Fig. 9

Effects of LprB overexpression on MAH tolerance to drugs and biofilm formation. a In vitro time-kill curves for MAH LprB overexpressed clone with and without exposure to bactericidal concentrations of AMK and CLA over 4 days. Results represent means ± standard errors of three independent experiments. b MAH control and LprB overexpression clones (3 × 10⁸ CFU/ml) were inoculated in PVC 96-well plates with HBSS for 15 days in presence or absence of AMK. The biofilm was evaluated using crystal violet stain as described in Materials and Methods. The A570 readings from three experiments are shown and values are means ± standard deviations. c The time dependent phagocytosis assay of MAH by THP-1 macrophages shows significant differences between the uptake of LprB overexpression clone and the wild type control over time. The experiment was performed in triplicate, repeated three times and means ± SDs were determined. The significance between control and experimental groups is **p < 0.01. d The visualization of RFP fusion LprB protein by fluorescent microscopy, in contrast to MAH control expressing only RFP. e Western blot analysis of soluble and insoluble fractions of MAH expressing 6×-His tagged proteins. Soluble proteins were first extracted by mechanical disruption in HBSS. Insoluble proteins were then extracted in a denaturing urea solution. Cell envelope and membrane localizing proteins tend to remain insoluble in HBSS, while cytoplasmic and secreted proteins are readily extracted in HBSS. (1) Cell fractionation shows that LprB protein is found in the insoluble and soluble fraction of MAH, while RFP protein remains only in soluble portion. (2) Biofilms were formed in HBSS at 37 °C for 7 days. Supernatants were gently removed and total proteins of the biofilm biomass were extracted in HBSS. In contrast to MAH control expressing only RFP, we observe that LprB lipoprotein is translocated into the MAH biofilm matrix