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

Measurement of <i>rna</i>III transcription.

<p>Real – time qPCR results of 19 strains consisting of the positive and negative control RN6390B and RN6911 respectively, the 3 negative vesicle lysis test strains (MSSA 71, MRSA 378 and MSSA 707), 13 non-haemolytic strains and the haemolytic strain MRSA 325. Illustrates <i>rna</i>III transcription in those strains designated <i>agr</i> positive by vesicle method and no transcription is evident in those 3 strains which show no lysis of vesicles.</p

Effect of purified toxins on lipid vesicles and T – cells.

<p><b>A)</b> Vesicle rupture as a result of incubation with 10 µM synthetic PSM and delta peptides for 30 min <b>B)</b> Lysis of vesicles and <b>C)</b> T-cells subjected to selected concentrations of purified delta toxin. <b>D)</b> Lysis of vesicles and <b>E)</b> T-cells subjected to selected concentrations of purified PSM3α toxin. The vesicle system is highly sensitive to both toxins at low concentrations, while PSM3α is more potent at lysing both vesicles and T-cells than delta toxin. T-cells were incubated for 15 min at 37°C with purified toxins.</p

Key characteristics of CAMP and VLT assayed strains.

<p>Abbreviations: CC; Clonal complex, CAMP; synergistic haemolysis plate assay, VLT; vesicle lysis test.</p>¶<p>Values shown in normalised fluorescence units.</p>*<p>qRT-PCR after 8 hours.</p

SDS-PAGE of concentrated <i>agr</i> regulated peptides.

<p>Concentrated and extracted proteins from several <i>S. aureus</i> strains, showing the presence of protein bands, indicating a mix of delta and PSM peptides. Purified delta peptide and RN6390B used as a positive control and RN6911 as a negative control. Figure shows the absence of bands in those strains which cause no lysis of vesicles.</p

Differences in <i>agr</i> activity observed using two methods.

<p><b>A)</b> Delta haemolysin plate assay of <i>agr</i> positive (RN6390B) and negative (RN6911) strains, 17 <i>S. aureus</i> clinical isolates, 16 which are designated as <i>agr</i> – negative, one <i>agr</i> positive isolate and <i>agr</i> – positive LAC and its corresponding isogenic <i>hld</i> mutant strain, signifying the effects of delta toxin and PSM on the haemolysin plate assay. <b>B)</b> Normalized fluorescence measurements of 89 clinical <i>S. aureus</i> strains using the vesicle–supernatant method, highlighting the three strains causing no vesicle lysis.</p

Bacterial strains, peptide sequences and primers used in this study.

<p><b>Abbreviations:</b><i>agr</i>, accessory gene regulator; <i>hla</i>, gene for α-toxin; <i>pvl</i>, gene for Panton-Valentine Leucocidin; <i>hld</i>, delta hemolysin; <i>luk</i>, leukocidin; <i>hlg</i>, gamma haemolysin; PSM, phenol soluble modulin; <i>gyr</i>B, DNA gyrase B.</p

Eap is a virulence factor in a murine bacteremia model.

<p>Mice were challenged with Newman wild-type (WT), mAH12 (<i>eap-</i>), mAH12 (pll39) and mAH12 (pll<i>eap</i>) and their weight monitored over 7 days. Error bars represent the standard deviation of the mean. Values that are significantly different (p<0.05) from mAH12 (<i>eap-</i>) are highlighted with an *, and those significantly different (p<0.05) from mAH12 (pll39) with a #.</p

Protein A mediates increased attachment to endothelial cells via a mechanism involving gC1qR/p33.

<p>A selection of strains deficient in cell-surface proteins Protein A (spa), Clumping factor A (<i>clf</i>), Coagulase (<i>coa</i>)) or capsular polysaccharide (<i>cps</i>) were assessed for their ability to bind to either TNFα pre-treated or untreated endothelial cells (A). Attachment of <i>S. aureus</i> Newman to TNFΑ-treated cells in the absence (Ctl) or presence of various antibodies, including α5 (α5) and β1 (β1) integrin subunits, ICAM-1 (IC), Fibronectin (Fn), Tissue factor (TF) and gC1qR/p33 (C1) was also determined (B). Values indicate the mean average of 3 independent experiments performed in duplicate. Error bars represent the standard deviation of the mean. Values that are significantly different (p<0.05) from experiments where endothelial cells were incubated in the absence of TNFα (A) or antibodies (B) are indicated (*).</p