Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.     

Expression patterns and adaptive functional diversity of vertebrate myoglobins

Recent years have witnessed a new round of research on one of the most studied proteins - myoglobin (Mb), the oxygen (O₂) carrier of skeletal and heart muscle. Two major discoveries have stimulated research in this field: 1) that Mb has additional protecting functions, such as the regulation of in vivo levels of the signaling molecule nitric oxide (NO) by scavenging and generating NO during normoxia and hypoxia, respectively; and 2) that Mb in vertebrates (particularly fish) is expressed as tissue-specific isoforms in other tissues than heart and skeletal muscle, such as vessel endothelium, liver and brain, as found in cyprinid fish. Furthermore, Mb has also been found to protect against oxidative stress after hypoxia and reoxygenation and to undergo allosteric, O₂-linked S-nitrosation, as in rainbow trout. Overall, the emerging evidence, particularly from fish species, indicates that Mb fulfills a broader array of physiological functions in a wider range of different tissues than hitherto appreciated. This new knowledge helps to better understand how variations in Mb structure and function may correlate with differences in animals' lifestyles and hypoxia-tolerance. This review integrates old and new results on Mb expression patterns and functional properties amongst vertebrates and discusses how these may relate to adaptive variations in different species. This article is part of a special issue entitled: Oxygen Binding and Sensing Proteins.     

Urea’s Effect on the Ribonuclease A Catalytic Efficiency: A Kinetic, 1H NMR and Molecular Orbital Study

Understanding of protein–urea interactions is one of the greatest challenges to modern structural protein chemistry. Based in enzyme kinetics experiments and ¹H NMR spectroscopic analysis we proposed that urea, at low concentrations, directly interacts with the protonated histidines of the active center of RNase A, following a simple model of competitive inhibition. These results were supported by theoretical analysis based on the frontier molecular orbital theory and suggest that urea might establish a favorable interaction with the cationic amino acids. Our experimental evidence and theoretical analysis indicate that the initials steps of the molecular mechanism of Urea–RNase A interaction passes through the establishment of a three center four electron adduct. Also, our results would explain the observed disruption of the ¹H NMR signals corresponding to H12 and H119 (involved in catalysis) of the RNase A studied in the presence of urea. Our interaction model of urea–amino acids (cationic) can be extended to explain the inactivation of other enzymes with cationic amino acids at the active site.     

1H, 13C and 15N chemical shift assignments for an intracellular proteinase inhibitor of Bacillus subtilis

Intracellular proteinases (ISPs) are the main component of the bacilli degradome and a distinctive class in different bacilli. An intracellular proteinase inhibitor of the bacteria Bacillus subtillis was shown to regulate the activity of ISP-1. To study the structure of this inhibitor, we report the resonance assignment for this protein with 119 amino acid. The data will allow us to perform structural study on this inhibitor to understand its mechanism for ISP-1 inhibition.     

Impaired Eye Region Search Accuracy in Children with Autistic Spectrum Disorders

To explore mechanisms underlying reduced fixation of eyes in autism, children with Autistic Spectrum Disorders (ASD) and typically developing children were tested in five visual search experiments: simple color feature; color-shape conjunction; face in non-face objects; mouth region; and eye region. No group differences were found for reaction time profile shapes in any of the five experiments, suggesting intact basic search mechanics in children with ASD. Contrary to early reports in the literature, but consistent with other more recent findings, we observed no superiority for conjunction search in children with ASD. Importantly, children with ASD did show reduced accuracy for eye region search (p = .005), suggesting that eyes contribute less to high-level face representations in ASD or that there is an eye region-specific disruption to attentional processes engaged by search in ASD.     

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.     

Evidence for the Circulation and Inter-Hemispheric Movement of the H14 Subtype Influenza A Virus

Three H14 influenza A virus (IAV) isolates recovered in 2010 during routine virus surveillance along the Mississippi Migratory Bird Flyway in Wisconsin, U.S.A. raised questions about the natural history of these rare viruses. These were the first H14 IAV isolates recovered in the Western Hemisphere and the only H14 IAV isolates recovered since the original four isolates in 1982 in Asia. Full length genomic sequencing of the 2010 H14 isolates demonstrated the hemagglutinin (HA) gene from the 1982 and 2010 H14 isolates showed 89.6% nucleotide and 95.6% amino acid similarity and phylogenetic analysis of these viruses placed them with strong support within the H14 subtype lineage. The level of genomic divergence observed between the 1982 and 2010 viruses provides evidence that the H14 HA segment was circulating undetected in hosts and was not maintained in environmental stasis. Further, the evolutionary relationship observed between 1982 H14 and the closely related H4 subtype HA segments were similar to contemporary comparisons suggesting limited adaptive divergence between these sister subtypes. The nonstructural (NS) segment of one 2010 isolate was placed in a NS clade isolated infrequently over the last several decades that includes the NS segment from a previously reported 1982 H14 isolate indicating the existence of an unidentified pool of genomic diversity. An additional neuraminidase reassortment event indicated a recent inter-hemispheric gene flow from Asia into the center of North America. These results demonstrate temporal and spatial gaps in the understanding of IAV natural history. Additionally, the reassortment history of these viruses raises concern for the inter-continental spread of IAVs and the efficacy of current IAV surveillance efforts in detecting genomic diversity of viruses circulating in wild birds.     

Dynamic Activity of miR-125b and miR-93 during Murine Neural Stem Cell Differentiation In Vitro and in the Subventricular Zone Neurogenic Niche

Several microRNAs (miRNAs) that are either specifically enriched or highly expressed in neurons and glia have been described, but the identification of miRNAs modulating neural stem cell (NSC) biology remains elusive. In this study, we exploited high throughput miRNA expression profiling to identify candidate miRNAs enriched in NSC/early progenitors derived from the murine subventricular zone (SVZ). Then, we used lentiviral miRNA sensor vectors (LV.miRT) to monitor the activity of shortlisted miRNAs with cellular and temporal resolution during NSC differentiation, taking advantage of in vitro and in vivo models that recapitulate physiological neurogenesis and gliogenesis and using known neuronal- and glial-specific miRNAs as reference. The LV.miRT platform allowed us monitoring endogenous miRNA activity in low represented cell populations within a bulk culture or within the complexity of CNS tissue, with high sensitivity and specificity. In this way we validated and extended previous results on the neuronal-specific miR-124 and the astroglial-specific miR-23a. Importantly, we describe for the first time a cell type- and differentiation stage-specific modulation of miR-93 and miR-125b in SVZ-derived NSC cultures and in the SVZ neurogenic niche in vivo, suggesting key roles of these miRNAs in regulating NSC function.