Platelet activation and aggregation by the opportunistic pathogen <i>Cutibacterium (Propionibacterium) acnes</i>

<div><p><i>Cutibacterium</i> (<i>Propionibacterium</i>) <i>acnes</i>, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of <i>C</i>. <i>acnes</i> with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of <i>C</i>. <i>acnes</i> with platelets and plasma proteins. We report that the ability of <i>C</i>. <i>acnes</i> to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with <i>C</i>. <i>acnes</i>. Pretreatment of <i>C</i>. <i>acnes</i> with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on <i>C</i>. <i>acnes</i> is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on <i>C</i>. <i>acnes</i>, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased <i>C</i>. <i>acnes</i>-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of <i>C</i>. <i>acnes</i> and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of <i>C</i>. <i>acnes</i> and platelets in the aggregates. Here, we have demonstrated the ability of <i>C</i>. <i>acnes</i> to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.</p></div

<i>C</i>. <i>acnes</i> aggregation of platelets is dose, type and donor-dependent.

<p><i>C</i>. <i>acnes</i> isolate AS12 was incubated with platelets at different ratios (0.11:1–1.8:1; <i>C</i>. <i>acnes</i>:platelet) in plasma and aggregation measured using an aggregometer (Chronolog) (A). The ability of different types of <i>C</i>. <i>acnes</i> (B), presence of bacteriophages (C), or donors (D), to influence aggregation was investigated. Failure to induce aggregation is indicated by the maximum lag time (t = 25 min). Each dot in the figures represents one experiment (e.g. one bacterial strain). For phylotype and phage dependence, a single donor was used. All experiments were performed three independent times and are presented as medians with range where appropriate. Due to the non-parametric nature of the samples, Kruskal-Wallis (B, D), and Mann-Whitney (C) were used for statistical evaluation.</p

<i>C</i>. <i>acnes</i> activation of platelets is donor-dependent and does not affect bacterial viability.

<p>Platelet activation was measured by platelet surface-bound CD62P (A; FACS) and PAC-1 expression on platelets (B; FACS), in three different donors using four strains of <i>C</i>. <i>acnes</i> (AS12, AS13, AD1, KPA171202), and the difference between the donors was investigated using Kruskal Wallis (A; p = 0.0076, B; p = 0.0132). The ability of <i>C</i>. <i>acnes</i> strain AS12 to survive in the presence of PRP with or without LL37 was measured at three different time points (0, 25, and 120 minutes after mixture) after vortex (C) or combining vortex and sonication (D) before serial dilution and cfu counting. Bacterial count did not change significantly over time as assessed by Kruskal Wallis (n.s.). In all activation experiments, collagen, ADP and thrombin were used as controls where applicable, and PRP and HEPES buffer as references. All experiments were performed three independent times and presented as medians with range.</p

IgG and fibrinogen are critical mediators for <i>C</i>. <i>acnes</i> platelet aggregation.

<p>Pretreatment of <i>C</i>. <i>acnes</i> strain AS12 with plasma (PPP) or serum significantly reduces the lag time before aggregation of PRP (A; p = 0.03), while pretreatment with heat-inactivated serum does not reduce the lag time. Inhibition of IgG-mediated aggregation of platelets through pre-incubation of PRP with IdeS and AT10 resulted in significant inhibition in aggregation (B; p = 0.03). IdeS will inhibit IgG mediated signaling through specific cleavage of IgG in the hinge region, while AT10 mediates its effect through FcRγIIA antagonist properties. PRP was pre-treated with Abciximab in order to investigate the importance of fibrinogen in platelet aggregation of <i>C</i>. <i>acnes</i> stimulated platelets (C; CD62P; p = 0.04). All experiments were performed four independent times and presented as medians with range and analyzed using Mann Whitney.</p

Fluorescence microscopy indicates co-localization of <i>C</i>. <i>acnes</i> and platelets.

<p><i>C</i>. <i>acnes</i> isolate AS12 was incubated with PPP or PRP, and fluorescent probes against the bacterium (DAPI), platelets (TxRed; Alexa Fluor 594), IgG (Cy5; Alexa Fluor 647), and fibrinogen (GFP; FITC), used to visualize the presence and localization of targets. PBS, PPP and PRP alone were used as negative controls, and collagen I as a positive control for platelet aggregation (A). Co-localization of <i>C</i>. <i>acnes</i> and platelets was analyzed by merging the signal for DAPI (<i>C</i>. <i>acnes</i>) and TxRed (platelets) (B). The co-localization of <i>C</i>. <i>acnes</i> (Oregon Green) and platelets (TxRed) after 25 minutes (T25) and at starting point (T0) was compared and analyzed using Pearson's correlation (p = 0.017)(C). All micrographs are at 40x magnification.</p

Mass spectrometry analysis of abundant plasma proteins deposited on <i>C</i>. <i>acnes</i>.

<p><i>C</i>. <i>acnes</i> strains KPA171202 (black) and AS12 (gray) were grown in the presence of 2% plasma after which they were thoroughly washed, adherent proteins released by trypsin digestion, and analyzed using Shotgun mass spectrometry. Only the most abundant identified proteins are displayed and presented as means (± SD) from three biological replicates.</p