Solution structure of a repeated unit of the ABA-1 nematode polyprotein allergen of ascaris reveals a novel fold and two discrete lipid-binding sites

Parasitic nematode worms cause serious health problems in humans and other animals. They can induce allergic-type immune responses, which can be harmful but may at the same time protect against the infections. Allergens are proteins that trigger allergic reactions and these parasites produce a type that is confined to nematodes, the nematode polyprotein allergens (NPAs). These are synthesized as large precursor proteins comprising repeating units of similar amino acid sequence that are subsequently cleaved into multiple copies of the allergen protein. NPAs bind small lipids such as fatty acids and retinol (Vitamin A) and probably transport these sensitive and insoluble compounds between the tissues of the worms. Nematodes cannot synthesize these lipids, so NPAs may also be crucial for extracting nutrients from their hosts. They may also be involved in altering immune responses by controlling the lipids by which the immune and inflammatory cells communicate. We describe the molecular structure of one unit of an NPA, the well-known ABA-1 allergen of Ascaris and find its structure to be of a type not previously found for lipid-binding proteins, and we describe the unusual sites where lipids bind within this structur

Staphylococcus aureus Keratinocyte Invasion Is Dependent upon Multiple High-Affinity Fibronectin-Binding Repeats within FnBPA

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α5β1 integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA

Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs

Citation: Liang, X. W., Garcia, B. L., Visai, L., Prabhakaran, S., Meenan, N. A. G., Potts, J. R., . . . Hook, M. (2016). Allosteric Regulation of Fibronectin/alpha(5)beta(1) Interaction by Fibronectin-Binding MSCRAMMs. Plos One, 11(7), 17. doi:10.1371/journal.pone.0159118Adherence ofmicrobes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with alpha(5)beta(1) integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/alpha(5)beta(1) integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/alpha(5)beta(1) integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/alpha(5)beta(1) on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic alpha(5)beta(1) interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/alpha(5)beta(1) affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Keratinocyte expression of α₅ and β₁ integrins is lower than endothelial cells.

<p>HaCat keratinocytes (Kera) or EA. Hy926 endothelial (Endo) cells were harvested from flasks, lysed and the lysate examined by SDS-PAGE and Western-immunoblot to determine relative α₅ or β₁ integrin expression levels.</p

Over-expression of FnBPR1 or FnBPR6–8 fails to trigger maximal invasion of Keratinocytes.

<p>Expression of FnBPA variant constructs on the surface of <i>L. lactis</i> was performed using a nisin-inducible system. Invasion of <i>L. lactis</i> expressing FnBPAR1–11 (circles), FnBPAR1,10,11 (triangles), FnBPAR1 (squares) or FnBPAR6–8 (diamonds) was determined at 3 different levels of induction (0, 10 or 100 ng ml⁻¹ nisin). Experiments were performed 3 times in duplicate. Error bars represent the standard deviation of the mean. MOI = 100. Values that are significantly different (p = <0.05) from <i>L. lactis</i> FnBPAR1–11 at identical nisin concentrations are indicated (*).</p

<i>S. aureus</i> adhesion to, and invasion of, keratinocyte and endothelial cells over time.

<p>The total number of <i>S. aureus</i> 8325.4 (DU5883 Δ<i>fnb</i>A/B pFnBA4 expressing full length FnBPA) associated (adherent and internalized; circles) and internalized (squares) with each cell type was determined over a period of 90 minutes. Values for adhesion to, and invasion of, endothelial cells that are statistically significantly different from those obtained with HaCat cells are denoted (*). Experiments were performed 3 times in duplicate. Multiplicity of infection (MOI) = 20. Error bars represent the standard deviation of the mean.</p

Keratinocyte invasion occurs via a similar mechanism to endothelial cells.

<p>HaCat cells were pre-incubated (60 min) with inhibitors of cell function before the addition of <i>S. aureus</i> FnBPAR1–11 (DU5883 Δ<i>fnb</i>A/B pFnBA4) and invasion determined after 90 mins. Inhibitors used were cytochalasin D (CD, inhibits actin polymerization), wortmannin (WRT, inhibits PI3-Kinase activity), genistein (GEN, inhibits tyrosine kinase activity), PP2 (PP2, Src kinase inhibitor), colchicine (COL, interferes with microtubule organisation), cycloheximide (CHX, inhibits eukaryotic protein synthesis) and methyl-β-cyclodextrine (MCD, depletes membrane cholesterol). Inhibitor-free medium was used as a positive control (CTL). Experiments were performed three times in duplicate. MOI = 20. Error bars represent the standard deviation of the mean. Values that are significantly different (p = <0.05) from control are indicated (*).</p

A recombinantly-expressed FnBR peptide inhibits <i>S. aureus</i> invasion.

<p>The adhesion to (circles), and invasion (squares) of, keratinocytes by <i>S. aureus</i> FnBPR1–11 (DU5883 Δ<i>fnb</i>A/B pFnBA4) in the presence of various concentrations of recombinant FnBPA peptide (R9,10). Values that statistically significantly difference from those obtained in the absence of peptide are indicated (*). MOI = 20. Experiments were performed 3 times in duplicate. Error bars represent the standard deviation of the mean.</p