41 research outputs found
Homology modelling of transferrin-binding protein A from Neisseria meningitidis
Neisseria meningitidis, a causative agent of bacterial
meningitis, obtains transferrin-bound iron by expressing
two outer membrane located transferrin-binding proteins,
TbpA and TbpB. TbpA is thought to be an integral outer
membrane pore that facilitates iron uptake. Evidence suggests
that TbpA is a useful antigen for inclusion in a vaccine
effective against meningococcal disease, hence the identification
of regions involved in ligand binding is of paramount
importance to design strategies to block uptake of iron. The
protein shares sequence and functional similarities to the
Escherichia coli siderophore receptors FepA and FhuA,
whose structures have been determined. These receptors
are composed of two domains, a 22-stranded b-barrel and
an N-terminal plug region that sits within the barrel and
occludes the transmembrane pore. A three-dimensional
TbpA model was constructed using FepA and FhuA structural
templates, hydrophobicity analysis and homology
modelling. TbpA was found to possess a similar architecture
to the siderophore receptors. In addition to providing
insights into the highly immunogenic nature of TbpA and
allowing the prediction of potentially important ligandbinding
epitopes, the model also reveals a narrow channel
through its entire length. The relevance of this channel and
the spatial arrangement of external loops, to the mechanism
of iron translocation employed by TbpA is discussed
Imaging of Disease Dynamics during Meningococcal Sepsis
Neisseria meningitidis is a human pathogen that causes septicemia and meningitis with high mortality. The disease progression is rapid and much remains unknown about the disease process. The understanding of disease development is crucial for development of novel therapeutic strategies and vaccines against meningococcal disease. The use of bioluminescent imaging combined with a mouse disease model allowed us to investigate the progression of meningococcal sepsis over time. Injection of bacteria in blood demonstrated waves of bacterial clearance and growth, which selected for Opa-expressing bacteria, indicating the importance of this bacterial protein. Further, N. meningitidis accumulated in the thyroid gland, while thyroid hormone T4 levels decreased. Bacteria reached the mucosal surfaces of the upper respiratory tract, which required expression of the meningococcal PilC1 adhesin. Surprisingly, PilC1 was dispensable for meningococcal growth in blood and for crossing of the blood-brain barrier, indicating that the major role of PilC1 is to interact with mucosal surfaces. This in vivo study reveals disease dynamics and organ targeting during meningococcal disease and presents a potent tool for further investigations of meningococcal pathogenesis and vaccines in vivo. This might lead to development of new strategies to improve the outcome of meningococcal disease in human patients
Outer membrane protein size and LPS O-antigen define protective antibody targeting to the Salmonella surface
Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs
Genome Wide Expression Profiling Reveals Suppression of Host Defence Responses during Colonisation by Neisseria meningitides but not N. lactamica
Both Neisseria meningitidis and the closely related bacterium Neisseria lactamica colonise human nasopharyngeal mucosal surface, but only N. meningitidis invades the bloodstream to cause potentially life-threatening meningitis and septicaemia. We have hypothesised that the two neisserial species differentially modulate host respiratory epithelial cell gene expression reflecting their disease potential. Confluent monolayers of 16HBE14 human bronchial epithelial cells were exposed to live and/or dead N. meningitidis (including capsule and pili mutants) and N. lactamica, and their transcriptomes were compared using whole genome microarrays. Changes in expression of selected genes were subsequently validated using Q-RT-PCR and ELISAs. Live N. meningitidis and N. lactamica induced genes involved in host energy production processes suggesting that both bacterial species utilise host resources. N. meningitidis infection was associated with down-regulation of host defence genes. N. lactamica, relative to N. meningitidis, initiates up-regulation of proinflammatory genes. Bacterial secreted proteins alone induced some of the changes observed. The results suggest N. meningitidis and N. lactamica differentially regulate host respiratory epithelial cell gene expression through colonisation and/or protein secretion, and that this may contribute to subsequent clinical outcomes associated with these bacteria
Clinical and biological roles of Kelch-like family member 7 in breast cancer: a marker of poor prognosis
Background: The functions of many proteins are tightly regulated with a complex array of cellular functions including ubiquitination. In cancer cells, aberrant ubiquitination may promote the activity of oncogenic pathways with subsequent tumour progression. Kelch-like family member 7 (KLHL7) is involved in the regulation of ubiquitination and may play a role in breast cancer (BC). Present study aims to evaluate the biological and clinical usefulness of KLHL7 in BC utilising large well-characterised cohorts with long-term follow-up.
Methods: The relationships between KLHL7 gene copy number alteration (CNA) and mRNA expression and clinicopathological variables and clinical outcomes were evaluated in 1980 patients from the METABRIC BC cohort. Prognostic signifcance of KLHL7 mRNA was validated using the Breast Cancer Gene-Expression Miner v4.0 datasets (n=5206). KLHL7 protein expression was assessed using immunohistochemistry in a large annotated series of early-stage BC (n=917) with long-term follow-up.
Results: KLHL7 CNA was signifcantly correlated with its mRNA expression. KLHL7 mRNA expression was higher in luminal B and basal-like molecular subtypes and in higher grade tumours. Increased KLHL7 protein expression was signifcantly correlated with features of aggressive phenotype including lymphovascular invasion, high histological grade, hormonal receptor negativity, high PIK3CA and p53 expression. Outcome analysis showed that high KLHL7 expression is an independent predictor of shorter survival (p=0.0011).
Conclusions: KLHL7 appears to play an important role in BC progression. High KLHL7 protein expression identifed a subgroup of BC with aggressive behaviour and provided independent prognostic information
Expression and purification of functional recombinant meningococcal transferrin-binding protein A
Pathogenic bacteria of the genus Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and human transferrin (hTf), a serum protein. In the meningococcus, this uptake system is dependent on two surface-exposed, transferrin-binding proteins (Tbps), TbpA and TbpB. TbpA is highly conserved among meningococcal strains, and is thought to be a porin-like integral protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is more variable in size, lipidated and fully surface-exposed. Given its location on the cell surface, its role in pathogenicity and interstrain sequence conservation, TbpA is currently being regarded for inclusion in a meningococcal vaccine effective against all serogroups. This requires gaining knowledge of the ligand-receptor interactions. In the present study we have optimized a procedure for obtaining purified, functionally active recombinant TbpA at a level and stability necessary for the initiation of such studies
The clinical and biological significance of HER2 over-expression in breast ductal carcinoma in situ: a large study from a single institution
BACKGROUND: Previous studies have reported up to 50% of ductal carcinoma in situ (DCIS), is HER2 positive, but the frequency of HER2-positive invasive breast cancer (IBC) is lower. The aim of this study is to characterise HER2 status in DCIS and assess its prognostic value. METHODS: HER2 status was evaluated in a large series of DCIS (n = 868), including pure DCIS and DCIS associated with IBC, prepared as tissue microarrays (TMAs). HER2 status was assessed using immunohistochemistry (IHC) and chromogenic in situ hybridisation (CISH). RESULTS: In pure DCIS, HER2 protein was over-expressed in 9% of DCIS (3+), whereas 15% were HER2 equivocal (2+). Using CISH, the final HER2 status was positive in 20%. In mixed DCIS, HER2 amplification of the DCIS component was detected in 15% with amplification in the invasive component of only 12%. HER2-positive DCIS was associated with features of aggressiveness (p < 0.0001) and more frequent local recurrence (p = 0.03). On multivariate analysis, combined HER2+/Ki67+ profile was an independent predictor of local recurrence (p = 0.006). CONCLUSIONS: The frequency of HER2 positivity in DCIS is comparable to IBC- and HER2-positive DCIS is associated with features of poor prognosis. The majority of HER2 over-expression in DCIS is driven by gene amplification