Synthesis of a Dicarba Analogue of Human β-Defensin-1 Using a Combined Ring Closing Metathesis–Native Chemical Ligation Strategy

We herein describe the first synthesis of the native antimicrobial protein HBD-1 making use of an orthogonal thiol protection strategy and a novel dicarba analogue thereof. The robust hydrocarbon linkage was installed by replacement of one disulfide bond using on-resin ring closing metathesis. The unprecedented 59-membered C-terminal cysteine macrocyclic fragment thus formed then engages in native chemical ligation allowing convergent access to this unique synthetic protein analogue

Synthesis and purification of M1:HBcAg VLPs.

<p>M1 was coupled to HBcAg (residues 1–142) using the heterobifunctional cross-linker sMBS (Pierce). Western Blot indicating the coupled product is recognised by both the anti-M1 and anti-HBcAg antibodies. (B) Following conjugation, the M1:HBcAg was purified from unbound M1 protein by ultracentrifugation. The resulting coupled product was analysed by SDS-PAGE under reducing conditions followed by silver stain. Following purification no contaminating M1 was observed in the purified product. HBcAg (C) and M1:HBcAg (D) were stained with uranyl acetate and analysed by electron microscopy. The morphology of HBcAg was retained following coupling and purification.</p

Colocalisation of M1:HBcAg and Endocytic markers.

<p>Day 6 immature DCs were incubated with M1:HBcAg on ice to allow binding and the HBcAg was fluorescently labelled. The cells were subsequently incubated at 37°C to allow internalisation. At the indicated timepoints, cells were harvested, fixed, permeabilised and stained for endocytic markers (in green) for analysis by confocal microscopy. M1:HBcAg colocalised with the early endosome marker EEA1 within 10 minutes and the late endosomal marker LAMP1 from 30 minutes. Following internalisation, M1:HBcAg was shown to colocalise with MHC I. The frequency of pixel colocalisation (on the left) was determined by Manders coefficient from 4–8 cells per treatment. Statistical significance was determined by the unpaired student <i>t</i>-test; *p&lt;0.05, **p&lt;0.005.</p

M1-conjugation improves CD4⁺ responses to the universal T_H-epitope TTp2.

<p>TTp2 was coupled to M1 under oxidising conditions and coupling confirmed by SDS-PAGE (A). PBMC were treated with M1:TTp2 or molar equivalents of M1, TTp2 or mixed M1 and TTp2 in an IFNγ Elispot Assay (BD Biosciences). Responses from 10 individuals are shown (B). Statistically significant differences in the response observed for M1-coupled peptide and controls are denoted by * (determined by t Test). Activation of purified CD4⁺ cells (C) in the presence of the indicated antigen was assessed by Elispot assay and compared to PBMC depleted of CD4 cells in (D). Data representative of one individual's response is given showing the mean nd standard deviation of duplicate samples. Statistical significance is indicated by * where p&lt;0.05; statistical significance was determined by the student t-test.</p

M1:HBcAg is presented on MHCI and activates HBcAg-specific CD8+ T cells.

<p>Immature DCs were pulsed with M1:HBcAg or the control antigens for 60 minutes at room temperature. Following extensive washing, the cells were incubated overnight at 37°C. Antigen-loaded cells or untreated cells (UT) were incubated with HBc_18–27 cells for 6 hours in the presence of Brefeldin A and monensin. CD107a (A&amp;C) expression and IFNγ (B&amp;D) production were measured in parallel. Results in A and B indicate the T cell activation as a percentage of the positive control of DCs loaded with the molar equivalent of C_18–27 peptide. Data are representative of 4 individual experiments. M1:HBcAg treatment resulted in significantly more T cell clones expressing CD107a and IFNγ than the control antigens. *Denotes statistical significance determined by student <i>t</i>-test, &lt;0.05. A representative data set from one individual is presented in C &amp; D showing mean and standard deviation of duplicate samples.</p

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) period-prevalence rates for clinical isolates in New Zealand, 2005–2011, stratified by (A) age, and (B) ethnicity.

<p>Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) period-prevalence rates for clinical isolates in New Zealand, 2005–2011, stratified by (A) age, and (B) ethnicity.</p

Relative proportions of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) clones circulating in New Zealand, 2005–2011.

<p>Relative proportions of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) clones circulating in New Zealand, 2005–2011.</p

Sociodemographic and molecular epidemiological associations of community-associated MRSA (CA-MRSA) and healthcare-associated MRSA (HCA-MRSA) in New Zealand, 2005–2011.

<p>Note: Data are number (%) of patients unless stated otherwise.</p>a<p>Mann-Whitney <i>U</i> test.</p><p>Abbreviations: OR, odds ratio; CI, confidence interval; IQR, interquartile range; ST, sequence type, NZDep, New Zealand Deprivation Index score; MRSA, methicillin-resistant <i>Staphylococcus aureus</i>.</p

Period-prevalence rates of community-associated methicillin-resistant <i>Staphylococcus aureus</i> (CA-MRSA), healthcare-associated community-onset MRSA (HCA-CO MRSA) and healthcare-associated hospital-onset (HCA-HO MRSA) in New Zealand, 2005–2011.

<p>Period-prevalence rates of community-associated methicillin-resistant <i>Staphylococcus aureus</i> (CA-MRSA), healthcare-associated community-onset MRSA (HCA-CO MRSA) and healthcare-associated hospital-onset (HCA-HO MRSA) in New Zealand, 2005–2011.</p

Staphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors

<div><p><i>Staphylococcus aureus</i> is an opportunistic pathogen that produces many virulence factors. Two major families of which are the staphylococcal superantigens (SAgs) and the Staphylococcal Superantigen-Like (SSL) exoproteins. The former are immunomodulatory toxins that induce a Vβ-specific activation of T cells, while the latter are immune evasion molecules that interfere with a wide range of innate immune defences. The superantigenic properties of Staphylococcal enterotoxin-like X (SElX) have recently been established. We now reveal that SElX also possesses functional characteristics of the SSLs. A region of SElX displays high homology to the sialyl-lactosamine (sLacNac)-specific binding site present in a sub-family of SSLs. By analysing the interaction of SElX with sLacNac-containing glycans we show that SElX has an equivalent specificity and host cell binding range to the SSLs. Mutation of key amino acids in this conserved region affects the ability of SElX to bind to cells of myeloid origin and significantly reduces its ability to protect <i>S</i>. <i>aureus</i> from destruction in a whole blood killing (WBK) assay. Like the SSLs, SElX is up-regulated early during infection and is under the control of the <i>S</i>. <i>aureus</i> exotoxin expression (Sae) two component gene regulatory system. Additionally, the structure of SElX in complex with the sLacNac-containing tetrasaccharide sialyl Lewis X (sLeX) reveals that SElX is a unique single-domain SAg. In summary, SElX is an ‘SSL-like’ SAg.</p></div