Nuclease activity and protein A release of Staphylococcus aureus clinical isolates determine the virulence in a murine model of acute lung infection

Abstract

Staphylococcus aureus is a common cause of hospital-acquired pneumonia associated with high mortality. Adequate clinical treatment is impeded by increasing occurrence of antibiotic resistances. Understanding the underlying mechanisms of its virulence during infections is a prerequisite to finding alternative treatments. Here, we demonstrated that an increased nuclease activity of a S. aureus isolate from a person with cystic fibrosis confers a growth advantage in a model of acute lung infection compared to the isogenic strain with low nuclease activity. Comparing these CF-isolates with a common MRSA-USA300 strain with similarly high nuclease activity but significantly elevated levels of Staphylococcal Protein A (SpA) revealed that infection with USA300 resulted in a significantly increased bacterial burden in a model of murine lung infection. Replenishment with the cell wall-bound SpA of S. aureus, which can also be secreted into the environment and binds to tumor necrosis factor receptor -1 (TNFR-1) to the CF-isolates abrogated these differences. In vitro experiments confirmed significant differences in spa-expression between USA300 compared to CF-isolates, thereby influencing TNFR-1 shedding, L-selectin shedding, and production of reactive oxygen species through activation of ADAM17.

Keywords: L-selectin shedding; SpA; Staphylococcus aureus; TNFR shedding; lung infection; neutrophil extracellular traps; neutrophil recruitment.

Figure 1

Increased nuclease activity confers a growth advantage in vitro and in vivo. (A) Representative agarose gel showing calf thymus DNA samples after incubation with the supernatant of S. aureus cultures USA300, low₁₇ or high₈₁ or BHI medium as control. (B) Densitometric analysis of agarose gels with digested calf thymus DNA following incubation with S. aureus supernatants. n=3. (C) Representative ImageStream^®X images of MPO⁺/Sytox⁺ BMDNs following incubation with S. aureus low₁₇ and high₈₁. BMDNs were co-cultivated with S. aureus CF-isolates low₁₇ or high₈₁ at an MOI of 10. (D) Ly6G⁺ neutrophils were analyzed by flow cytometry for the percentage of cells stained positive for MPO and Sytox as a readout for NET release. (E) The supernatant of these co-cultures was used for a NET-specific ELISA (capturing: H3-cit, abcam; detection: anti-DNA, Roche). (F) Number of CFUs at the respective time points divided by the number of CFUs seeded at the beginning of the co-incubation are presented as relative growth after 1 h or 2 h of co-cultivation. Data are mean +/- sem; n=5-7; *p < 0.05, **p < 0.01, Two-way ANOVA. (G–L) Analysis of CFU and neutrophil recruitment 24 h after intratracheal instillation of 6x10⁸ bacteria. Serial dilutions of BAL (G), lung homogenate (H) or blood (I) were plated to assess the number of CFUs. Flow cytometry analysis revealed the number of neutrophils recruited to the alveoli (J) or the lung tissue (K). The protein content in the BAL was analyzed using a BCA protein assay (L). Data are presented as mean +/- sem; n=8-10; *p < 0.05, ***p < 0.001, ****p < 0.0001; One-way ANOVA or student's t-test.

Figure 2

Mice infected with USA300 develop significantly worsened pneumonia compared to mice infected with CF-isolates low₁₇ and high_81. Analysis of neutrophil recruitment and CFUs 4h (A–D) or 24 h (E–H) after intratracheal instillation of S. aureus (6x10⁸/mouse). Neutrophil recruitment to the alveoli (A, E) or the lung (B, F) and bacterial burden in the alveoli (C, G) or the lung (D, H) was determined by flow cytometry and plating serial dilutions. The protein content in the BAL was assessed using a BCA assay (I, J). Number of moribund or dead mice are illustrated by a Kaplan-Meier Survival curve (K). Data are presented as mean +/- sem; n = 6-10; *p < 0.05 One-way ANOVA.

Figure 3

Release of protein A correlates with the amount of sL-sel and sTNFR. (A) Amount of Protein A in the supernatant of S. aureus cultures after 24 h of cultivation. (B) Flow cytometric analysis of bound IgG2a antibody binding to Ly6G⁺ neutrophils. (C) Flow cytometry analysis of L-Selectin expression on Ly6G⁺ neutrophils left untreated or after incubation with water as vehicle control or 1 µg purified SpA for the indicated timepoints. (D–G) sL-sel ELISA of BAL and plasma samples of S. aureus infected mice 4h (D, E) or 24h (F, G) after infection. (H–K) sTNFR ELISA of BAL and plasma samples of S. aureus infected mice 4 h (H, I) or 24 h (J, K) after infection. Data are presented as mean +/- sem; n = 6-10; *p < 0.05, **p < 0.01, ***p < 0.001 One-way ANOVA.

Figure 4

TNFα-primed BMDNs challenged with USA300 produce significantly more ROS compared to BMDNs challenged with CF-isolates. O₂^- release of BMDNs in response to TNFα, fibrinogen, and S. aureus. BMDNs were pre-incubated with (A) vehicle control H₂O (7.2% v/v), (B) SpA (1 µg/1x10⁵ cells), (C) vehicle control DMSO (0.5%), or (D) ADAM17 inhibitor GM6001 (50 µM); n=4. (E–H) Analysis of neutrophil recruitment and bacterial burden 24 h after intratracheal instillation of 6x10⁸ USA300 or S. aureus low₁₇ and high₈₁ supplemented with 50 µg SpA per mouse. Neutrophil recruitment to the alveoli (E), to the lung (F) and bacterial burden within the BAL (G) or the lung (H) were analyzed. Data are presented as mean +/- sem; n = 3-5; *p < 0.05, **p < 0.01, ***p < 0.001 One-way ANOVA; Dunnett’s Multiple Comparison Test (for A–D).