Fig 1.
Staphylococcus aureus LukAB induces cell death in human monocytic cells.
(A) THP1 cells were infected with the indicated S. aureus strains at a multiplicity of infection (MOI) of 50 for 2 hours and culture supernatants were analyzed for LDH release as a measurement of cell lysis. (B) Culture filtrates, acquired from log-phase growth of S. aureus strains listed, were used to intoxicate THP1 cells at the indicated concentrations for 4 hours and LDH release was analyzed. (C and D) THP1 cells were intoxicated with culture filtrates from S. aureus Newman or the indicated isogenic mutants for 4 hours and LDH release was analyzed. (E) THP1 cells were infected with S. aureus Newman or the indicated isogenic mutants at an MOI of 50 and LDH release was analyzed after 2 hours. (F) THP1 cells were incubated with propidium iodide and intoxicated with culture filtrates from S. aureus Newman, lukAB mutant, or complemented strain at 1% (v/v) for 4 hours then analyzed by flow cytometry. THP1 cells (G) and primary CD14+ human monocytes (H) were infected with S. aureus Newman, USA300 (LAC) or USA400 (MW2) or the respective lukAB-deficient mutants and complemented strains at an MOI of 50 (G) or 25 (H) and LDH release was analyzed after 2 hours. Error bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Experiments with primary cells include at least three independent donors. Asterisks indicate significance at a p-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.
Fig 2.
LukAB targets CD11b on human monocytes to potently induce cell death.
(A) THP1 cells were intoxicated with titrations of the indicated purified toxins for 1 hour and LDH release was analyzed. (B) THP1 cells were transduced with either non-targeting shRNA or shRNA against CD11b and surface CD11b levels were evaluated by flow cytometry. (C) THP1 cells described in panel B were intoxicated with titrations of the indicated toxins for 1 hour and LDH release was analyzed. (D) THP1 cells were intoxicated with the indicated concentration of LukAB for 1 hour and analyzed by flow cytometry for permeability to propidium iodide. (E) Primary CD14+ human monocytes were intoxicated with titrations of the indicated toxins for 1 hour and analyzed by flow cytometry for permeability to propidium iodide. EC50 values are also shown. Error bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Primary cell experiments include three independent donors. Asterisks indicate significance at a p-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.
Fig 3.
LukAB induces necrotic cell death and secretion of pro-inflammatory cytokines IL-1β and IL-18.
(A) THP1 cells were intoxicated with culture filtrate from S. aureus Newman (10% v/v), isogenic lukAB mutant or culture media for 1 hour. Cells were collected, prepared and imaged by transmission electron microscopy (see methods). (B) THP1 cells were intoxicated with 1% (v/v) culture filtrate from S. aureus Newman or the indicated isogenic mutants and after 4 hours supernatants were collected and analyzed by immunoblot for HMGB1 release. (C) THP1 cells were intoxicated with purified LukAB at the indicated concentrations for 1 hour and supernatants were analyzed by immunoblot for HMGB1 release. (D) THP1 cells were intoxicated with culture filtrates (1% v/v) from the indicated strains and supernatants were collected and analyzed for secretion of the indicated cytokines. THP1 cells (E) and primary CD14+ human monocytes (F) were primed for production of pro-IL-1β with 500 ng/mL LTA for 3 hours followed by intoxication with LukAB (THP1 with 50 ng/mL and CD14+ monocytes with 30 ng/mL) for 1 hour then supernatants were analyzed for secretion of the indicated cytokines. Error bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Primary cell experiments include three independent donors. Asterisks indicate significance at a p-value of < = 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.
Fig 4.
LukAB is a potent activator of Caspase 1.
(A) THP1 cells were intoxicated with 50 ng/mL LukAB for 1 hour and cell lysates were analyzed by immunoblot for Caspase 1 cleavage, which indicates activation. (B and C) THP1 cells were incubated with FLICA-1 (660-YVAD-FMK) then intoxicated with culture filtrates (1% v/v) from the indicated S. aureus stains. After 1 hour, cells were washed, fixed and analyzed by flow cytometry. Panel B shows a representative flow plot from one experiment, and panel C shows the corresponding mean florescence intensities (MFI). (D) THP1 cells were incubated with FLICA-1, intoxicated with purified LukAB (50 ng/mL) for 1 hour, then washed, fixed and analyzed by flow cytometry. (E) FLICA-1 was added to THP1 cells then LukAB (50 ng/mL) was added for time-course samples in descending order. All samples were washed and fixed at the same time, corresponding to different LukAB incubation times, then analyzed by flow cytometry. THP1 cells (F) and primary CD14+ human monocytes (G) were incubated with FLICA-1 and intoxicated with a dose titration of the indicated purified S. aureus leukotoxins for 1 hour. Cells were washed, fixed and analyzed by flow cytometry. Bars represent the standard error of the mean of triplicate samples. EC50 values are also shown. Each graph is representative of three experiments. Primary cell experiments include three independent donors. Asterisks indicate significance at a p-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.
Fig 5.
LukAB activates the NLRP3 inflammasome leading to cell death and cytokine secretion.
(A) THP1 cells were transduced with shRNA against NLRP3, ASC or a non-targeting control. Cell lysates were analyzed by immunoblot to confirm knock down of NLRP3 and ASC. (B) Surface CD11b levels were evaluated by flow cytometry. (C) THP1 shRNA cells were incubated in the presence of FLICA-1 then intoxicated with LukAB (50 ng/mL) for 1 hour then analyzed by flow cytometry. (D) THP1 shRNA cells were primed with 500 ng/mL LTA for 3 hours followed by intoxication with LukAB (50ng/mL) for one hour. Culture supernatants were collected and analyzed for secretion of the indicated cytokines. (E) THP1 shRNA cells were intoxicated with LukAB (50 ng/mL) for 1 hour and culture supernatants were collected for analysis of LDH release. (F) THP1 shRNA cells were incubated with propidium iodide then intoxicated with LukAB (50 ng/mL) for 1 hour then analyzed by flow cytometry. (G and H) THP1 cells were incubated in media supplemented with an additional 25 mM NaCl or KCl. Cells were incubated with FLICA-1 (G) or propidium iodide (H) then intoxicated with LukAB (50 ng/mL) for 1 hour and analyzed by flow cytometry. Bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Asterisks indicate significance at a p-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.
Fig 6.
Genetic or pharmacologic disruption of Caspase 1 blocks LukAB-induced cytokine secretion but not cell death.
(A) THP1 cells were transfected with siRNA against Caspase 1, ASC or a non-targeting sequence. Cell lysates were collected and analyzed by immunoblot to confirm knockdown of pro-Caspase 1 and ASC. (B) THP1 siRNA cells were incubated with propidium iodide and intoxicated with 50 ng/mL LukAB for 1 hour then analyzed by flow cytometry. (C and D) THP1 siRNA cells were either primed with LTA (500ng/mL) (C) or untreated (D) then intoxicated with LukAB (50ng/mL) for 1 hr before culture supernatants were analyzed for release of IL-1β (C) or IL-18 (D). (E and F) Primary CD14+ human monocytes were incubated with the indicated concentration of z-YVAD-FMK or VX-765 for 30 minutes. Primary monocytes were incubated in the presence of propidium iodide (E) then intoxicated with LukAB (50ng/mL) and analyzed by flow cytometry. (F) Primary monocytes were intoxicated with LukAB (50ng/mL) and culture supernatants were analyzed for release of IL-18. Propidium iodide staining and IL-18 secretion are reported as a fraction of measurement in primary cells not treated with inhibitor. Bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Asterisks indicate significance at a p-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.
Fig 7.
LukAB produced by extracellular or phagocytized S. aureus kills human monocytes.
(A) THP1 cells were infected with S. aureus Newman strains at an MOI of 10 for 120 min under non-phagocytosing conditions (extracellular infection; see methods), stained with the fixable viability dye eFluor 450, a membrane damage and cell death marker, and analyzed by flow cytometry. Representative histogram is shown. (B-D) THP1 shRNA cells were infected with S. aureus Newman, MOI 10 for 120 min, followed by flow cytometry analysis for maximal eFluor 450 staining (B) and FLICA-1 activation (C). (D) Culture supernatants were collected from extracellular infections and analyzed for IL-1β secretion. (E and F) THP1 cells were infected with GFP-expressing S. aureus Newman strains at an MOI of 10 for 45 min under phagocytosing conditions (intracellular infection; see methods). (E) THP1 cells were stained with the fixable viability dye eFluor 450. After infection, THP1 cells were analyzed by flow cytometry and GFP-positive THP1 cells were selected, indicative of S. aureus phagocytosis. Maximal eFluor 450 incorporation was gated among GFP-positive cells, indicative of THP1 death by intracellular S. aureus. Very few cells appear in the first plot corresponding to the background autofluorescent uninfected cells. Representative plots are shown. (F) Representative histogram of GFP fluorescence in S. aureus infected and uninfected THP1 cells is shown. (G-H) Purified primary CD14+ human monocytes were infected with S. aureus Newman and USA300-BK18807 strains, along with respective isogenic lukAB mutants, at an MOI of 5 for 45 min then stained with eFluor 450 (G) or FLICA-1 (H), and analyzed by flow cytometry. Graphs reflect the fraction of cells that were GFP positive. (I-L) THP1 shRNA cells were infected under phagocytosing conditions with S. aureus Newman, MOI 10 for 45 min, and analyzed by flow cytometry for (I) GFP fluorescence indicating phagocytosis; (J) maximal eFluor 450 staining indicating cell death; and (K) FLICA-1 activation. (L) Culture supernatants were collected and analyzed for IL-1β secretion. Bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Experiments with primary cell experiments include at least three independent donors. Asterisks indicate significance at a p-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.
Table 1.
Staphylococcus aureus strains used in this study.