid its toxic accumulation or deprivation. H. pylori possesses original mechanisms for nickel uptake, distribution, storage and trafficking that will be discussed in this review. During evolution, acquisition of nickel transporters and specific nickel-binding proteins has been a decisive event to allow Helicobacter species to become able to colonize the stomach. Accordingly, many of the factors involved in these mechanisms are required for mouse colonization by H. pylori. These mechanisms are controlled at different levels including protein interaction networks, transcriptional, post-transcriptional and post-translational regulation. Bismuth is another metal used in combination with antibiotics to efficiently treat H. pylori infections. Although the precise mode of action of bismuth is unknown, many targets have been identified in H. pylori and there is growing evidence that bismuth interferes with the essential nickel pathways. Understanding the metal pathways will help improve treatments against H. pylori and other pathogens.

Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues.

Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction-modification (R-M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni2+ over Mg2+. Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first Histidine residue of the HNH motif to Alanine does not abolish the enzymatic activity, but instead causes loss of fidelity compared with wildtype HpyAII. Previous studies have shown that mutation of the first Histidine residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. The present study provides further insights into the evolution of restriction enzymes.

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.