Fig. 2: HNP1 promotes A. baumannii adhesion to host cells in vitro and on mouse skin wounds in vivo.
A Section and side views of overnight adhesion of 19606 to host cells (scale bar = 10 μm) representative of three independent experiments. Red arrows indicate the locations of bacteria. After washing with DMEM, the cells were added with 19606 in the presence or absence of HNP1, and incubated for indicated times. The adhering bacteria were visualized by confocal microscopy and quantified by colony counting. Colony counting results showing that HNP1 significantly promoted adhesion of 19606 to A549 (B) and J774a.1 (C) cells after 4 h of incubation, while the bacterial internalization remained low after 4–8 h of incubation plus 1 h of gentamicin treatment. Results are mean ± SD, representative of at least three experiments (n = 6 biological replicates per experiment). D Schematic of the murine wound infection assay. C57BL/6J mice were anaesthetized, the dorsal side was shaved and depilated, and two 5-mm-diameter circular punches were made per mouse. The wounds were inoculated with 20 μl of bacterial suspension containing ~3 × 106 CFU of 19606 pretreated with 0 or 10 μM HNP1, and then covered with waterproof bandages and tissue adhesive. Mice were sacrificed 2-, 4-, and 6-days post-infection, and the wounds were collected for bacterial load quantification and SEM observation. E Colony counting showing a higher bacterial load on murine wounds in the presence of 10 μM HNP1. Results are mean ± SEM (four mice per time point per group, two wounds per mouse). F SEM images of bacteria on murine wounds (Scale bar = 10 μm) representative of four mice in one experiment. Statistical significances for B and C were evaluated by two-way ANOVA with Tukey correction for multiple comparison. Statistical significances for (E) were evaluated by multiple unpaired t-tests (two-sided). See also Supplementary Figs. S3 and S4.
