Fig 9. K2 treatment increases access of a hydrophobic probe to the bacterial membrane of K. pneumoniae.

A. In order to assess the efficacy with which the LPS shielded the bacterial membrane, bacteria were exposed to the fluorescent probe 1-N-phenylnaphthylamine (NPN), and fluorescence was recorded, providing a measure of the insertion of NPN in the bacterial membrane. The membrane of ΔwaaQ mutant K. pneumoniae was more accessible to NPN than that of WT and ΔwbbM mutant. In K2-treated bacteria, access of NPN increased in WT and in ΔwbbM but not in ΔwaaQ bacteria (mean ± SEM; *: p<0.05; Kruskal-Wallis test; N = 5 independent experiments). B. Three chemical analogs of K2 (dT = deoxythymidine, dU = deoxyuridine and 5-EdU = 5-Ethynyl-2’-deoxyuridine) were tested for their ability to increase the membrane accessibility of NPN. K2 was the only compound that increased significantly the accessibility of the outer membrane of K. pneumoniae to NPN (mean ± SEM; *: p<0.05; Kruskal-Wallis test; DMSO, K2: N = 6; and N = 5 independent experiments for the three analogs of K2). C, D. The effect of K2 was tested on three different strains of K. pneumoniae (KpGE, Kp21 and Kp52145) as well as Escherichia coli (E.c), Pseudomonas aeruginosa (P.a), Bacillus subtilis (B.s) and Microccocus luteus (M.l). K2 increased NPN incorporation in K. pneumoniae, but exhibited little or no effect on other bacteria. (mean ± SEM; *: p<0.05; Mann-whitney test. N = 8 independent experiments).