Development of a robust and quantitative high-throughput screening method for assessing phenotypic variation in large Neisseria meningitidis isolate collections

Genome-wide association studies are a powerful approach for identifying determinants of disease. For infectious diseases, high throughput assays are required for measuring the variance in multiple virulence-related phenotypes of large bacterial isolate collections and for association of this phenotypic variance with genotype. The primary limiting factors are cost, effectiveness and a standardized inoculum. A method was developed to create an inoculum array of multiple isolates that could be used for a series of high-throughput multi-isolate phenotypic investigations in a laboratory setting. A key starting point was the standardisation of the inoculum by production of identical batches of each isolate from cells grown to mid-log phase. Cultures with pre-determined optical densities were aliquoted in set patterns into multiple multi-well plates containing 50% glycerol and stored at -80 °C. Prior to a specific assay, an inoculum plate was defrosted and subjected to a brief period of incubation. Control strains can be placed on each plate in order to control for intra-assay variability. A high throughput screen is described in detail for quantification of biofilm formation. This example utilised the crystal violet staining method and multi-assay stock plates containing 16 meningococcal isolates. • Multi-assay stock plate of exponentially growing isolates is cost-effective and simple to implement in a laboratory setting. • This method would predict realistic standard deviations for multiple isolates in phenotypic assays and generate data for performance of power calculations for genotyping. • This method has the potential to identify both known and unknown genetic determinants of phenotypic variability for each tested isolate when paired with genetic analysis of whole genome sequencing data

Phase Variation of NadA in Invasive Neisseria meningitidis Isolates Impacts on Coverage Estimates for 4C-MenB, a MenB Vaccine.

A recombinant NadA protein is one of the four major protective antigens of 4C-MenB (Bexsero), a vaccine developed for serogroup B Neisseria meningitidis (MenB). The meningococcal antigen typing system (MATS) is utilized as a high-throughput assay for assessing the invasive MenB strain coverage of 4C-MenB. Where present, the nadA gene is subject to phase-variable changes in transcription due to a 5'TAAA repeat tract located in a regulatory region. The promoter-containing intergenic region (IGR) sequences and 5'TAAA repeat numbers were determined for 906 invasive meningococcal disease isolates possessing the nadA gene. Exclusion of the 5'TAAA repeats reduced the number of IGR alleles from 82 to 23. Repeat numbers were associated with low and high levels of NadA expression by Western blotting and enzyme-linked immunosorbent assay (ELISA). Low-expression repeat numbers were present in 83% of 179 MenB isolates with NadA-2/3 or NadA-1 peptide variants and 68% of 480 MenW ST-11 complex isolates with NadA-2/3 peptide variants. For isolates with vaccine-compatible NadA variants, 93% of MATS-negative isolates were associated with low-expression repeat numbers, whereas 63% of isolates with MATS relative potency (RP) scores above the 95% confidence interval for the positive bactericidal threshold had high-expression repeat numbers. Analysis of 5'TAAA repeat numbers has potential as a rapid, high-throughput method for assessing strain coverage for the NadA component of 4C-MenB. A key application will be assessing coverage in meningococcal disease cases where confirmation is by PCR only and MATS cannot be applied

Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates

Meningococcal lipoprotein, Factor H binding protein (FHbp), is the sole antigen of the Trumenba vaccine (Pfizer) and one of four antigens of the Bexsero vaccine (GSK) targeting Neisseria meningitidis serogroup B isolates. Lipidation of FHbp is assumed to occur for all isolates. We show in the majority of a collection of United Kingdom isolates (1742/1895) non-synonymous single nucleotide polymorphisms (SNPs) in the signal peptide (SP) of FHbp. A single SNP, common to all, alters a polar amino acid that abolishes processing: lipidation and SP cleavage. Whilst some of the FHbp precursor is retained in the cytoplasm due to reduced binding to SecA, remarkably some is translocated and further surface-localized by Slam. Thus we show Slam is not lipoprotein-specific. In a panel of isolates tested, the overall reduced surface localization of the precursor FHbp, compared to isolates with an intact SP, corresponded with decreased susceptibility to antibody-mediated killing. Our findings shed new light on the canonical pathway for lipoprotein processing and translocation of important relevance for lipoprotein-based vaccines in development and in particular for Trumenba

Potentiation of phase variation in multiple outer membrane proteins during spread of the hyperinvasive Neisseria meningitidis serogroup W ST-11 lineage.

BACKGROUND: Since 2009, increases in invasive meningococcal disease have occurred in the United Kingdom due to a sub-lineage of the Neisseria meningitidis serogroup W ST-11 clonal complex (the 'original-UK' strain). In 2013, a descendent sub-strain (the '2013-strain') became the dominant disease-causing variant. Multiple outer membrane proteins (OMP) of meningococci are subject to phase-variable switches in expression due to hypermutable simple sequence repeats (SSR). We investigated whether alterations in phase-variable genes may have impacted on the relative prevalence of the original-UK and 2013 sub-strains using multiple disease and carriage isolates. METHODS: Repeat numbers were determined by either bioinformatic analysis of whole-genome sequencing data or PCR-amplification and sizing of fragments from genomic DNA extracts. Immunoblotting or sequence-translation identified expression states. RESULTS: Significant increases in repeat number were detected between the original-UK and 2013-strains in genes encoding PorA, NadA and two Opa variants. Invasive and carriage isolates exhibited similar repeat numbers but the absence of pilC gene expression was frequently associated with disease. CONCLUSIONS: Elevated repeat numbers in OMP genes of the 2013-strain are indicative of higher phase variation rates suggesting that rapid expansion of this strain was due to a heightened ability to evade host immune responses during transmission and asymptomatic carriage

Gastro-duodenal disease in Africa: Literature review and clinical data from Accra, Ghana.

Gastroduodenal disease (GDD) was initially thought to be uncommon in Africa. Amongst others, lack of access to optimal health infrastructure and suspicion of conventional medicine resulted in the reported prevalence of GDD being significantly lower than that in other areas of the world. Following the increasing availability of flexible upper gastro-intestinal endoscopy, it has now become apparent that GDD, especially peptic ulcer disease (PUD), is prevalent across the continent of Africa. Recognised risk factors for gastric cancer (GCA) include Helicobater pylori (H. pylori), diet, Epstein-Barr virus infection and industrial chemical exposure, while those for PUD are H. pylori, non-steroidal anti-inflammatory drug (NSAID)-use, smoking and alcohol consumption. Of these, H. pylori is generally accepted to be causally related to the development of atrophic gastritis (AG), intestinal metaplasia (IM), PUD and distal GCA. Here, we perform a systematic review of the patterns of GDD across Africa obtained with endoscopy, and complement the analysis with new data obtained on pre-malignant gastric his-topathological lesions in Accra, Ghana which was compared with previous data from Maputo, Mozambique. As there is a general lack of structured cohort studies in Africa, we also considered endoscopy-based hospital or tertiary centre studies of symptomatic individuals. In Africa, there is considerable heterogeneity in the prevalence of PUD with no clear geographical patterns. Furthermore, there are differences in PUD within-country despite universally endemic H. pylori infection. PUD is not uncommon in Africa. Most of the African tertiary-centre studies had higher prevalence of PUD when compared with similar studies in western countries. An additional intriguing observation is a recent, ongoing decline in PUD in some African countries where H. pylori infection is still high. One possible reason for the high, sustained prevalence of PUD may be the significant use of NSAIDs in local or over-the-counter preparations. The prevalence of AG and IM, were similar or modestly higher over rates in western countries but lower than those seen in Asia. . In our new data, sampling of 136 patients in Accra detected evidence of pre-malignant lesions (AG and/or IM) in 20 individuals (14.7%). Likewise, the prevalence of pre-malignant lesions, in a sample of 109 patients from Maputo, were 8.3% AG and 8.3% IM. While H. pylori is endemic in Africa, the observed prevalence for GCA is rather low. However, cancer data is drawn from country cancer registries that are not comprehensive due to considerable variation in the availability of efficient local cancer reporting systems, diagnostic health facilities and expertise. Validation of cases and their source as well as specificity of outcome definitions are not explicit in most studies further contributing to uncertainty about the precise incidence rates of GCA on the continent. We conclude that evidence is still lacking to support (or not) the African enigma theory due to inconsistencies in the data that indicate a particularly low incidence of GDD in African countries

Recombination of the phase variable spnIII locus is independent of all known pneumococcal site-specific recombinases.

Streptococcus pneumoniae is one of the world's leading bacterial pathogens, causing pneumonia, septicaemia and meningitis. In recent years it has been shown that genetic rearrangements in a type I restriction-modification system (SpnIII) can impact colony morphology and gene expression. By generating a large panel of mutant strains, we have confirmed a previously reported result that the CreX (also known as IvrR and PsrA) recombinase found within the locus is not essential for hsdS inversions. In addition, mutants of homologous recombination pathways also undergo hsdS inversions. In this work we have shown that these genetic rearrangements, which result in different patterns of genome methylation, occur across a wide variety of serotypes and sequence types including two strains (a 19F and a 6B strain) naturally lacking CreX. Our gene expression analysis, by RNAseq, confirm that the level of creX expression is impacted by these genomic rearrangements. In addition, we have shown that the frequency of hsdS recombination is temperature dependent. Most importantly we have demonstrated that the other known pneumococcal site-specific recombinases XerD, XerS and SPD_0921 are not involved in spnIII recombination, suggesting a currently unknown mechanism is responsible for the recombination of these phase variable type I systems.ImportanceStreptococcus pneumoniae is a leading cause of pneumonia, septicaemia and meningitis. The discovery that genetic rearrangements in a type I restriction modification locus can impact gene regulation and colony morphology have led to a new understanding of how this pathogen switches from harmless coloniser to invasive pathogen. These rearrangements, which alter the DNA specificity of the type I restriction modification enzyme, occur across many different pneumococcal serotypes and sequence types, and in the absence of all known pneumococcal site-specific recombinases. This finding suggests that this is a truly global mechanism of pneumococcal gene regulation and the need for further investigation of mechanisms of site specific recombination