Staphylococcal Complement Evasion Protein Sbi Stabilises C3d Dimers by Inducing an N-Terminal Helix Swap.

Staphylococcus aureus is an opportunistic pathogen that is able to thwart an effective host immune response by producing a range of immune evasion molecules, including S. aureus binder of IgG (Sbi) which interacts directly with the central complement component C3, its fragments and associated regulators. Recently we reported the first structure of a disulfide-linked human C3d17C dimer and highlighted its potential role in modulating B-cell activation. Here we present an X-ray crystal structure of a disulfide-linked human C3d17C dimer, which undergoes a structurally stabilising N-terminal 3D domain swap when in complex with Sbi. These structural studies, in combination with circular dichroism and fluorescence spectroscopic analyses, reveal the mechanism underpinning this unique helix swap event and could explain the origins of a previously discovered N-terminally truncated C3dg dimer isolated from rat serum. Overall, our study unveils a novel staphylococcal complement evasion mechanism which enables the pathogen to harness the ability of dimeric C3d to modulate B-cell activation.

Insights Into the Structure-Function Relationships of Dimeric C3d Fragments.

Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.

A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia.

Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia.

A comparison of nucleosome organization in Drosophila cell lines.

Changes in the distribution of nucleosomes along the genome influence chromatin structure and impact gene expression by modulating the accessibility of DNA to transcriptional machinery. However, the role of genome-wide nucleosome positioning in gene expression and in maintaining differentiated cell states remains poorly understood. Drosophila melanogaster cell lines represent distinct tissue types and exhibit cell-type specific gene expression profiles. They thus could provide a useful tool for investigating cell-type specific nucleosome organization of an organism's genome. To evaluate this possibility, we compared genome-wide nucleosome positioning and occupancy in five different Drosophila tissue-specific cell lines, and in reconstituted chromatin, and then tested for correlations between nucleosome positioning, transcription factor binding motifs, and gene expression. Nucleosomes in all cell lines were positioned in accordance with previously known DNA-nucleosome interactions, with helically repeating A/T di-nucleotide pairs arranged within nucleosomal DNAs and AT-rich pentamers generally excluded from nucleosomal DNA. Nucleosome organization in all cell lines differed markedly from in vitro reconstituted chromatin, with highly expressed genes showing strong nucleosome organization around transcriptional start sites. Importantly, comparative analysis identified genomic regions that exhibited cell line-specific nucleosome enrichment or depletion. Further analysis of these regions identified 91 out of 16,384 possible heptamer sequences that showed differential nucleosomal occupation between cell lines, and 49 of the heptamers matched one or more known transcription factor binding sites. These results demonstrate that there is differential nucleosome positioning between these Drosophila cell lines and therefore identify a system that could be used to investigate the functional significance of differential nucleosomal positioning in cell type specification.

Remotely acting SMCHD1 gene regulatory elements: in silico prediction and identification of potential regulatory variants in patients with FSHD.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of the D4Z4 macro-satellite repeat on chromosome 4q35 (FSHD1) or mutations in the SMCHD1 gene (FSHD2). Recent studies have shown that the clinical manifestation of FSHD1 can be modified by mutations in the SMCHD1 gene within a given family. The absence of either D4Z4 contraction or SMCHD1 mutations in a small cohort of patients suggests that the disease could also be due to disruption of gene regulation. In this study, we postulated that mutations responsible for exerting a modifier effect on FSHD might reside within remotely acting regulatory elements that have the potential to interact at a distance with their cognate gene promoter via chromatin looping. To explore this postulate, genome-wide Hi-C data were used to identify genomic fragments displaying the strongest interaction with the SMCHD1 gene. These fragments were then narrowed down to shorter regions using ENCODE and FANTOM data on transcription factor binding sites and epigenetic marks characteristic of promoters, enhancers and silencers. RESULTS: We identified two regions, located respectively ~14 and ~85 kb upstream of the SMCHD1 gene, which were then sequenced in 229 FSHD/FSHD-like patients (200 with D4Z4 repeat units <11). Three heterozygous sequence variants were found ~14 kb upstream of the SMCHD1 gene. One of these variants was found to be of potential functional significance based on DNA methylation analysis. Further functional ascertainment will be required in order to establish the clinical/functional significance of the variants found. CONCLUSIONS: In this study, we propose an improved approach to predict the possible locations of remotely acting regulatory elements that might influence the transcriptional regulation of their associated gene(s). It represents a new way to screen for disease-relevant mutations beyond the immediate vicinity of the specific disease gene. It promises to be useful for investigating disorders in which mutations could occur in remotely acting regulatory elements.

Sealing properties of mineral trioxide aggregate orthograde apical plugs and root fillings in an in vitro apexification model.

One-visit mineral trioxide aggregate (MTA) apexification is gaining in popularity over the use of calcium hydroxide barriers. This study examined the sealing properties of two MTA apexification procedures using an in vitro apexification model. White MTA was introduced into instrumented single-rooted canals with standardized artificially created open apices to form 3- to 5-mm-thick orthograde apical plugs or complete orthograde fillings of the root segments. The remaining canal spaces in the former group were backfilled with thermoplasticized gutta-percha without a sealer. A Flodec fluid filtration device was used to record fluid flow across filled root canals at 48 hours and after 4 weeks of immersion in phosphate-buffered saline (PBS). Although MTA root fillings exhibited a better seal than MTA apical plugs at 48 hours, seals of these two groups were not significantly different after 4 weeks. Interaction of MTA with PBS may result in apatite deposition that improves the seal of MTA apical plugs with time.