Mycobacterium ulcerans cytotoxicity in an adipose cell model

Abstract

An adipose cell (SW872) model was developed to observe cellular necrosis and apoptosis upon Mycobacterium ulcerans infection and treatment with mycobacterial exudate. Apoptosis was likely due to secreted proteins, while necrosis was likely due to mycolactone. Our data suggest that additional factors in M. ulcerans may be involved in Buruli ulcer pathogenesis.

FIG. 1

EM of human adipose cells (SW872) infected with M. ulcerans for 24 h. (A) SW872 cells incubated at 32.5°C undergoing necrosis and apoptosis. Magnification, ×2,950. Arrows denote nucleosome condensation, cell blebbing, and a vacuole, respectively (top to bottom). (B) SW872 cells incubated at 37°C undergoing necrosis and apoptosis. Magnification, ×2,950. Arrows denote a highly granulated and vacuolated cell and a cell with numerous vacuoles, respectively (top to bottom). (C) Uninfected SW872 cells. Magnification, ×2,950.

FIG. 2

Quantitative analysis of necrosis and apoptosis in adipose cell monolayers treated with MUCF or mycolactone. (A) Optical densities of intracellular histone-DNA complexes from the cell lysates of SW872 cell monolayers. (B) Percent LDH release from SW872 cell monolayers. Data shown are the means of triplicate experiments, and error bars represent the standard deviations of these data. The values obtained were normalized by subtraction of background levels of intracellular histone-DNA complexes and of LDH release of uninfected SW872 cell monolayers at each time point. Solid bars, 20 μg of native 94-816 MUCF; horizontally striped bars, 20 μg of heat-denatured 94-816 MUCF; vertically striped bars, 20 μg of proteinase K-treated and heat-denatured 94-816 MUCF; checkered bars, 1.0 μg of mycolactone.

FIG. 3

Analysis of the MUCF. (A) Resolution of the ASL fraction of the MUCF by MALDI-TOF mass spectrometry. The double asterisk indicates the presence of a mycolactone-related product. (B) Resolution of the MUCF proteins by two-dimensional gel electrophoresis and staining with silver. Proteins are focused horizontally by pI (range of 4 to 6.5) and vertically by size (range of 100 to 10 kDa). M, molecular weight marker lane, with marker sizes in thousands shown to the left. The circled protein region represents the serodiagnostic 38-kDa protein of the MUCF. The arrow denotes a high-molecular-weight lipoprotein complex in the MUCF.