Genomic and global approaches to unravelling how hypermutable sequences influence bacterial pathogenesis

Rapid adaptation to fluctuations in the host milieu contributes to the host persistence and virulence of bacterial pathogens. Adaptation is frequently mediated by hypermutable sequences in bacterial pathogens. Early bacterial genomic studies identified the multiplicity and virulence-associated functions of these hypermutable sequences. Thus, simple sequence repeat tracts (SSRs) and site-specific recombination were found to control capsular type, lipopolysaccharide structure, pilin diversity and the expression of outer membrane proteins. We review how the population diversity inherent in the SSR-mediated mechanism of localised hypermutation is being unlocked by the investigation of whole genome sequences of disease isolates, analysis of clinical samples and use of model systems. A contrast is presented between the problematical nature of analysing simple sequence repeats in next generation sequencing data and in simpler, pragmatic PCR-based approaches. Specific examples are presented of the potential relevance of this localized hypermutation to meningococcal pathogenesis. This leads us to speculate on the future prospects for unravelling how hypermutable mechanisms may contribute to the transmission, spread and persistence of bacterial pathogens

Phase Variation of PorA, a Major Outer Membrane Protein, Mediates Escape of Bactericidal Antibodies by Neisseria meningitides

Several outer membrane proteins of Neisseria meningitidis are subject to phase variation due to alterations in simple sequence repeat tracts. The PorA protein is a major outer membrane protein and a target for protective host immune responses. Phase variation of PorA is mediated by a poly-G repeat tract present within the promoter, leading to alterations in protein expression levels. N. meningitidis strain 8047 was subjected to serial passage in the presence of P1.2, a PorA-specific bactericidal monoclonal antibody. Rapid development of resistance to bactericidal activity was associated with a switch in the PorA repeat tract from 11G to 10G. Phase variants with a 10G repeat tract exhibited a 2-fold reduction in surface expression of PorA protein. A mutS mutant of strain 8047, with an elevated phase variation rate, exhibited a higher rate of escape and an association of escape with 10G and 9G variants, the latter having a 13-fold reduction in surface expression of PorA. We conclude that graduated reductions in the surface expression of outer membrane proteins mediated by phase variation enable meningococci to escape killing in vitro by bactericidal antibodies. These findings indicate how phase variation could have a major impact on immune escape and host persistence of meningococci

Men B : lack of coverage or vaccine failure?

Men B : lack of coverage or vaccine failure

Destabilization of tetranucleotide repeats in Haemophilus influenzae mutants lacking RnaseHI or the Klenow domain of PolI.

A feature of Haemophilus influenzae genomes is the presence of several loci containing tracts of six or more identical tetranucleotide repeat units. These repeat tracts are unstable and mediate high frequency, reversible alterations in the expression of surface antigens. This process, termed phase variation (PV), enables H.influenzae to rapidly adapt to fluctuations in the host environment. Perturbation of lagging strand DNA synthesis is known to destabilize simple sequence repeats in yeast and Escherichia coli. By using a chromosomally located reporter construct, we demonstrated that the mutation of an H.influenzae rnhA (encoding RnaseHI) homologue increases the mutation rates of tetranucleotide repeats ∼3-fold. Additionally, deletion of the Klenow domain of DNA polymerase I (PolI) resulted in a ∼35-fold increase in tetranucleotide repeat-mediated PV rates. Deletion of the PolI 5′>3′ exonuclease domain appears to be lethal. The phenotypes of these mutants suggest that delayed or mutagenic Okazaki fragment processing destabilizes H.influenzae tetranucleotide repeat tracts

Phage-Resistant Phase-Variant Sub-populations Mediate Herd Immunity Against Bacteriophage Invasion of Bacterial Meta-Populations

Hypermutable loci are widespread in bacteria as mechanisms for rapid generation of phenotypic diversity within a population that enables survival of fluctuating, often antagonistic, selection pressures. Localized hypermutation can mediate phase variation and enable survival of bacteriophage predation due to high frequency, reversible alterations in the expression of phage receptors. As phase variation can also generate population-to-population heterogeneity, we hypothesized that this phenomenon may facilitate survival of spatially-separated bacterial populations from phage invasion in a manner analogous to herd immunity to infectious diseases in human populations. The lic2A gene of Haemophilus influenzae is subject to “ON” and “OFF” switches in expression mediated by mutations in a 5′CAAT repeat tract present within the reading frame. The enzyme encoded by lic2A mediates addition of a galactose moiety of the lipopolysaccharide. This moiety is required for attachment of the HP1C1 phage such that the ON state of the lic2A gene is associated with HP1c1 susceptibility while the OFF state is resistant to infection. We developed an “oscillating prey assay” to examine phage spread through a series of sub-populations of Haemophilus influenzae whose phage receptor is in an ON or OFF state. Phage extinction was frequently observed when the proportion of phage-resistant sub-populations exceeded 34%. In silico modeling indicated that phage extinction was interdependent on phage loss during transfer between sub-populations and the frequency of resistant sub-populations. In a fixed-area oscillating prey assay, heterogeneity in phage resistance was observed to generate vast differences in phage densities across a meta-population of multiple bacterial sub-populations resulting in protective quarantining of some sub-populations from phage attack. We conclude that phase-variable hypermutable loci produce bacterial “herd immunity” with resistant intermediary-populations acting as a barricade to reduce the viral load faced by phage-susceptible sub-populations. This paradigm of meta-population protection is applicable to evolution of hypermutable loci in multiple bacteria-phage and host-pathogen interactions

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

High Throughput Method for Analysis of Repeat Number for 28 Phase Variable Loci of Campylobacter jejuni Strain NCTC11168

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method—the 28-locus-CJ11168 PV-analysis assay—for rapidly determining ON/OFF states of 28 of these phase-variable loci in a large number of individual colonies from C. jejuni strain NCTC11168. The method combines a series of multiplex PCR assays with a fragment analysis assay and automated extraction of fragment length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. Application of this method to analysis of the 28 polyG/polyC tracts in 90 C. jejuni colonies detected a 2.5-fold increase in slippage products as tracts lengthened from G8 to G11 but no difference between tracts of similar length indicating that flanking sequence does not influence slippage rates. Comparison of this observed slippage to previously measured mutation rates for G8 and G11 tracts in C. jejuni indicates that PCR amplification of a DNA sample will over-estimate phase variation frequencies by 20-35-fold. An important output of the 28-locus-CJ11168 PV-analysis assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species

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

Variation in the genomic locations and sequence conservation of STAR elements among staphylococcal species provides insight into DNA repeat evolution.

Background:Staphylococcus aureus Repeat (STAR) elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. Results:Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. Conclusions:The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis

Random sorting of Campylobacter jejuni phase variants due to a narrow bottleneck during colonization of broiler chickens.

Phase variation (PV), involving stochastic switches in gene expression, is exploited by the human pathogen Campylobacter jejuni to adapt to different environmental and host niches. Phase-variable genes of C. jejuni modulate expression of multiple surface determinants, and hence may influence host colonization. Population bottlenecks can rapidly remove the diversity generated by PV, and strict single-cell bottlenecks can lead to propagation of PV states with highly divergent phenotypes. Using a combination of high-throughput fragment size analysis and comparison with in vivo and in silico bottleneck models, we have characterized a narrow population bottleneck during the experimental colonization of broiler chickens with C. jejuni strain 81-176. We identified high levels of variation in five PV genes in the inoculum, and subsequently, massively decreased population diversity following colonization. Each bird contained a dominant five-gene phasotype that was present in the inoculum indicative of random sorting through a narrow, non-selective bottleneck during colonization. These results are evidence of the potential for confounding effects of PV on in vivo studies of Campylobacter colonization factors and poultry vaccine studies. Our results are also an argument for population bottlenecks as mediators of stochastic variability in the propensity to survive through the food chain and cause clinical human disease