Protein Disulfide Isomerase and Extracellular Adherence Protein Cooperatively Potentiate Staphylococcal Invasion into Endothelial Cells

ABSTRACT Invasion of host cells is an important feature of Staphylococcus aureus. The main internalization pathway involves binding of the bacteria to host cells, e.g., endothelial cells, via a fibronectin (Fn) bridge between S. aureus Fn binding proteins and α5β1-integrin, followed by phagocytosis. The secreted extracellular adherence protein (Eap) has been shown to promote this cellular uptake pathway of not only S. aureus, but also of bacteria otherwise poorly taken up by host cells, such as Staphylococcus carnosus. The exact mechanisms are still unknown. Previously, we demonstrated that Eap induces platelet activation by stimulation of the protein disulfide isomerase (PDI), a catalyst of thiol-disulfide exchange reactions. Here, we show that Eap promotes PDI activity on the surface of endothelial cells, and that this contributes critically to Eap-driven staphylococcal invasion. PDI-stimulated β1-integrin activation followed by increased Fn binding to host cells likely accounts for the Eap-enhanced uptake of S. aureus into non-professional phagocytes. Additionally, Eap supports the binding of S. carnosus to Fn-α5β1 integrin, thereby allowing its uptake into endothelial cells. To our knowledge, this is the first demonstration that PDI is crucial for the uptake of bacteria into host cells. We describe a hitherto unknown function of Eap—the promotion of an enzymatic activity with subsequent enhancement of bacterial uptake—and thus broaden mechanistic insights into its importance as a driver of bacterial pathogenicity. IMPORTANCE Staphylococcus aureus can invade and persist in non-professional phagocytes, thereby escaping host defense mechanisms and antibiotic treatment. The intracellular lifestyle of S. aureus contributes to the development of infection, e.g., in infective endocarditis or chronic osteomyelitis. The extracellular adherence protein secreted by S. aureus promotes its own internalization as well as that of bacteria that are otherwise poorly taken up by host cells, such as Staphylococcus carnosus. In our study, we demonstrate that staphylococcal uptake by endothelial cells requires catalytic disulfide exchange activity by the cell-surface protein disulfide isomerase, and that this critical enzymatic function is enhanced by Eap. The therapeutic application of PDI inhibitors has previously been investigated in the context of thrombosis and hypercoagulability. Our results add another intriguing possibility: therapeutically targeting PDI, i.e., as a candidate approach to modulate the initiation and/or course of S. aureus infectious diseases.


Supplemental Fig. S1: Eap enhances staphylococcal adherence to and internalization in endothelial cells. (A)
Adhesion of S. aureus 8325-4 and (B) S. carnosus TM300 to HMEC-1 in absence and presence of 20 µg/ml recombinant Eap. Cells were incubated with an MOI of 50. One hour post infection unbound bacteria were washed away with PBS. Host cells were lysed with icecold distilled water in order to release extra-and intracellular bacteria and number of bacteria was assessed by plate counting. Data represent the means ± SD from absolute numbers of four (S. aureus) or three (S. carnosus) independent experiments. *p<0.05, unpaired t test. (C) Internalization of S. aureus 8325-4 and (D) S. carnosus TM300 in HMEC-1 in absence and presence of 20 µg/ml recombinant Eap. Cells were incubated with an MOI of 50. One hour post infection, extracellular staphylococci were removed by lysostaphin treatment. Host cells were lysed with ice-cold distilled water in order to release intracellular bacteria and number of bacteria was assessed by plate counting. Data represent the means ± SD from absolute numbers from five independent experiments. ***p≤0.001, **p≤0.01, unpaired t test.
(E) Internalization of S. carnosus TM300 in HMEC-1 in absence and presence of 20 µg/ml native Eap. Numbers of bacteria in control cells were set to 100%. Data represent the means ± SD from absolute numbers from three independent experiments, unpaired t test. (F) Internalization of S. aureus strain 8325-4 (absolute numbers) and (G) (numbers of bacteria in control cells were set to 100%) in EA.hy926, recombinant Eap. (H) Internalization of S. carnosus TM300 (absolute numbers) and (I) (numbers of bacteria in control cells were set to 100%) in EA.hy926 cells, recombinant Eap. (J) Internalization of S. carnosus TM300 in EA.hy926 in absence and presence of 20 µg/ml native Eap. Numbers of bacteria in control cells were set to 100%. Data represent the means ± SD from at least three independent experiments. ***p≤0.001, **p≤0.01, *p<0.05 unpaired t test.

Supplemental Fig. S2: Eap promotes protein disulfide activity on the surface of eukaryotic cells. (A)
Eap-stimulated abundance of free ecto-sulfhydryls on the surface of EA.hy926 cells was detected by the binding of the thiol-reactive dye Alexa Flour 488 C5 maleimide and measured by flow cytometry. Native Eap, data represent the mean ± SD of three independent experiments. ***p≤0.001; one-way ANOVA followed by Dunnett's multiplecomparison test. (B) Inhibition of Eap-promoted abundance of ecto-sulfhydryls on EA.hy926 by anti-PDI antibody RL90 (10µg/ml) detected with Alexa Flour 488 C5 maleimide. Recombinant Eap, data represent the mean ± SD of three independent experiments. *p<0.05, two-way ANOVA followed by Bonferroni posttest. (C) Influence of Eap on kinetics of PDI-catalyzed oxidative refolding of scrambled RNAse A, determined as ∆A260nm. Eap was preincubated with soluble PDI before PDI was incubated with scrambled RNAse A. Recombinant Eap, data represent the mean ± SD of three independent experiments. ***p≤0.001, *p<0.05; two-way ANOVA followed by Bonferroni posttest, comparison to 0 µg/ml Eap. (D) Influence of Eap (20 µg/ml) on kinetics of Di-E-GSSG reduction to E-GSH catalyzed by soluble PDI (200 nM) or by fibronectin (50 µg/ml). Native Eap, data represent the mean ± SD of three independent experiments. ***p≤0.001, **p≤0.01; two-way ANOVA followed by Bonferroni posttest; for better readability, only significant differences between PDI and PDI + Eap are indicated.

Supplemental Fig. S3: Inhibition of protein disulfide isomerase reverses the enhancing effect of Eap on staphylococcal internalization.
Internalization of S. aureus 8325-4 and S. carnosus TM300 as indicated in HMEC-1 cells in absence and presence of 20 µg/ml recombinant Eap. Cells were pre-incubated with (A/ B) bacitracin (10mM), (C/ D) dithiobis-nitrobenzoic acid (DTNB, 10 mM; DMSO, 1% v/v as vehicle control), (E/ F) Rutin (60 nM; 0.6% v/v as vehicle control) 30 minutes before stimulation with Eap. In case of bacitracin, host cells were washed before addition of bacteria to remove bacitracin. Cells were incubated with an MOI of 50. One hour post infection, extracellular staphylococci were removed by lysostaphin treatment. Number of intracellular bacteria was assessed by plate counting. Numbers of bacteria in control cells were set to 100%. Recombinant Eap, data represent the mean ± SD of at least three independent experiments. ***p≤0.001, **p≤0.01, *p<0.05; one-way ANOVA followed by Bonferroni posttest.

Supplemental Fig. S4: Eap enhances activation of β1 integrin to cells in a thiol isomerase-dependent manner, affecting staphylococcal uptake (A)
Binding of FITC-labelled antibody against activated anti-integrin β1 to detached HMEC-1 cells was determined by flow cytometry after incubation of cells with native Eap native Eap and/ or or bacitracin (10mM) added to the cells 15 min before addition of Eap, binding of antibody to control (0 µg/ml Eap) was set to 100%. Data represent the means ± SD of at least three independent experiments, ***p≤0.001, two-way ANOVA followed by Bonferroni posttest. (B) Internalization of S. carnosus TM300 HMEC-1 cells in absence and presence of 20 µg/ml Eap. Cells were pre-incubated 10 µM ATN161 30 minutes before stimulation with Eap. Cells were incubated with an MOI of 50. One hour post infection, extracellular staphylococci were removed by lysostaphin treatment. Number of intracellular bacteria was assessed by plate counting. Numbers of bacteria in control cells were set to 100%. Recombinant Eap, data represent the mean ± SD of three independent experiments. ***p≤0.001, *p<0.05; one-way ANOVA followed by Bonferroni posttest. (C) siRNA P4HB knock down (10 pM siRNA) in HMEC-1 cells was confirmed by Western Blotting. Data are means ± SD from Western blot quantification of three independent experiments. ***p≤0.001; one-way ANOVA followed by Bonferroni posttest.