Phasevarion Mediated Epigenetic Gene Regulation in Helicobacter pylori

Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M) system, controls expression of a phase-variable regulon of genes (a “phasevarion”), via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates). Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis

Origin of the Diversity in DNA Recognition Domains in Phasevarion Associated modA Genes of Pathogenic Neisseria and Haemophilus influenzae

Phase variable restriction-modification (R-M) systems have been identified in a range of pathogenic bacteria. In some it has been demonstrated that the random switching of the mod (DNA methyltransferase) gene mediates the coordinated expression of multiple genes and constitutes a phasevarion (phase variable regulon). ModA of Neisseria and Haemophilus influenzae contain a highly variable, DNA recognition domain (DRD) that defines the target sequence that is modified by methylation and is used to define modA alleles. 18 distinct modA alleles have been identified in H. influenzae and the pathogenic Neisseria. To determine the origin of DRD variability, the 18 modA DRDs were used to search the available databases for similar sequences. Significant matches were identified between several modA alleles and mod gene from distinct bacterial species, indicating one source of the DRD variability was via horizontal gene transfer. Comparison of DRD sequences revealed significant mosaicism, indicating exchange between the Neisseria and H. influenzae modA alleles. Regions of high inter- and intra-allele similarity indicate that some modA alleles had undergone recombination more frequently than others, generating further diversity. Furthermore, the DRD from some modA alleles, such as modA12, have been transferred en bloc to replace the DRD from different modA alleles

Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System

Bacterial Type Three Secretion System (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C-terminus of the Shigella flexneri effector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles

Detection of intracellular bacteria in exfoliated urothelial cells from women with urge incontinence

The role of subclinical infection in patients with urge incontinence has been largely ignored. The aim of this study was to test for the presence of intracellular bacteria in exfoliated urothelial cells obtained from the urine of patients with detrusor overactivity or mixed incontinence +/- a history of UTI, and compare this to a control group of patients with stress incontinence and no history of infection. Bacterial cystitis was assessed by routine microbiology and compared to microscopic analysis of urine by Wright staining. Subsequent analysis of urothelial cells by confocal microscopy was performed to determine the existence of intracellular bacteria. Bacterial cystitis was seen in 13% of patients based on routine microbiology. Wright staining of concentrated urothelial cells demonstrated the presence of bacteria in 72% of samples. Filamentous bacterial cells were observed in 51% of patients and were significantly more common in patients with detrusor overactivity. Intracellular Escherichia coli were observed by confocal microscopy. This study supports the possibility that a subset of patients with urge incontinence may have unrecognised chronic bacterial colonisation, maintained via an intracellular reservoir. In patients with negative routine microbiology, application of the techniques used in this study revealed evidence of infection, providing further insights into the aetiology of urge incontinence

Diagrammatical representation of the 18 <i>modA</i> alleles of <i>H. influenzae</i> and pathogenic <i>Neisseria</i>.

<p>A. The 18 <i>modA</i> DNA recognition domains are shown as coloured lines and the colour scheme established in Fox <i>et al.</i> (14) was used here for each representative allele: 1, <i>H. influenzae</i> R3327; 2, <i>H. influenzae</i> 723; 3, <i>H. influenzae</i> R3366; 4, <i>H. influenzae</i> R3579; 5, <i>H. influenzae</i> 1268; 6, <i>H. influenzae</i> C1626; 7, <i>H. influenzae</i> R3265; 8, <i>H. influenzae</i> C505; 9, <i>H. influenzae</i> 1209; 10, <i>H. influenzae</i> R3157; 11, <i>N. meningitidis</i> BZ83; 12, <i>N. gonorrhoeae</i> 01DO64; 13, <i>H. influenzae</i> R3023; 14, <i>H. influenzae</i> R1527; 15, <i>H. influenzae</i> R3570; 16, <i>H. influenzae</i> ATCC9007; 17, <i>H. influenzae</i> ATCC9833; 18, <i>N. meningitidis</i> NGE28. Each <i>modA</i> DNA recognition domain has been assigned a unique colour to permit visual identification of possible recombination events between different <i>modA</i> DNA recognition domains. BLASTn matches longer than 13 nucleotides and >80% identity between the 18 <i>mod</i> alleles were mapped onto the corresponding allele in the appropriate colour. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032337#pone.0032337.s001" target="_blank">Table S1</a> contains the nucleotide coordinates for each BLASTn match. B. Diagrammatical representation of the tBLASTn match between <i>modA10</i> and <i>modA14</i>. Horizontal bars represent the <i>modA</i> alleles 10 and 14 described in panel A, and are coloured according to the same scheme. The vertical bars between the coloured lines indicate the nucleotide match according to BLAST, ranging from light grey (>80% nt identity) to black (100% nt identity).</p

Diagrammatical representation of significant matches.

<p>A. Diagrammatical representation of the <i>H. influenzae</i> and <i>Helicobacter acinonychis mod</i> alignment. The tBLASTn local alignment identified between the DNA recognition domain of <i>mod</i>A4 and <i>Helicobacter acinonychis</i> was further extended to include the entire <i>modA</i> gene of <i>H. influenzae</i>. A nucleotide alignment was completed to include the regions flanking the DRD. The yellow arrow represents entire <i>modA</i> gene and the green arrow the match in <i>H. acinonychis</i>. B. Diagrammatical representation of the <i>H. influenzae</i> and <i>Moraxella catarrhalis mod</i> alignment. The tBLASTn local alignment identified between the DNA recognition domain of <i>mod</i>A5 and <i>Moraxella catarrhalis</i> was further extended to include the entire <i>modA</i> gene of <i>H. influenzae</i>. A nucleotide alignment was completed to include the regions flanking the DRD. The purple arrow represents the entire <i>modA</i> gene in <i>H. influenzae</i> and the pink arrow the match in <i>M. catarrhalis</i>. The nucleotides are represented as vertical blue bars (dark blue >80% identity; light blue >50% identity; white >50% identity or gap). Strain and accession numbers that define the matches are shown to the left. The position of nucleotide that the match occurs is indicated above the coloured arrows and beside the amino acid alignment.</p

Alignment profile for 77 <i>N. meningitidis modA12</i> alleles and flanking regions.

<p>The DRD variable domain, 330 nt upstream and 450 nt downstream were aligned using ClustalW and visualized using Jalview. The nucleotides are represented as vertical bars coloured according to consensus identity (dark blue >90% identity; light blue >50% identity; white <50% identity). Highlighted in red is a block of sequence indicative of recombination, where some sequences are identical to equivalent regions of <i>H. influenzae</i> PittEE, <i>modA6</i>.</p

An unrooted phylogram shows the relationship between 5′ regions of <i>modA</i> genes (ATG to repeat, 410 nt).

<p>Phylogram of the 410 nt <i>modA</i> 5′ nucleotide sequence produced using the Neighbor-joining algorithm, as implemented in PHYLIP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032337#pone.0032337-Felsenstein1" target="_blank">[32]</a>. Taxa that do not cluster according to their cognate <i>mod</i> allele are highlighted: <i>N. meningitidis</i> and <i>N. gonorrhoeae modA11</i> (red), <i>modA12</i> (purple) and <i>modA13</i> (green). Bootstrap values from 1000 replications are shown as percentages for nodes with more than 50% support. Scale bar shows distances in nucleotide substitutions per site.</p

Sequence analysis of <i>N. meningitidis modA12</i> genes.

a<p>Ns = Non-synonymous sites with more than 2 sequences contain a Non-synonymous substitution.</p

Diagrammatical representation of <i>modA</i> and <i>res</i> genes of <i>H. influenzae</i>, <i>N. meningitidis</i> and <i>N. gonorrhoeae</i>.

<p>A. The methyltransferase (<i>modA</i>) genes, and restriction endonuclease (<i>res</i>) genes, with the repeat regions that mediate phase variation. The DNA recognition domain is represented by the striped box <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032337#pone.0032337-Fox1" target="_blank">[14]</a>. B. The variable regions for each of the 18 <i>modA</i> alleles in the multiple sequence alignment were aligned in ClustalW and visualised with JalView using the overview feature. The nucleotides are represented as vertical bars coloured according to consensus identity (dark blue >80% identity; light blue >50% identity; white <50% identity or gap). The <i>mod</i> alleles are as follows: <i>mod</i>A1 <i>Hi</i> R3327 (126508350); <i>mod</i>A2 <i>Hi</i> 723 (126508378); <i>mod</i>A3 <i>Hi</i> R3366 (126508386); <i>mod</i>A4 <i>Hi</i> 3579 (126508396); <i>mod</i>A5 <i>Hi</i> 1268 (126508406); <i>mod</i>A6 <i>Hi</i> C1626 (126508412); <i>mod</i>A7 <i>Hi</i> R3265 (126508420); <i>mod</i>A8 <i>Hi</i> C505 (126508426); <i>mod</i>A9 <i>Hi</i> 1209 (126508430); <i>mod</i>A10 <i>Hi</i> R3157 (126508438); <i>mod</i>A11 <i>Nm</i> BZ83 (257124056); <i>mod</i>A12 <i>Nm</i> 129E (257124138); <i>mod</i>A13 <i>Ng</i> 1291 (26506021); <i>mod</i>A14 <i>Hi</i> R1527 (126508442); <i>mod</i>A15 <i>Hi</i> R3570 (126508452); <i>mod</i>A16 <i>Hi</i> ATCC9007 (126508448); <i>mod</i>A17 <i>Hi</i> ATCC9833 (126508446); <i>mod</i>A18 <i>Nm</i> NGE28 (257124039). As each <i>mod</i> allele is a different length, the number of base pairs is indicated to the right of the nucleotide alignment.</p