Staphylococcal Bi-component Toxins Research Articles

Cytotoxicity analysis of staphylococcal bi-component β-pore forming toxins using the CHO cells expressing human lymphocyte receptor CCR5.

CCR5-mediated cytotoxicity of staphylococcal bi-component toxins was investigated using human CCR5-expressing CHO cells. Cytotoxicity of rim domain loop-exchange mutants between LukE and Hlg2 indicated that loop-4 of LukE is essential for cytotoxicity in combination with LukD. Interestingly, Hlg2 showed LukF-dependent CCR5-mediated cytotoxicity, suggesting that the F-components of toxins also play a role in the cell-specific cytotoxicity.

Intermolecular ionic interactions serve as a possible switch for stem release in the staphylococcal bi-component toxin for β-barrel pore assembly

The β-strand stem release system of staphylococcal β-barrel pore-forming toxin γ-hemolysin was investigated. Mutations at K15 and R16 in the cap domain of Hlg2 decreased hemolytic activity more markedly than their effect on erythrocyte binding. In addition, D122N mutation of LukF prestem lost the activity with Hlg2 R16A, indicating that electrostatic interactions between residues in the Hlg2 cap and prestem of adjacent LukF in the ring-shaped complex might serve as a switch for stem release.

Characterization of the stoichiometry of the complex formed by Staphylococcal toxin LukSF and human C5a receptor

Staphylococcus aureus causes diseases ranging from superficial skin and soft tissue infections (SSTI) to severe invasive diseases like osteomyelitis and necrotizing pneumonia. Panton Valentine Leukocidin (PVL) is a powerful leukocidal toxin produced by multiple S. aureus isolates. It is a pro-phage encoded bi-component, β-barell pore-forming toxin (β-PFT) comprising the protein subunits LukS-PV and LukF-PV. Binding of LukS-PV to the surface of target cells induces secondary binding of LukF-PV, initiating the assembly of lytic pore-forming hetero-octamers. The stoichiometric analysis of LukS/LukF complex suggests that the leukocidin exists as an octamer consisting of 4-plus-4 subunits. In this complex only LukS is known to interact with the human C5a receptor (C5aR, CD88) that is a G-protein coupled seven-transmembrane receptor (GPCR). However, binding of LukS alone on the receptor is not sufficient for the lysis of the cell but requires simultaneous interaction between both of the leukocidin subunits and C5aR. Here, multiple possible subunit and receptor combinations are theoretically possible and the exact stoichiometry between LukS, LukF and C5aR is not yet known. The aim of this study is to determine the stoichiometry of the complexes formed by Staphylococcal bi-component toxin LukSF and human C5aR. By using novel techniques of fluorescence microscopy such as total-internal-reflection fluorescence (TIRF) combined with single molecule photobleaching analysis we were able to obtain super-resolution pictures and identify single molecules within LukSF-C5aR complex on living eukaryotic cells. Our results suggest that in addition to determination of subunit stoichiometry this technique can be used to measure the kinetics of the PFT assembly on receptors within lipid membranes of living cells. These studies may facilitate our understanding of these crucial virulence factors and their interactions with host cells.

Mode of action of β-barrel pore-forming toxins of the staphylococcal α-hemolysin family

Staphylococcal α-hemolysin is the prototype of a family of bacterial exotoxins with membrane-damaging function, which share sequence and structure homology. These toxins are secreted in a soluble form which finally converts into a transmembrane pore by assembling an oligomeric β-barrel, with hydrophobic residues facing the lipids and hydrophilic residues facing the lumen of the channel. Besides α-hemolysin the family includes other single chain toxins forming homo-oligomers, e.g. beta-toxin of Clostridium perfringens, hemolysin II and cytotoxin K of Bacillus cereus, but also the staphylococcal bi-component toxins, like γ-hemolysins and leucocidins, which are only active as the combination of two similar proteins which form hetero-oligomers. The molecular basis of membrane insertion has become clearer after the determination of the crystal structure of both the oligomeric pore and the soluble monomer. Studies on this family of β-barrel pore-forming toxins are important for many aspects: (i) they are involved in serious pathologies of humans and farmed animals, (ii) they are a good model system to investigate protein-membrane interaction and (iii) they are the basic elements for the construction of nanopores with biotechnological applications in various fields.

Composition of staphylococcal bi-component toxins determines pathophysiological reactions.

Staphylococcus aureus produces numerous bi-component toxins, e.g., Panton-Valentine leukocidin (Luk-PVL) and gamma-haemolysin, which consist of type S and F proteins. Previous studies showed that Luk-PVL induces inflammatory mediator release from human granulocytes that might reflect the in-vivo effects, e.g., dermonecrosis by Luk-PVL. Clinical isolates not only harbour the two genes coding for Luk-PVL (S-protein: LukS-PVL, F-protein: LukF-PVL) but also the three genes encoding gamma-haemolysin (S-protein: HlgA, HlgB; F-protein: HlgC). The interaction of all the possible potential toxins with human granulocytes was studied with regard to the generation of oxygen metabolites (chemiluminescence response), enzyme activity (beta-glucuronidase) and histamine release as well as interleukin (IL)-8 generation. The data clearly show that the individual subunits (S, F) differ in their activities. The following activities were obtained for the S components: LukS-PVL > HlgC > HlgA; the F components LukF-PVL and HlgB were similarly active. Thus, the toxins LukS-PVL/LukF-PVL and LukS-PVL/HlgB were the most potent inducers of inflammatory mediator release from human granulocytes, followed by HlgC/LukF-PVL and HlgC/HlgB and to a lesser degree by the toxins HlgA/LukF-PVL and HlgA/HlgB. The data indicate that class S components and class F components are interchangeable and give toxins with genuine biological activities.

黄色ブドウ球菌のロイコシジンとガンマヘモリジンの構造と活性

黄色ブドウ球菌のロイコシジンとガンマヘモリジンの構造と活性

The two Staphylococcal bi-component toxins, leukocidin and gamma-hemolysin, share one component in common

Staphylococcal bi-component toxins, leukocidin and γ-hemolysin, consist of two protein components, i.e. F and S for leukocidin and HγI and HγII for γ-hemolysin. In this study we purified HγI and HγII to homogeneity from the culture medium of Staphylococcus aureus RIMD 310925 and compared their properties with those of F and S purified from the same source. The N-terminal 59- and C-terminal 2-residue amino acid sequences, apparent molecular mass, and isoelectri point of purified HγI were the same as those of F. In an Ouchterlony double diffusion test a fused line without spur was formed between F and HγI using either anti-F or anti-HγI antibodies. A synergistic action of F and HγII caused hemolysis of human red blood cells, and HγI acted synergistically with S to exhibit leukocidin activity. We conclude that the two toxins share one protein component (F = HγI) in common and leukocidin- and γ-hemolysin-specific activities are determined by S and HγII, respectively. It is also reported that the N-terminal 58-residue sequence of HγII is 72% similar to the corresponding sequence of S.