Microenvironment of Mycobacterium smegmatis Culture to Induce Cholesterol Consumption Does Cell Wall Remodeling and Enables the Formation of Granuloma-Like Structures

Abstract

Pathogenic species of mycobacteria are known to use the host cholesterol during lung infection as an alternative source of carbon and energy. Mycobacteria culture in minimal medium (MM) has been used as an in vitro experimental model to study the consumption of exogenous cholesterol. Once in MM, different species of mycobacteria start to consume the cholesterol and initiate transcriptional and metabolic adaptations, upregulating the enzymes of the methylcitrate cycle (MCC) and accumulating a variety of primary metabolites that are known to be important substrates for cell wall biosynthesis. We hypothesized that stressful pressure of cultures in MM is able to induce critical adaptation for the bacteria which win the infection. To identify important modifications in the biosynthesis of the cell wall, we cultured the fast-growing and nonpathogenic Mycobacterium smegmatis in MM supplemented with or without glycerol and/or cholesterol. Different from the culture in complete medium Middlebrook 7H9 broth, the bacteria when cultured in MM decreased growth and changed in the accumulation of cell wall molecules. However, the supplementation of MM with glycerol and/or cholesterol recovered the accumulation of phosphatidylinositol mannosides (PIMs) and other phospholipids but maintained growth deceleration. The biosynthesis of lipomannan (LM) and of lipoarabinomannan (LAM) was significantly modulated after culture in MM, independently of glycerol and/or cholesterol supplementation, where LM size was decreased (LM_13-25KDa) and LAM increased (LAM_37-100KDa), when compared these molecules after bacteria culture in complete medium (LM_17-25KDa and LAM_37-50KDa). These changes modified the cell surface hydrophobicity and susceptibility against H₂O₂. The infection of J774 macrophages with M. smegmatis, after culture in MM, induced the formation of granuloma-like structures, while supplementation with cholesterol induced the highest rate of formation of these structures. Taken together, our results identify critical changes in mycobacterial cell wall molecules after culture in MM that induces cholesterol accumulation, helping the mycobacteria to increase their capacity to form granuloma-like structures.