In-silico prediction and deep-DNA sequencing validation indicate phase variation in 115 Neisseria meningitidis genes.

BACKGROUND: The Neisseria meningitidis (Nm) chromosome shows a high abundance of simple sequence DNA repeats (SSRs) that undergo stochastic, reversible mutations at high frequency. This mechanism is reflected in an extensive phenotypic diversity that facilitates Nm adaptation to dynamic environmental changes. To date, phase-variable phenotypes mediated by SSRs variation have been experimentally confirmed for 26 Nm genes. RESULTS: Here we present a population-scale comparative genomic analysis that identified 277 genes and classified them into 52 strong, 60 moderate and 165 weak candidates for phase variation. Deep-coverage DNA sequencing of single colonies grown overnight under non-selective conditions confirmed the presence of high-frequency, stochastic variation in 115 of them, providing circumstantial evidence for their phase variability. We confirmed previous observations of a predominance of variable SSRs within genes for components located on the cell surface or DNA metabolism. However, in addition we identified an unexpectedly broad spectrum of other metabolic functions, and most of the variable SSRs were predicted to induce phenotypic changes by modulating gene expression at a transcriptional level or by producing different protein isoforms rather than mediating on/off translational switching through frameshifts. Investigation of the evolutionary history of SSR contingency loci revealed that these loci were inherited from a Nm ancestor, evolved independently within Nm, or were acquired by Nm through lateral DNA exchange. CONCLUSIONS: Overall, our results have identified a broader and qualitatively different phenotypic diversification of SSRs-mediated stochastic variation than previously documented, including its impact on central Nm metabolism.

Genetic characterization and estimated 4CMenB vaccine strain coverage of 284 Neisseria meningitidis isolates causing invasive meningococcal disease in Argentina in 2010-2014.

Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.

The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.

The respiratory syncytial virus (RSV) prefusion F-protein functional antibody repertoire in adult healthy donors.

Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre-existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell-sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F-protein (cross-binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross-binder nAbs used a wide array of gene rearrangements, while preF-binder nAbs derived mostly from the expansion of B-cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F-protein, identifying an important site of pathogen vulnerability.

Dataset of antibody variable region sequence features inferred from a respiratory syncytial virus fusion protein-specific B cell receptor repertoire induced by natural infection of a healthy adult.

Respiratory syncytial virus (RSV) is the primary cause for acute lower respiratory syndrome in children younger than 5 years. Research on B cell repertoires and antibodies binding the RSV fusion protein (RSV F) is of major interest in the development of potential vaccine candidates and therapies. B cell receptors (BCRs) which have higher affinities for a specific antigen are preferentially selected for B cell clonal expansion in germinal center reactions. Consequently, antigen-specific BCR repertoires share common features, as for instance preferential variable gene usage, variable region mutation levels or lengths of the heavy chain complementarity-determining region 3. Since RSV repeatedly infects every person throughout life, memory B cells (MBC) expressing RSV F-binding BCRs circulate in the blood of healthy adults. This dataset of BCR variable region sequence features was derived from single cell-sorted RSV F-directed MBCs of a healthy adult blood donor [1]. The dataset was produced with publicly available data analysis software programs and scripts, which facilitates integration or comparison with antibody sequence repertoire data of different individuals derived with the same or comparable data analysis approaches and tools.

The respiratory syncytial virus fusion protein-specific B cell receptor repertoire reshaped by post-fusion subunit vaccination.

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory illness in children of less than 5 years of age which usually results in hospitalization or even in death. Vaccine development is hampered in consequence of a failed vaccine trial with fatalities in the 1960s. Even though research has been more focused on the RSV fusion protein in its pre-fusion conformation, maternal vaccination with post-fusion protein (post F) was considered as a promising vaccine strategy for passive immunization of babies, because post F preserves very potent neutralizing epitopes. We extensively analyzed post F-binding B cell receptor (BCR) repertoires of three vaccinees who received a post F-subunit vaccine in the context of a first-in-human, Phase 1, randomized, observer-blind, placebo-controlled clinical trial (ClinicalTrials.gov Identifier: NCT02298179). In order to compare the vaccine-induced BCR repertoires with BCR repertoires induced by natural infection, we also analyzed pre F- and post F-binding BCRs isolated from a healthy blood donor with relatively high F-binding memory B cell (MBC) frequencies. Analysis of the vaccine-induced repertoires revealed that preferentially VH4-encoded BCRs were expanded in response to vaccination. Estimation of antigen-driven selection further demonstrated that expanded BCRs accumulated positively selected replacement mutations which substantiated the hypothesis that post F-vaccination induces diversification of VH4-encoded BCRs in germinal centers. Comparison of the vaccine-induced BCR repertoires with clonally related pre and post F-binding BCRs of the healthy blood donor suggested that the vaccine expanded pre/post F cross-reactive MBCs. Interestingly, several vaccine-induced BCRs shared stereotypic VDJ gene junctions with known neutralizing Abs. Once expressed for functional characterization, the selected monoclonal Abs demonstrated the predicted neutralization activities in plaque reduction neutralization assays indicating that the post F-vaccine induced expansion of neutralizing BCRs.

Expanding the Helicobacter pylori genetic toolbox: modification of an endogenous plasmid for use as a transcriptional reporter and complementation vector.

Helicobacter pylori is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous H. pylori plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an Escherichia coli origin of replication, a kanamycin resistance cassette, a promoterless gfpmut3 gene, and a functional multiple cloning site to form pTM117. The promoters of amiE and pfr, two well-characterized Fur-regulated promoters, were fused to the promoterless gfpmut3, and green fluorescent protein (GFP) expression of the fusions in wild-type and delta fur strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing amiE, pfr, and fur expression in wild-type and delta fur strains carrying a wild-type copy of fur on the plasmid. Proper regulation of these genes was restored in the delta fur background under high- and low-iron conditions, signifying complementation of both iron-bound and apo Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of H. pylori.

Genomic Analysis Reveals Multi-Drug Resistance Clusters in Group B Streptococcus CC17 Hypervirulent Isolates Causing Neonatal Invasive Disease in Southern Mainland China.

Neonatal invasive disease caused by group B Streptococcus (GBS) represents a significant public health care concern globally. However, data related to disease burden, serotype distribution, and molecular epidemiology in China and other Asian countries are very few and specifically relative to confined regions. The aim of this study was to investigate the genetic characteristics of GBS isolates recovered from neonates with invasive disease during 2013-2014 at Guangzhou and Changsha hospitals in southern mainland China. We assessed the capsular polysaccharide type, pilus islands (PIs) distribution and hvgA gene presence in a panel of 26 neonatal clinical isolates, of which 8 were recovered from Early Onset Disease and 18 from Late Onset Disease (LOD). Among 26 isolates examined, five serotypes were identified. Type III was the most represented (15 cases), particularly among LOD strains (n = 11), followed by types Ib (n = 5), V (n = 3), Ia (n = 2) and II (n = 1). We performed whole-genome sequencing analysis and antimicrobial susceptibility testing on the 14 serotype III isolates belonging to the hypervirulent Clonal Complex 17 (serotype III-CC17). The presence of PI-2b alone was associated with 13 out of 14 serotype III-CC17 strains. Genome analysis led us to identify two multi-drug resistance gene clusters harbored in two new versions of integrative and conjugative elements (ICEs), carrying five or eight antibiotic resistance genes, respectively. These ICEs replaced the 16 kb-locus that normally contains the PI-1 operon. All isolates harboring the identified ICEs showed multiple resistances to aminoglycoside, macrolide, and tetracycline antibiotic classes. In conclusion, we report the first whole-genome sequence analysis of 14 GBS serotype III-CC17 strains isolated in China, representing the most prevalent lineage causing neonatal invasive disease. The acquisition of newly identified ICEs conferring multiple antibiotic resistance could in part explain the spread of this specific clone among Chinese neonatal isolates and underlines the need for a constant epidemiological surveillance.

A human short-chain dehydrogenase/reductase gene: structure, chromosomal localization, tissue expression and subcellular localization of its product.

We have previously described the cloning of Hep27, a short-chain dehydrogenase/reductase, which is synthesized in human hepatoblastoma HepG2 cells following growth arrest induced by butyrate treatment. The present report describes the cloning, the structure and the physical and cytogenetic mapping of the gene coding for Hep27. We also show that Hep27 is synthesized in a limited number of human normal tissues and that it is localized in the nuclei and cytoplasm of HepG2 cells.

Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk.

UNLABELLED: The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies. IMPORTANCE: Simultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease.

Whole-Genome Sequences of Nonencapsulated Haemophilus influenzae Strains Isolated in Italy.

Haemophilus influenzae is an important human pathogen involved in invasive disease. Here, we report the whole-genome sequences of 11 nonencapsulated H. influenzae (ncHi) strains isolated from both invasive disease and healthy carriers in Italy. This genomic information will enrich our understanding of the molecular basis of ncHi pathogenesis.

Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population.

The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl transferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.

An extended multi-locus molecular typing schema for Streptococcus pneumoniae demonstrates that a limited number of capsular switch events is responsible for serotype heterogeneity of closely related strains from different countries.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococcal strains are classified according to their capsular serotype and through a Multi-Locus Sequence Typing schema (MLST) based on the sequencing of seven housekeeping genes. However, strains with a defined allelic profile (Sequence Type, ST) can have different serotypes, suggesting that the micro-evolution of the MLST lineages leads to a considerable degree of phenotypic variability. To better investigate the genetic diversity within these lineages, we set-up and then validated an extended molecular typing schema (96-MLST) based on the sequencing of ninety-six genomic loci. 96-MLST loci were designed within core-genes in a collection of 39 complete genomes of S. pneumoniae. None of the capsular genes was included in the schema. When tested on a collection of 69 isolates, 96-MLST was able to partition strains with the same ST and diverse serotypes into groups that were homogenous for capsular serotype, improving our understanding of the evolution of epidemiologically relevant lineages. Phylogenetic sequence analysis showed that the capsular heterogeneity of three STs that were sampled more extensively could be traced back to a limited number of capsular switch events, indicating that changes of serotype occur occasionally during the short term expansion of clones. Moreover, a geographical structure of ST156 was identified, suggesting that the resolution guaranteed by this method is sufficient for phylogeographic studies. In conclusion, we showed that an extended typing schema was able to characterize the expansion of individual lineages in a complex species such as S. pneumoniae.

Sequence analysis of 96 genomic regions identifies distinct evolutionary lineages within CC156, the largest Streptococcus pneumoniae clonal complex in the MLST database.

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.