[PMC free article] [PubMed] [CrossRef] [Google Scholar] 13. attached layer surrounding the cell wall (7). Predominantly consisting of polysaccharides, the capsule is usually shed by under culture conditions that include detergents, which is the case in most studies (6). grown without detergent has been described to have a distinct phenotype, exhibiting pronounced characteristics regarding the MICs of antibiotics (8), drug tolerance (9), and gene expression (10). We show here that cultivation of in detergent-free broth causes retention of EsxA around the bacterial surface, resulting in a phenotype of that rapidly induces macrophage cell death. Similarly, lung surfactant, which has detergent-like properties, removes EsxA from the bacterial surface, suggesting a novel role for lung surfactant in antimycobacterial defense. RESULTS bacteria cultivated without detergent retain EsxA on their surface. Omitting detergent from the broth to preserve the capsule-like layer (6), we observed that EsxA accumulated at the bacterial surface after 3 to 6?days of incubation (Fig. 1A and ?andB).B). EsxA could not be detected when detergent was used in the broth, on EsxA-deficient cultivated without detergent, or in the control staining without primary antibody (Fig. 1A). The effect was more pronounced on bacterial aggregates consisting of two or more bacteria (the prevailing morphological structure in detergent-free cultures) (Fig. 1B). Immunogold labeling of EsxA followed by transmission electron microscopy (TEM) confirmed the presence of EsxA around the bacterial surface, while it was absent from the EsxA-deficient mutant and Tween 80 broth-cultivated wild-type bacteria (Fig. 1C and ?andDD). Open in a separate window FIG 1 EsxA can be detected on the surface of after cultivation in the absence of detergent or surfactant. (A and B) H37Rv wild type (left) or the EsxA-deleted strain H37Rv (right) was cultivated with or without Tween 80 and for the indicated times. Fixed bacteria were stained with anti-EsxA antibody and an Alexa Fluor 594-conjugated secondary antibody. The images in panel A are from bacteria cultivated for 6?days. In the samples for the antibody control images (fourth column), the anti-EsxA antibody was omitted. Images were obtained using a 100 (numerical aperture, 1.45) objective. Bars, 5?m. (B) EsxA-positive single bacteria or aggregates (2 bacteria) were expressed as a percentage of all single bacteria or aggregates. Day 0 indicates the initial stock culture which contained 0.05% Tween 80. Bars and error bars depict means and SEMs from three impartial experiments (on average, 79 bacteria or aggregates were analyzed per sample; range, 17 to 441). Significant differences between the time points were tested with 2-way analysis of variance comparing all time points to the day 0 time point using the Bonferroni test for multiple comparisons, and the earliest significant time point for each group is usually indicated by asterisks. (C and D) H37Rv or H37Rv was fixed after 6?days of cultivation with or without Tween 80, followed by immunogold labeling for EsxA and TEM analysis. (C) Representative images are shown. Arrows, EsxA-positive debris; arrowheads, immunogold particles in the bacterial cell wall. The unfavorable control (neg ctrl) was the wild type (wt) grown without detergent, where the EsxA antibody was omitted during the labeling procedure. (D) The amount of immunogold particles per bacterium was quantified from the TEM pictures in a TK05 blind fashion. Bars and Thy1 error bars show means and SEMs for 30 to 54 bacteria per sample. Significant differences were TK05 tested with a 1-way analysis of variance, followed by Tukeys test comparing all groups. (E) The H37Rv wild type was cultured in broth made up of TK05 0.05% Tween 80 or without detergents and increasing amounts of the bovine lung surfactant Curosurf. Bacteria were fixed, stained, imaged, and analyzed as described in the legend to panels A and B. Bars and error bars show means and SEMs from 5 experiments (on average, 106 bacteria were analyzed per.