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. 2013 Nov;94(5):971-9.
doi: 10.1189/jlb.0213080. Epub 2013 Sep 11.

The impact of α-toxin on host cell plasma membrane permeability and cytokine expression during human blood infection by CA-MRSA USA300

Tyler K Nygaard  1 , Kyler B Pallister, Oliwia W Zurek, Jovanka M Voyich
Affiliations

The impact of α-toxin on host cell plasma membrane permeability and cytokine expression during human blood infection by CA-MRSA USA300

Tyler K Nygaard et al. J Leukoc Biol. 2013 Nov.

Abstract

This investigation examines the influence of α-toxin (Hla) expression by CA-MRSA on host immune cell integrity and cytokine expression during infection of human blood. Flow cytometry analysis of human blood infected by Staphylococcus aureus PFGE type USA300 or a USA300Δhla demonstrated that Hla expression significantly increased plasma membrane permeability of human CD14(+) monocytes. The increased susceptibility of human CD14(+) monocytes to Hla toxicity paralleled the high cell-surface expression on these cell types of ADAM10. USA300 rapidly associated with PMNs and monocytes but not T cells following inoculation of human blood. Transcription analysis indicated a strong up-regulation of proinflammatory cytokine transcription following infection of human blood by USA300 and USA300Δhla. CBAs and ELISAs determined that IL-6, IL-10, TNF-α, IFN-γ, IL-1β, IL-8, and IL-4 are significantly up-regulated during the initial phases of human blood infection by USA300 relative to mock-infected blood but failed to distinguish any significant differences in secreted cytokine protein concentrations during infection by USA300Δhla relative to USA300. Collectively, these findings demonstrate that expression of Hla by USA300 has a significant impact on human CD14(+) monocyte plasma membrane integrity but is not exclusively responsible for the proinflammatory cytokine profile induced by USA300 during the initial stages of human blood infection.

Keywords: Staphylococcus aureus; T cell; chemokine; monocyte; pathogenesis; virulence.

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Figures

Figure 1.
Figure 1.. Flow cytometry analysis of PMN, CD3+ lymphocyte, and CD14+ monocyte cells following infection of human blood with USA300, USA300Δhla, USA300Δhla Comp, or DPBS control.
(A) Representative flow cytometry histograms for PI-stained human PMN, CD3+ lymphocyte, and CD14+ monocyte cells following infection with USA300 (dark, solid lines) relative to the uninfected DPBS control (shaded gray areas) at 3 h postinfection with 1 × 106 CFU/mL human blood. (B) Geometric mean PI+ signal of PMN, CD3+ lymphocyte, and CD14+ monocyte cells at 1 h and 3 h postinfection of human blood with 1 × 106 CFU/mL USA300, USA300Δhla (Δhla), USA300Δhla Comp (Comp), or uninfected DPBS control. Data represent four separate experiments using at least three different blood donors, with *P ≤ 0.05, as determined by paired two-tailed t-test relative to samples infected with USA300.
Figure 2.
Figure 2.. Flow cytometry analysis of host cell-surface ADAM10 expression.
(A) Representative flow cytometry histograms of human PMN, CD3+ lymphocyte, and CD14+ monocyte cells stained with anti-ADAM10 (shaded areas) relative to staining with an IgG control (dotted lines). (B) Compiled results comparing the geometric mean ADAM10+ signal of human PMN, CD3+ lymphocyte, and CD14+ monocyte cells. Data represent three separate experiments using different blood donors, with *P ≤ 0.05, as determined by one-way repeated-measures ANOVA with Tukey's post-test relative to CD14+ monocytes.
Figure 3.
Figure 3.. The association of USA300 with PMN, CD3+ lymphocyte, and CD14+ monocyte cells following infection of human blood.
(A) Representative flow cytometry histograms of PMNs, CD3+ lymphocyte, and CD14+ monocyte cells at 3 h postinfection with FITC-labeled USA300 (solid lines) relative to a DPBS control (shaded areas), with the percentage of FITC+ human cells indicated. (B) Compiled results illustrating the percentage of FITC+ PMN, CD3+ lymphocyte, and CD14+ monocyte cells at 1 h and 3 h postinfection with 1 × 106 CFU/mL FITC-labeled USA300, USA300Δhla, USA300Δhla Comp, or DPBS control. Data represent five separate experiments using at least three different blood donors, with **P ≤ 0.01, and ***P ≤ 0.001, as determined by paired two-tailed t-test relative to samples infected with USA300.
Figure 4.
Figure 4.. Hla expression modulates proinflammatory cytokine transcript abundance during USA300 infection of human blood.
Cytokine transcription profiles at 3 h postinfection of human blood with 1 × 105 CFU/mL USA300 or USA300Δhla relative to DPBS mock-infected blood. Only genes that exhibited at least a twofold change in transcript abundance and a P value < 0.05 for USA300- or USA300Δhla-infected blood relative to the DPBS mock-infected control are shown. Data represent compiled results from three separate experiments using different blood donors. Fold-changes and P values were calculated using SABiosciences web-based software, as described in Materials and Methods.
Figure 5.
Figure 5.. The influence of Hla on cytokine expression following infection of human blood with USA300.
(A) Concentrations of IL-1β and IL-8, as determined by ELISA at 5 h postinfection of human blood with 1 × 105 CFU/mL USA300, USA300Δhla (Δhla), USA300Δhla Comp (Comp), DPBS control, or USA300 incubated at ≥98°C for 20 min prior to infection (Heat tx). (B) CBA analysis of IL-6, IL-10, TNF-α, IFN-γ, IL-17A, IL-12p70, IL-2, and IL-4 expression in human blood at 5 h postinfection with 1 × 105 CFU/mL USA300, USA300Δhla, USA300Δhla Comp, or DPBS control. Data represent at least four separate experiments using at least three different blood donors, with *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, as determined by one-way repeated-measures ANOVA with Tukey's post-test relative to samples infected with USA300.
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