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.

Echoes of a distant past: The cag pathogenicity island of Helicobacter pylori.

This review discusses the multiple roles of the CagA protein encoded by the cag pathogenicity island of Helicobacter pylori and highlights the CagA degradation activities on p53. By subverting the p53 tumor suppressor pathway CagA induces a strong antiapoptotic effect. Helicobacter pylori infection has been always associated with an increased risk of gastric cancer. The pro-oncogenic functions of CagA also target the tumor suppressor ASPP2. In the absence of tumor suppressor genes, cells survive and proliferate at times and in places where their survival and proliferation are inappropriate.

Helicobacter pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53 (ASPP2) tumor suppressor pathway of the host.

Type I strains of Helicobacter pylori (Hp) possess a pathogenicity island, cag, that encodes the effector protein cytotoxin-associated gene A (CagA) and a type four secretion system. After translocation into the host cell, CagA affects cell shape, increases cell motility, abrogates junctional activity, and promotes an epithelial to mesenchymal transition-like phenotype. Transgenic expression of CagA enhances gastrointestinal and intestinal carcinomas as well as myeloid and B-cell lymphomas in mice, but the mechanism of the induced cancer formation is not fully understood. Here, we show that CagA subverts the tumor suppressor function of apoptosis-stimulating protein of p53 (ASPP2). Delivery of CagA inside the host results in its association with ASPP2. After this interaction, ASPP2 recruits its natural target p53 and inhibits its apoptotic function. CagA leads to enhanced degradation of p53 and thereby, down-regulates its activity in an ASPP2-dependent manner. Finally, Hp-infected cells treated with the p53-activating drug Doxorubicin are more resistant to apoptosis than uninfected cells, an effect that requires ASPP2. The interaction between CagA and ASPP2 and the consequent degradation of p53 are examples of a bacterial protein that subverts the p53 tumor suppressor pathway in a manner similar to DNA tumor viruses. This finding may contribute to the understanding of the increased risk of gastric cancer in patients infected with Hp CagA+ strains.

Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination.

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis (Nm), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm. Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.

Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species.

BACKGROUND: Streptococcus pneumoniae is one of the most important causes of microbial diseases in humans. The genomes of 44 diverse strains of S. pneumoniae were analyzed and compared with strains of non-pathogenic streptococci of the Mitis group. RESULTS: Despite evidence of extensive recombination, the S. pneumoniae phylogenetic tree revealed six major lineages. With the exception of serotype 1, the tree correlated poorly with capsular serotype, geographical site of isolation and disease outcome. The distribution of dispensable genes--genes present in more than one strain but not in all strains--was consistent with phylogeny, although horizontal gene transfer events attenuated this correlation in the case of ancient lineages. Homologous recombination, involving short stretches of DNA, was the dominant evolutionary process of the core genome of S. pneumoniae. Genetic exchange occurred both within and across the borders of the species, and S. mitis was the main reservoir of genetic diversity of S. pneumoniae. The pan-genome size of S. pneumoniae increased logarithmically with the number of strains and linearly with the number of polymorphic sites of the sampled genomes, suggesting that acquired genes accumulate proportionately to the age of clones. Most genes associated with pathogenicity were shared by all S. pneumoniae strains, but were also present in S. mitis, S. oralis and S. infantis, indicating that these genes are not sufficient to determine virulence. CONCLUSIONS: Genetic exchange with related species sharing the same ecological niche is the main mechanism of evolution of S. pneumoniae. The open pan-genome guarantees the species a quick and economical response to diverse environments.

Supramolecular organization of the repetitive backbone unit of the Streptococcus pneumoniae pilus.

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 A resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility.

The complete genome sequence of Helicobacter pylori strain G27.

Helicobacter pylori is a gram-negative pathogen that colonizes the stomachs of over half the world's population and causes a spectrum of gastric diseases including gastritis, ulcers, and gastric carcinoma. The H. pylori species exhibits unusually high levels of genetic variation between strains. Here we announce the complete genome sequence of H. pylori strain G27, which has been used extensively in H. pylori research.

Pilus operon evolution in Streptococcus pneumoniae is driven by positive selection and recombination.

BACKGROUND: The evolution of bacterial organelles involved in host-pathogen interactions is subject to intense and competing selective pressures due to the need to maintain function while escaping the host immune response. To characterize the interplay of these forces in an important pathogen, we sequenced the rlrA islet, a chromosomal region encoding for a pilus-like structure involved in adherence to lung epithelial cells in vitro and in colonization in a murine model of infection, in 44 clinical isolates of Streptococcus pneumoniae. RESULTS: We found that the rrgA and rrgB genes, encoding the main structural components of the pilus, are under the action of positive selection. In contrast, the rrgC gene, coding for a component present in low quantities in the assembled pilus, and the srtB, srtC and srtD genes, coding for three sortase enzymes essential for pilus assembly but probably not directly exposed to the host immune system, show no evidence of positive selection. We found several events of homologous recombination in the region containing these genes, identifying 4 major recombination hotspots. An analysis of the most recent recombination events shows a high level of mosaicism of the region coding for the rrgC, srtB, srtC and srtD genes. CONCLUSIONS: In the rlrA islet, the genes coding for proteins directly exposed to the host immune response are under the action of positive selection, and exist in distinct forms in the population of circulating strains. The genes coding for proteins not directly exposed on the surface of the bacterial cell are more conserved probably due to the homogenizing effect of recombination.

A second pilus type in Streptococcus pneumoniae is prevalent in emerging serotypes and mediates adhesion to host cells.

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.

Pneumococcal pili are composed of protofilaments exposing adhesive clusters of Rrg A.

Pili have been identified on the cell surface of Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide. In contrast to Gram-negative bacteria, little is known about the structure of native pili in Gram-positive species and their role in pathogenicity. Triple immunoelectron microscopy of the elongated structure showed that purified pili contained RrgB as the major compound, followed by clustered RrgA and individual RrgC molecules on the pilus surface. The arrangement of gold particles displayed a uniform distribution of anti-RrgB antibodies along the whole pilus, forming a backbone structure. Antibodies against RrgA were found along the filament as particulate aggregates of 2-3 units, often co-localised with single RrgC subunits. Structural analysis using cryo electron microscopy and data obtained from freeze drying/metal shadowing technique showed that pili are oligomeric appendages formed by at least two protofilaments arranged in a coiled-coil, compact superstructure of various diameters. Using extracellular matrix proteins in an enzyme-linked immunosorbent assay, ancillary RrgA was identified as the major adhesin of the pilus. Combining the structural and functional data, a model emerges where the pilus RrgB backbone serves as a carrier for surface located adhesive clusters of RrgA that facilitates the interaction with the host.

Pathogenomics: an updated European Research Agenda.

The emerging genomic technologies and bioinformatics provide novel opportunities for studying life-threatening human pathogens and to develop new applications for the improvement of human and animal health and the prevention, treatment, and diagnosis of infections. Based on the ecology and population biology of pathogens and related organisms and their connection to epidemiology, more accurate typing technologies and approaches will lead to better means of disease control. The analysis of the genome plasticity and gene pools of pathogenic bacteria including antigenic diversity and antigenic variation results in more effective vaccines and vaccine implementation programs. The study of newly identified and uncultivated microorganisms enables the identification of new threats. The scrutiny of the metabolism of the pathogen in the host allows the identification of new targets for anti-infectives and therapeutic approaches. The development of modulators of host responses and mediators of host damage will be facilitated by the research on interactions of microbes and hosts, including mechanisms of host damage, acute and chronic relationships as well as commensalisms. The study of multiple pathogenic and non-pathogenic microbes interacting in the host will improve the management of multiple infections and will allow probiotic and prebiotic interventions. Needless to iterate, the application of the results of improved prevention and treatment of infections into clinical tests will have a positive impact on the management of human and animal disease. The Pathogenomics Research Agenda draws on discussions with experts of the Network of Excellence "EuroPathoGenomics" at the management board meeting of the project held during 18-21 April 2007, in the Villa Vigoni, Menaggio, Italy. Based on a proposed European Research Agenda in the field of pathogenomics by the ERA-NET PathoGenoMics the meeting's participants updated the established list of topics as the research agenda for the future.

Streptococcus pneumoniae contains 3 rlrA pilus variants that are clonally related.

BACKGROUND: Pilus components of Streptococcus pneumoniae encoded by rlrA were recently shown to elicit protection in an animal model of infection. Limited data are available on the prevalence of the rlrA operon in pneumococci; therefore, we investigated its distribution and its antigenic variation among disease-causing strains. METHODS: The prevalence of rlrA and its association with serotype and genotype were evaluated in a global panel of 424 pneumococci isolates (including the 26 drug-resistant clones described by the Pneumococcal Molecular Epidemiology Network). RESULTS: The rlrA islet was found in 130 isolates (30.6%) of the defined collection. Sequence alignment of 15 rlrA islets defined the presence of 3 clade types, with an overall homology of 88%-92%. The presence or absence of a pilus-encoding operon correlated with S. pneumoniae genotype (P < .001), as determined by multilocus sequence typing, and not with serotype. Further investigation identified a positive trend of rlrA occurrence among antimicrobial-resistant pneumococci. CONCLUSIONS: On the basis of S. pneumoniae genotype, it is possible to predict the incidence of the rlrA pilus operon in a collection of pneumococcal isolates. This will facilitate the development of a protein vaccine.

Analysis of detergent-resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a role for MARK2/Par1b in CagA-mediated disruption of cellular polarity.

Detergent-resistant membranes of eukaryotic cells are enriched in many important cellular signalling molecules and frequently targeted by bacterial pathogens. To learn more about pathogenic mechanisms of Helicobacter pylori and to elucidate novel effects on host epithelial cells, we investigated how bacterial co-cultivation changes the protein composition of detergent-resistant membranes of gastric adenocarcinoma (AGS) tissue culture cells. Using iTRAQ (isobaric tags for relative and absolute quantification) analysis we identified several cellular proteins, which are potentially related to H. pylori virulence. One of the proteins, which showed a significant infection-dependent increase in detergent resistance, was the polarity-associated serine/threonine kinase MARK2 (EMK1/Par-1b). We demonstrate that H. pylori causes the recruitment of MARK2 from the cytosol to the plasma membrane, where it colocalizes with the bacteria and interacts with CagA. Using Mardin Darby Canine Kidney (MDCK) monolayers and a three-dimensional MDCK tissue culture model we showed that association of CagA with MARK2 not only causes disruption of apical junctions, but also inhibition of tubulogenesis and cell differentiation.

Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients.

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic beta cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between beta cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of beta cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect beta cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect beta cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.

Streptococcus pneumoniae pilus subunits protect mice against lethal challenge.

Streptococcus pneumoniae is a major public health threat worldwide. The recent discovery that this pathogen possesses pili led us to investigate their protective abilities in a mouse model of intraperitoneal infection. Both active and passive immunization with recombinant pilus subunits afforded protection against lethal challenge with the S. pneumoniae serotype 4 strain TIGR4.

Protein homology network families reveal step-wise diversification of Type III and Type IV secretion systems.

From the analysis of 251 prokaryotic genomes stored in public databases, the 761,260 deduced proteins were used to reconstruct a complete set of bacterial proteic families. Using the new Overlap algorithm, we have partitioned the Protein Homology Network (PHN), where the proteins are the nodes and the links represent homology relationships. The algorithm identifies the densely connected regions of the PHN that define the families of homologous proteins, here called PHN-Families, recognizing the phylogenetic relationships embedded in the network. By direct comparison with a manually curated dataset, we assessed that this classification algorithm generates data of quality similar to a human expert. Then, we explored the network to identify families involved in the assembly of Type III and Type IV secretion systems (T3SS and T4SS). We noticed that, beside a core of conserved functions (eight proteins for T3SS, seven for T4SS), a variable set of accessory components is always present (one to nine for T3SS, one to five for T4SS). Each member of the core corresponds to a single PHN-Family, while accessory proteins are distributed among different pure families. The PHN-Family classification suggests that T3SS and T4SS have been assembled through a step-wise, discontinuous process, by complementing the conserved core with subgroups of nonconserved proteins. Such genetic modules, independently recruited and probably tuned on specific effectors, contribute to the functional specialization of these organelles to different microenvironments.

Inhibitors of Helicobacter pylori ATPase Cagalpha block CagA transport and cag virulence.

With the steadily increasing occurrence of antibiotic resistance in bacteria, there is a great need for new antibacterial compounds. The approach described here involves targeting virulence-related bacterial type IV secretion systems (TFSSs) with small-molecule inhibitors. The cag TFSS of Helicobacter pylori was chosen as a model, and novel inhibitors directed against the cag VirB11-type ATPase Cagalpha were identified. The cag genes encode proteins that are components of a contact-dependent secretion system used by the bacterium to translocate the effector molecule CagA into host cells. Translocated CagA is associated with severe gastritis, and carcinoma. Furthermore, functional TFSSs and immunodominant CagA play a role in interleukin (IL)-8 induction, which is an important factor for chronic inflammation. Inhibitors of Cagalpha were identified by high-throughput screening of chemical libraries that comprised 524 400 small molecules. The ATPase activity of Cagalpha was inhibited by the selected compounds in an in vitro enzymic assay using the purified enzyme. The most active compound, CHIR-1, reduced TFSS function to an extent that cellular effects on AGS cells mediated by CagA were virtually undetectable, while reduced levels of IL-8 induction were observed. Gastric colonization by CHIR-1-pre-treated bacteria was found to be impaired in a dose-dependent manner using a mouse model of infection. Small-molecule Cagalpha inhibitors, the first described inhibitors of a TFSS, are potential candidates for the development of new antibacterial compounds that may lead to alternative medical treatments. The compounds are expected to impose weak selective pressure, since they target virulence functions. Moreover, the targeted virulence protein is conserved in a variety of bacterial pathogens. Additionally, TFSS inhibitors are potent tools to study the biology of TFSSs.

Microbial genomes and vaccine design: refinements to the classical reverse vaccinology approach.

The advent of whole-genome sequencing of bacteria and advances in bioinformatics have revolutionized the study of bacterial pathogenesis, enabling the targeting of possible vaccine candidates starting from genomic information. Nowadays, the availability of hundreds of bacterial genomes enables identification of the genetic differences across several genomes from the same species. The unexpected degree of intra-species diversity suggests that a single genome sequence is not entirely representative and does not offer a complete picture of the genetic variability of a species. The practical consequence is that, in many cases, a universal vaccine is possible only by including a combination of antigens and this combination must take into account the pathogen population structure.