Staphylococcal bicomponent pore-forming toxins: targets for prophylaxis and immunotherapy

Abstract

Staphylococccus aureus represents one of the most challenging human pathogens as well as a common colonizer of human skin and mucosal surfaces. S. aureus causes a wide range of diseases from skin and soft tissue infection (SSTI) to debilitating and life-threatening conditions such as osteomyelitis, endocarditis, and necrotizing pneumonia. The range of diseases reflects the remarkable diversity of the virulence factors produced by this pathogen, including surface antigens involved in the establishment of infection and a large number of toxins that mediate a vast array of cellular responses. The staphylococcal toxins are generally believed to have evolved to disarm the innate immune system, the first line of defense against this pathogen. This review focuses on recent advances on elucidating the biological functions of S. aureus bicomponent pore-forming toxins (BCPFTs) and their utility as targets for preventive and therapeutic intervention. These toxins are cytolytic to a variety of immune cells, primarily neutrophils, as well as cells with a critical barrier function. The lytic activity of BCPFTs towards immune cells implies a critical role in immune evasion, and a number of epidemiological studies and animal experiments relate these toxins to clinical disease, particularly SSTI and necrotizing pneumonia. Antibody-mediated neutralization of this lytic activity may provide a strategy for development of toxoid-based vaccines or immunotherapeutics for prevention or mitigation of clinical diseases. However, certain BCPFTs have been proposed to act as danger signals that may alert the immune system through an inflammatory response. The utility of a neutralizing vaccination strategy must be weighed against such immune-activating potential.

Figure 1

Comparison of hemolytic versus leukotoxic activities of (A) PVL; (B) LukED; (C) HlgAB, and (D) HlgCB. Dose response cytotoxicity assays were performed in HL-60 derived human PMNs or rabbit red blood cells (RRBC). Data are shown as percent cell lysis compared to no toxin control. Method: PMN Cytotoxicity assays were carried out as described previously [21] with minor modification. Briefly, DMSO (1.5%)-induced HL-60 cells (5 × 105 cells/well) were incubated with different homologous combinations of subunits for 24 h at 37 °C in an atmosphere of 5% CO₂–95% air. The cell survival was measured after adding 100 ug/mL of XTT (Sigma–Aldrich, St. Louis, MO, USA) and further incubating cells for another 16 h. A colorimetric readout at OD470 nm is used to calculate % cell lysis. For the hemolysis assay, toxin combinations were incubated with 2% rabbit blood at 37 °C for 30 mins. The suspension was centrifuged and 100 uL of the supernatants were transferred into ELISA plate. Hemolysis was measured at OD416 nm.

Figure 2

Cross combination of various leukotoxin subunits leads to qualitatively different toxin profiles in rabbit RBC hemolysis assay. OD₄₁₆ reflects the degree of hemolysis (hemolysis method same as Figure 1). Non-canonical pairing of LukD conveys novel hemolytic activity to LukS-PV (A) and HlgC (B). Replacement of HlgB in HlgAB with LukD results in a toxin (HlgA+LukD) with substantially enhanced hemolytic activity (C).

Figure 3

(A) Neutralization of PMN cytotoxic activity in supernatants of PVL positive (USA300 and USA400) and PVL negative (Newman and 8325-4) strains by antibodies to LukS-PV; (B) Neutralization of hemolytic activity of S. aureus strain Newman by antibodies to LukS-PV. Method: PMN cytotoxicity assays were carried out as described in Figure 1. For PMN TNA, purified rabbit anti-LukS IgG were incubated with serially diluted bacterial culture supernatant (grown O/N in BHI and filter sterilized) as a source of toxins. The mixtures were incubated for 1 hour at 37 °C in an atmosphere of 5% CO₂–95% air. Then, DMSO (1.5%)-induced HL-60 cells (5 × 105 cells/well) were added and incubated for 24 h at 37 °C. Cell survival was measured as described previously (Figure 1). For hemolysis TNA assay, purified rabbit anti-LukS IgG/control IgG were incubated with serially diluted Newman culture supernatant (grown O/N in BHI and filter sterilized) as a source of toxins for 10 mins at room temperature. Two percent rabbit blood was added and further incubated at 37 °C for 30 mins. The suspension was centrifuged and 100 uL of the supernatants were transferred into the ELISA reading plate. Hemolysis was measured as described in Figure 1.