Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells

Neisseria meningitidis is a human pathogen, which is a major cause of sepsis and meningitis. The bacterium colonizes the upper respiratory tract of approximately 10% of humans where it lives as a commensal. On rare occasions, it crosses the epithelium and reaches the bloodstream causing sepsis. From the bloodstream it translocates the blood-brain barrier, causing meningitis. Although all strains have the potential to cause disease, a subset of them, which belongs to hypervirulent lineages, causes disease more frequently than others. Recently, we described NadA, a novel antigen of N. meningitidis, present in three of the four known hypervirulent lineages. Here we show that NadA is a novel bacterial invasin which, when expressed on the surface of Escherichia coli, promotes adhesion to and invasion into Chang epithelial cells. Deletion of the N-terminal globular domain of recombinant NadA or pronase treatment of human cells abrogated the adhesive phenotype. A hypervirulent strain of N. meningitidis where the nad A gene was inactivated had a reduced ability to adhere to and invade into epithelial cells in vitro. NadA is likely to improve the fitness of N. meningitidis contributing to the increased virulence of strains that belong to the hypervirulent lineages.     

The Influence of Clinical Information in the Histopathologic Diagnosis of Melanocytic Skin Neoplasms

Background

We tested the relevance of clinical information in the histopathologic evaluation of melanocytic skin neoplasm (MSN).

Methods

Histopathologic specimens from 99 clinically atypical MSN were circulated among ten histopathologists; each case had clinical information available in a database with a five-step procedure (no information; age/sex/location; clinical diagnosis; clinical image; dermoscopic image); each step had a histopathologic diagnosis (D1 through D5); each diagnostic step had a level of diagnostic confidence (LDC) ranging from 1 (no diagnostic certainty) to 5 (absolute diagnostic certainty). The comparison of the LDC was employed with an analysis of variance (ANOVA) for repeated measures.

Findings

In D1 (no information), 36/99 cases (36.3%) had unanimous diagnosis; in D5 (full information available), 51/99 cases (51.5%) had unanimous diagnosis (p for difference between proportions <0.001). The observer agreement expressed as kappa increased significantly from D1 to D5. The mean LDC linearly increased for each observer from D1 through D5 (p for linear trend <0.001). On average, each histopathologist changed his initial diagnosis in 7 cases (range: 2–23). Most diagnostic changes were in D2 (age/sex/location).

Interpretation

The histopathologic criteria for the diagnosis of MSN can work as such, but the final histopathologic diagnosis is a clinically-aided interpretation. Clinical data sometimes reverse the initial histopathologic evaluation.     

Biotechnology and vaccines: application of functional genomics to Neisseria meningitidis and other bacterial pathogens

Since its introduction, vaccinology has been very effective in preventing infectious diseases. However, in several cases, the conventional approach to identify protective antigens, based on biochemical, immunological and microbiological methods, has failed to deliver successful vaccine candidates against major bacterial pathogens. The recent development of powerful biotechnological tools applied to genome-based approaches has revolutionized vaccine development, biological research and clinical diagnostics. The availability of a genome provides an inclusive virtual catalogue of all the potential antigens from which it is possible to select the molecules that are likely to be more effective. Here, we describe the use of "reverse vaccinology", which has been successful in the identification of potential vaccines candidates against Neisseria meningitidis serogroup B and review the use of functional genomics approaches as DNA microarrays, proteomics and comparative genome analysis for the identification of virulence factors and novel vaccine candidates. In addition, we describe the potential of these powerful technologies in understanding the pathogenesis of various bacteria.     

A deficient TLR2 signaling promotes airway mucin production in Mycoplasma pneumoniae-infected allergic mice

The original hygiene hypothesis suggests that early childhood respiratory infections preceding allergen exposure may decrease the prevalence of allergic diseases. We have recently demonstrated that Mycoplasma pneumoniae infection preceding allergen exposure reduced allergic responses in mice. However, the molecular mechanisms underlying the protective role of M. pneumoniae in allergic responses, particularly airway mucin production, remain unclear. Wild-type and Toll-like receptor 2 (TLR2)-deficient mice with a respiratory M. pneumoniae infection preceding allergen (ovalbumin) challenge were utilized to determine the regulatory role of TLR2-IFN-gamma signaling pathway in airway mucin expression. Furthermore, air-liquid interface cultures of mouse primary tracheal epithelial cells were performed to examine the effects of IFN-gamma on mucin expression. In wild-type mice, M. pneumoniae infection preceding allergen challenge significantly reduced airway mucins but increased IFN-gamma. In sharp contrast, in TLR2-deficient mice, M. pneumoniae preceding allergen challenge resulted in increased mucin protein without a noticeable change of IFN-gamma. In cultured mouse primary tracheal epithelial cells, IFN-gamma was shown to directly inhibit mucin expression in a dose-dependent manner. Our study demonstrates for the first time that a respiratory M. pneumoniae infection preceding allergen challenge reduces airway epithelial mucin expression in part through TLR2-IFN-gamma signaling pathway. A bacterial infection in asthmatic subjects with weakened TLR2-IFN-gamma signaling may result in an exaggerated airway mucin production.     

SARS--beginning to understand a new virus

The 114-day epidemic of the severe acute respiratory syndrome (SARS) swept 29 countries, affected a reported 8,098 people, left 774 patients dead and almost paralyzed the Asian economy. Aggressive quarantine measures, possibly aided by rising summer temperatures, successfully terminated the first eruption of SARS and provided at least a temporal break, which allows us to consolidate what we have learned so far and plan for the future. Here, we review the genomics of the SARS coronavirus (SARS-CoV), its phylogeny, antigenic structure, immune response and potential therapeutic interventions should the SARS epidemic flare up again.     

Bound conformations for ligands for G-protein coupled receptors

The conformation of the C-terminus of the -subunit of transducin, the G-protein of vision, has been determined by transfer NOE when bound to activated (MII) rhodopsin. One hundred three new NOE constraints are apparent when light is shown on a mixture of rhodopsin bilayers and the undecapeptide. Analogs of the -peptide with covalent constraints were designed restricting the bound conformation; they stabilize MII thus supporting the deduced structure. The NMR structure of a complex of the intracellular loops of rhodopsin facilitates docking of the -peptide and also shows proximity of residues known by mutational analysis to interact to generate the activated rhodopsin-transducin interface. This constrains the location of transmembrane helices in the structure of activated rhodopsin. Methods for the prediction of affinity have been used to estimate the relative binding constants of peptide analogs with the loop complex and show strong correlation with experimental data. Various models of the rhodopsin-transmembrane helical segments have been computationally fused with distance geometry to determine the overall model which best fits the experimental data on the rhodopsin-transducin interface.     

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.     

Synthesis and evaluation of a new series of Neuropeptide S receptor antagonists

Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood–brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited ¹²⁵I-NPS binding in the CNS when administered to rats.