Figures & data
Figure 1. RIC protects S. aureus from phagocyte producing ROS. Macrophages J774A.1 were infected, at an MOI ∼ 5, with S. aureus JE2 (black bar), JE2 ∆ric (white bar) and JE2 Δric carrying the complementation vector (pMK4-RIC– striped bar) following activation by INF-γ/LPS (A), and in the presence of the mammalian iNOS inhibitor L-NMMA (B) or the NADPH phagocyte oxidase inhibitor apocynin (C). Bacterial counts were determined at 0.5 or 6 h post-infection. In (D) is depicted the quantification of intracellular bacteria after infection of lung epithelial A549 cells with S. aureus JE2 (black bar) and JE2 Δric (white bar), for 2 h. Data represent means of three (A, B and C) and two (D) biological samples analysed in triplicate, with standard error and unpaired Student's t-test (###P < 0.0005; #P < 0.05; ns: not significant).
Figure 2. S. aureus ric mutant has decreased ability to infect Galleria mellonella. A. Survival of G. mellonella after infection with S. aureus JE2 at the following bacterial CFU per larva: 103 (solid line), 105 (dashed line), and 108 (dotted line).B. G. mellonella was infected with S. aureus JE2 (WT; black line), and the isogenic ric mutant strain (Δric; grey line) (###P < 0.0001). For complementation, G. mellonella was also infected by S. aureus JE2 Δric carrying the vector pMK4-RIC (black dashed line), or with the empty vector pMK4 (grey dashed line) (##P < 0.005). In B, approximately 107 bacterial cells were injected per larvae. The survival curves were compared using Mantel-Cox test. Ten larvae were analysed in each condition and larval survival was monitored daily. In all cases, no larval death was observed upon administration of PBS.
Figure 3. S. aureus Δric mutant has lower intracellular survival in G. mellonella. A. Bacterial load was analysed in the larvae hemolymph following incubation with S. aureus JE2 (black bar), JE2 Δric (white bar) and JE2 Δric carrying pMK4-RIC (striped bar) for 4 h and 8 h. Data represent means of four independent assays analysed with standard error and unpaired Student's t-test (##P < 0.001; ns: not significant). B. Quantitative RT-PCR analysis of the ric expression done in total RNA extracted from G. mellonella following incubation with S. aureus JE2, for 4 h and 8 h, and relative to the initial expression level (time zero of infection). Data was normalized to the expression of the constitutive 16S rRNA gene. Data represent mean values analysed with standard error (n = 6).
