The host cell membrane attacking toxins of <i>Staphylococcus aureus</i> and their roles beyond host cell lysis.

<p><b>(A)</b> Phagocytosis of invading bacteria is followed by fusing of the phagosome to the lysosome, resulting in destruction of the bacteria. <i>S</i>. <i>aureus</i> alpha (α) and phenol-soluble modulin (PSM) toxins inhibit fusing of the lysosome. This enables the bacteria to escape from the phagosome into the cytoplasm, allowing intracellular niche establishment and replication. <b>(B)</b> PSM toxins target cohabiting bacterial species within established niches, aiding in competition for resources and competitive exclusion of nonkin isolates. <b>(C)</b> PSM toxins have surfactant properties in vitro, enabling sliding movement across agar surfaces in the absence of traditional mobility structures such as flagella and pili. <b>(D)</b> Pore-forming toxins are involved at each step of <i>S</i>. <i>aureus</i> biofilm formation. During the initial cell attachment phase, alpha-toxin is involved in establishing cell-to-cell contacts, enabling the formation of secondary biofilm structures. In the later stages of the biofilm lifestyle, extracellular matrices develop, surrounding the cells within the biofilm. In the presence of extracellular DNA (eDNA), beta-toxin covalently cross-links with itself, adding to this extracellular nucleoprotein biofilm matrix and contributing to the formation of complex biofilm secondary structuring. Detachment from the mature biofilm allows for dispersal to new sites of infection. PSM toxins are involved in this stage of the biofilm lifestyle, aiding release of cell clusters from the main body of the biofilm.</p

Contribution of pertussis toxin (PT) and adenylate cyclase toxin (ACT) to pathogenicity of <i>Bordetella pertussis</i>.

<p>The adenylate cyclase (AC)-affecting toxins of <i>B</i>. <i>pertussis</i> contribute to disease progression via: <b>(A)</b> PT is endocytosed into a cell and, following intracellular processing by the endoplasmic reticulum, the alpha subunit is released into the cytosol. This subunit ADP-ribosylates the alpha subunit of G proteins, disassociating it from its G protein coupled receptor (GPCR) on the cell surface inhibiting recruitment of immune cells to the site of infection. <b>(B)</b> ACT interacts with cell surface complement receptor (CR3) on macrophages and neutrophils, affecting antigen presentation and recruitment of the downstream adaptive immune response. The AC domain translocates to the cell cytoplasm and is stimulated upon calmodulin binding, leading to increased cAMP levels, inhibiting pro-inflammatory cytokine release and complementing mediated phagocytosis, and interfering with immune cell recruitment. <b>(C)</b> PT released into the bloodstream from cells growing on ciliated epithelial lung cells has been shown to contribute to development of leukocytosis. The mechanism is unclear but several have been proposed including <b>(C1)</b> PT inhibiting migration of lymphocytes across epithelium layers, <b>(C2)</b> PT interfering with GPCR signalling, effecting immune cell recruitment, <b>(C3)</b> PT inhibiting GPCRs required for leukocytes to stick to lymph nodes, interfering with extravasation, and <b>(C4)</b> PT stimulating the expansion of normal naïve immune cells and not proliferation of activated cells. <b>(D)</b> ACT inhibits biofilm formation by interfering with filamentous haemagglutinin–filamentous haemagglutinin (FHA-FHA) interactions between cells. The AC domain of the toxin binds to the mature C-terminal domain (MCD) at the distal tip of the FHA protein, blocking its function in biofilm.</p

Inhibition of bacterial adherence and rClfB binding to immobilized ligands.

<p>(A) <i>L. lactis</i> NZ9800 (pNZ8037), <i>L. lactis</i> NZ9800 (pNZ8037:<i>clf</i>B) and <i>L. lactis</i> NZ9800 (pNZ8037:<i>clf</i>BQ235A) were added to wells containing immobilized GST-tagged Hlor, MLor, HK10, MK10 (0.5 µM). Bacterial adherence was measured by staining with crystal violet and measurement of the absorbance at 570 nm. The data shown is representative of two individual experiments (B). <i>S. aureus</i> Newman pre-incubated with GST or HK10 (2 µM) was added to loricrin-coated microtitre wells (0.5 µM). Bacterial adherence was measured by staining with crystal violet and measurement of the absorbance at 570 nm and was expressed as a percentage of total binding. Values represent the mean ± SD of triplicate wells. The data shown is representative of two individual experiments. (C) Recombinant ClfB N23_201–542 was pre-incubated with GST, HK10 or L2v (14 µM) before being added to loricrin-coated microtitre wells (0.5 µM). Bound protein was detected using HRP-conjugated anti-his antibodies and was expressed as a percentage of total bound protein. Values represent the mean ± SD of triplicate wells. The values shown are representative of 3 individual experiments. Statistical analysis was performed using an unpaired t-test. *** p<0.0005 versus binding of ClfB-expressing bacteria (A) or pre-incubation with GST (B, C).</p

HLor region L2v blocks ClfB-mediated adherence of <i>S. aureus</i> to human desquamated epithelial cells.

<p><i>S. aureus</i> strains were grown to exponential phase in TSB (A) or in RPMI (B). Washed cells were incubated with recombinant GST or recombinant L2v-GST, or just resuspended in PBS, before being incubated with human nasal epithelial cells. Adherent bacteria were enumerated by microscopy and were expressed as a percentage of the positive control. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003092#s2" target="_blank">Results</a> are expressed as the mean ± SD of 3 independent experiments. Statistical analysis was performed using an unpaired t test.</p

Nasal Colonisation by <em>Staphylococcus aureus</em> Depends upon Clumping Factor B Binding to the Squamous Epithelial Cell Envelope Protein Loricrin

<div><p><em>Staphylococcus aureus</em> asymptomatically colonises the anterior nares, but the host and bacterial factors that facilitate colonisation remain incompletely understood. The <em>S. aureus</em> surface protein ClfB has been shown to mediate adherence to squamous epithelial cells <em>in vitro</em> and to promote nasal colonisation in both mice and humans. Here, we demonstrate that the squamous epithelial cell envelope protein loricrin represents the major target ligand for ClfB during <em>S. aureus</em> nasal colonisation. <em>In vitro</em> adherence assays indicated that bacteria expressing ClfB bound loricrin most likely by the “dock, lock and latch” mechanism. Using surface plasmon resonance we showed that ClfB bound cytokeratin 10 (K10), a structural protein of squamous epithelial cells, and loricrin with similar affinities that were in the low µM range. Loricrin is composed of three separate regions comprising GS-rich omega loops. Each loop was expressed separately and found to bind ClfB, However region 2 bound with highest affinity. To investigate if the specific interaction between ClfB and loricrin was sufficient to facilitate <em>S. aureus</em> nasal colonisation, we compared the ability of ClfB⁺<em>S. aureus</em> to colonise the nares of wild-type and loricrin-deficient (Lor^−/−) mice. In the absence of loricrin, <em>S. aureus</em> nasal colonisation was significantly impaired. Furthermore a ClfB⁻ mutant colonised wild-type mice less efficiently than the parental ClfB⁺ strain whereas a similar lower level of colonisation was observed with both the parental strain and the ClfB⁻ mutant in the Lor^−/− mice. The ability of ClfB to support nasal colonisation by binding loricrin <em>in vivo</em> was confirmed by the ability of <em>Lactococcus lactis</em> expressing ClfB to be retained in the nares of WT mice but not in the Lor^−/− mice. By combining <em>in vitro</em> biochemical analysis with animal model studies we have identified the squamous epithelial cell envelope protein loricrin as the target ligand for ClfB during nasal colonisation by <em>S. aureus</em>.</p> </div

Systemic infection in Lor^−/− mice.

<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003092#s2" target="_blank">Results</a> expressed as mean Log CFU/ml of fluid or homogenised tissue ± SEM, n = 4 per group.</p

Surface Plasmon Resonance analysis of the interaction of ClfB with loricrin and keratin.

<p>Representative sensorgrams display binding of rClfB_201–542 to and dissocation from (A) GST-HLor, (B) GST-HK10, (C) GST-MLor and (D) GST-MK10 in a single cycle kinetics assay. GST-tagged ligands were captured onto a CM5 chip coated with anti-GST IgG and were exposed to increasing concentrations of rClfB_201–542. Binding is measured as response units (RU) against time. The affinities were calculated from curve fitting to a plot of the RU values against concentrations of rClfB_201–542. Arrows indicate the time at which rClfB_201–542 is injected. The data shown is representative of 3 individual experiments.</p

Affinities of ClfB N2N3_201–542 for loricrin, keratin and fibrinogen using surface plasmon resonance.

*<p>Data representative of n = 3 individual experiments.</p

Nasal colonisation of <i>L. lactis</i> expressing ClfB and Newman Δ<i>clfB</i>⁻ in the FVB wild-type and Lor^−/− mice.

<p>(A) Mice were inoculated intra-nasally with <i>L. lactis</i> MG1363 (pKS80) or <i>L. lactis</i> MG1363 (pKS80:<i>clf</i>B) (2×10¹¹ CFU). Mice were euthanized after 24 hours and the bacterial burden in noses was established. Inoculation with the empty vector (pKS80) did not result in significant colonisation (>5 CFU per nose) in either WT or Lor^−/− mice. Statistical analysis was performed using the Mann-Whitney test. (B) Mice were inoculated intra-nasally with Newman or Newman Δ<i>clfB</i> (2×10⁸ CFU). After 10 days, mice were euthanized and bacterial burden in the noses was established. Each dot indicates the number of CFU/nose for a single mouse. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003092#s2" target="_blank">Results</a> expressed as Log CFU per nose, median indicated by bar (n = 15–20 per group). Statistical analysis was performed using the Krustal-wallis test and Dunns Multiple Comparisons test. *p<0.05, **p<0.005.</p

Relative Contribution of Th1 and Th17 Cells in Adaptive Immunity to <i>Bordetella pertussis</i>: Towards the Rational Design of an Improved Acellular Pertussis Vaccine

<div><p>Whooping cough caused by <i>Bordetella pertussis</i> is a re-emerging infectious disease despite the introduction of safer acellular pertussis vaccines (Pa). One explanation for this is that Pa are less protective than the more reactogenic whole cell pertussis vaccines (Pw) that they replaced. Although Pa induce potent antibody responses, and protection has been found to be associated with high concentrations of circulating IgG against vaccine antigens, it has not been firmly established that host protection induced with this vaccine is mediated solely by humoral immunity. The aim of this study was to examine the relative contribution of Th1 and Th17 cells in host immunity to infection with <i>B. pertussis</i> and in immunity induced by immunization with Pw and Pa and to use this information to help rationally design a more effective Pa. Our findings demonstrate that Th1 and Th17 both function in protective immunity induced by infection with <i>B. pertussis</i> or immunization with Pw. In contrast, a current licensed Pa, administered with alum as the adjuvant, induced Th2 and Th17 cells, but weak Th1 responses. We found that IL-1 signalling played a central role in protective immunity induced with alum-adsorbed Pa and this was associated with the induction of Th17 cells. Pa generated strong antibody and Th2 responses, but was fully protective in IL-4-defective mice, suggesting that Th2 cells were dispensable. In contrast, Pa failed to confer protective immunity in IL-17A-defective mice. Bacterial clearance mediated by Pa-induced Th17 cells was associated with cell recruitment to the lungs after challenge. Finally, protective immunity induced by an experimental Pa could be enhanced by substituting alum with a TLR agonist that induces Th1 cells. Our findings demonstrate that alum promotes protective immunity through IL-1β-induced IL-17A production, but also reveal that optimum protection against <i>B. pertussis</i> requires induction of Th1, but not Th2 cells.</p> </div