Figure 1

E.coli overexpressing type 1 fimbriae yielded more intracellular survivors in macrophages.

(a) Timeline of the gentamicin assay for the measurement of intracellular survivors. Gentamicin was applied as a 15 minute pulse of 100 μg/ml to kill extracellular bacteria and was then reduced to 10 μg/ml for long term co-cultivation. (b) Fimbriae expression affected the number of viable bacteria extracted from macrophages. A composite image of 3 randomly chosen agar plates is shown from macrophage lysates after incubation with fim↑, wt fim and ∆fim, respectively, using the protocol as sketched in (a). (c) Total colony forming units recovered from gentamicin protection assays 0.5 hours post infection showed increased survivor numbers of fim↑ compared to wt fim and ∆fim bacteria, using the protocol as sketched in (a). RAW macrophages were seeded to 10⁵ cells/cm² and incubated with the three different E.coli strains, respectively, at a bacteria-to-macrophage ratio of 10:1. Box plot whiskers indicate the S.D. The variance of population means was analyzed using a one way ANOVA. Upper-case letters mark significant differences based on post hoc Tukey and Bonferroni tests. Pairs of samples that have no letter in common have significantly different means at p < 0.01; i.e., samples with the label A are significantly different from samples with the label B. (d) Fold changes of colony forming units, normalized to 0.5 hours post infection, respectively, indicated higher survival chances of fim↑ bacteria compared to wt fim and ∆fim. Error bars are S.D. The variance of population means was analyzed in the same way as in (c). (e) Staining for lysosomal acidification did not yield significant differences in the LysoID Red fluorescence between fim↑ and ∆fim (p = 1). Scale bar is 5 μm. fim↑, fimbriae overexpression strain; wt, fimbriae wild type strain; ∆fim, fimbriae knockout strain; 3-MA, 3-methyladenine; h, hours