Variety of repeat types found in pathogenic and commensal <i>Neisseria ssp</i>.

<p>Variety of repeat types found in pathogenic and commensal <i>Neisseria ssp</i>.</p

Range of phase variable genes identified in each species.

<p>Data shown the median, range, upper and lower quartile number of PV genes, as indicated by presence of a repeat tract. These data exclude gene groupings which contain dinucleotide repeat tracts, due to the insufficient evidence of phase variation associated with dinucleotide repeats in the literature, and the loci discussed herein. Statistical analysis were performed with a Kruskal-Wallis test with Dunn’s multiple comparisons. NS; not significant, ***; p-value of <0.0005.</p

Flowchart and visual output of Phasome<i>It</i>.

<p><b>(A)</b> Outputs of Phasome<i>It</i> can be viewed visually on the index page. Green bars indicate there is an homopolymeric tract within the open reading frame; orange bars indicate there is an SSR close to the gene of interest (for example in a promoter region); grey bars indicate there is a non-PV gene homologous to a PV gene in that same homology grouping; the remaining coloured bars are indicative of SSRs other than homopolymers which can be further derived from the dataset below the visual output. <b>(B)</b> Gene groupings corresponding to the visual output are found in a table below. From here, functions, PV status in each strain and tract entries can be obtained for the grouping of interest. The full dataset from which this figure is derived, containing further phasome information not discussed in this manuscript are available (<a href="https://figshare.com/s/d31b7b0b6ca4aeeb48df" target="_blank">https://figshare.com/s/d31b7b0b6ca4aeeb48df</a>). A red outline shows highlights both the graphical and interactive outputs for the <i>opa</i> loci as an example.</p

Distribution of phase variable genes between phase variable modules.

<p>Distribution of phase variable genes between phase variable modules.</p

Tract length distribution in different <i>Neisseria</i> species.

<p>Data are represented by heat maps. Colour intensity represents the percentage that a given tract length comprises of the total number of identified tracts of that type for each species. ‘-’ is indicative of no identified repeats of the given length. Information on the numbers of strains for each species, and numbers of tract lengths analysed can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196675#pone.0196675.t001" target="_blank">Table 1</a>.</p

Core phasome analysis of a subset of <i>Neisseria</i> species.

<p>Core phasome analysis of a subset of <i>Neisseria</i> species.</p

Phasome analysis of pathogenic and commensal Neisseria species expands the known repertoire of phase variable genes, and highlights common adaptive strategies

Pathogenic Neisseria are responsible for significantly higher levels of morbidity and mortality than their commensal relatives despite having similar genetic contents. Neisseria possess a disparate arsenal of surface determinants that facilitate host colonisation and evasion of the immune response during persistent carriage. Adaptation to rapid changes in these hostile host environments is enabled by phase variation (PV) involving high frequency, stochastic switches in expression of surface determinants. In this study, we analysed 89 complete and 79 partial genomes, from the NCBI and Neisseria PubMLST databases, representative of multiple pathogenic and commensal species of Neisseria using PhasomeIt, a new program that identifies putatively phase-variable genes and homology groups by the presence of simple sequence repeats (SSR). We detected a repertoire of 884 putative PV loci with maxima of 54 and 47 per genome in gonococcal and meningococcal isolates, respectively. Most commensal species encoded a lower number of PV genes (between 5 and 30) except N. lactamica wherein the potential for PV (36–82 loci) was higher, implying that PV is an adaptive mechanism for persistence in this species. We also characterised the repeat types and numbers in both pathogenic and commensal species. Conservation of SSR-mediated PV was frequently observed in outer membrane proteins or modifiers of outer membrane determinants. Intermittent and weak selection for evolution of SSR-mediated PV was suggested by poor conservation of tracts with novel PV genes often occurring in only one isolate. Finally, we describe core phasomes—the conserved repertoires of phase-variable genes—for each species that identify overlapping but distinctive adaptive strategies for the pathogenic and commensal members of the Neisseria genus

Exemplar gene groupings associated with in frame and read through phase variation.

<p>Exemplar gene groupings associated with in frame and read through phase variation.</p

PhasomeIt: an 'omics' approach to cataloguing the potential breadth of phase variation in the genus Campylobacter.

Hypermutable simple sequence repeats (SSRs) are drivers of phase variation (PV) whose stochastic, high-frequency, reversible switches in gene expression are a common feature of several pathogenic bacterial species, including the human pathogen Campylobacter jejuni. Here we examine the distribution and conservation of known and putative SSR-driven phase variable genes - the phasome - in the genus Campylobacter. PhasomeIt, a new program, was specifically designed for rapid identification of SSR-mediated PV. This program detects the location, type and repeat number of every SSR. Each SSR is linked to a specific gene and its putative expression state. Other outputs include conservation of SSR-driven phase-variable genes and the 'core phasome' - the minimal set of PV genes in a phylogenetic grouping. Analysis of 77 complete Campylobacter genome sequences detected a 'core phasome' of conserved PV genes in each species and a large number of rare PV genes with few, or no, homologues in other genome sequences. Analysis of a set of partial genome sequences, with food-chain-associated metadata, detected evidence of a weak link between phasome and source host for disease-causing isolates of sequence type (ST)-828 but not the ST-21 or ST-45 complexes. Investigation of the phasomes in the genus Campylobacter provided evidence of overlapping but distinctive mechanisms of PV-mediated adaptation to specific niches. This suggests that the phasome could be involved in host adaptation and spread of campylobacters. Finally, this tool is malleable and will have utility for studying the distribution and genic effects of other repetitive elements in diverse bacterial species

Clustered intergenic region sequences as predictors of factor H Binding Protein expression patterns and for assessing Neisseria meningitidis strain coverage by meningococcal vaccines

Factor H binding protein (fHbp) is a major protective antigen in 4C-MenB (Bexsero®) and Trumenba®, two serogroup B meningococcal vaccines, wherein expression level is a determinant of protection. Examination of promoter-containing intergenic region (IGR) sequences indicated that nine fHbp IGR alleles covered 92% of 1,032 invasive meningococcal strains with variant 1 fHbp alleles. Relative expression values for fHbp were determined for 79 meningococcal isolates covering ten IGR alleles by quantitative reverse transcriptase polymerase chain reaction (qRT PCR). Derivation of expression clusters of IGR sequences by linear regression identified five expression clusters with five nucleotides and one insertion showing statistically associations with differences in expression level. Sequence analysis of 273 isolates examined by the Meningococcal Antigen Typing Scheme, a sandwich ELISA, found that coverage depended on the IGR expression cluster and vaccine peptide homology combination. Specific fHbp peptide-IGR expression cluster combinations were designated as 'at risk' for coverage by 4C-MenB and were detected in multiple invasive meningococcal disease cases confirmed by PCR alone and occurring in partially-vaccinated infants. We conclude that sequence-based analysis of IGR sequences is informative for assessing protein expression and has utility for culture-independent assessments of strain coverage by protein-based vaccines