Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes
Fig 3
S. aureus aus mutants show altered virulence in epithelial cells and human neutrophils.
a) HeLa cells were left uninfected (control) or were infected with S. aureus JE2 wild type (WT), isogenic ausA and ausB mutants, a complemented ausB mutant (ausB compl), or mutants within the virulence regulators agrA and saeR. Kinetics of cytotoxicity was monitored over time (x-axis) by propidium iodide (PI) staining and flow cytometry (y-axis; HeLa cytotoxicity in %). The agrA mutant did not demonstrate detectable host cell death. saeR, ausA and ausB mutants were significantly diminished in their ability to kill host cells. Genetic complementation of ausB recovered the phenotype of the insertional mutant. The apparent reduction of cytotoxicity at 24 hours p.i. is mediated by extended fragmentation of cells, which therefore are outside of the FSC-SSC gate for viable cells. Statistical analysis was performed by linear modeling and ANOVA followed by Tukey’s post-hoc analysis; ***P < 0.001 (Wt vs. ausA or ausB; 8 h p.i.). Host cell death rates are reduced in (b) hPMN 4 hours p.i. or(c) primary human monocyte-derived macrophages (hMDM) 2 hours p.i. when the phagocytes were infected with aus mutants. Cell death was measured by LDH release as percentage relative to the complete lysate (positive control) and supernatant of uninfected cells (set to 0% cell death, negative control). d) The NRPS AusAB is involved in bacterial survival upon macrophage co-incubation. CFUs of wild type (WT), insertional mutants within ausA, ausB and agrA were recovered at the indicated time points and numbers were normalized to the 15 min time point. Phevalin (+Phe) was added to selected samples at a concentration of 10 μM to assess complementation with the synthetic dipeptide. Graphs show the mean of at least three independent experiments ± SD. Statistical analyses were performed by t-test (b,c) and Wilcoxon rank sum test (d). *P < 0.05; **P<0.01; ***P < 0.001.