Culture of Mycobacterium smegmatis in Different Carbon Sources to Induce In Vitro Cholesterol Consumption Leads to Alterations in the Host Cells after Infection: A Macrophage Proteomics Analysis.

During tuberculosis, Mycobacterium uses host macrophage cholesterol as a carbon and energy source. To mimic these conditions, Mycobacterium smegmatis can be cultured in minimal medium (MM) to induce cholesterol consumption in vitro. During cultivation, M. smegmatis consumes MM cholesterol and changes the accumulation of cell wall compounds, such as PIMs, LM, and LAM, which plays an important role in its pathogenicity. These changes lead to cell surface hydrophobicity modifications and H2O2 susceptibility. Furthermore, when M. smegmatis infects J774A.1 macrophages, it induces granuloma-like structure formation. The present study aims to assess macrophage molecular disturbances caused by M. smegmatis after cholesterol consumption, using proteomics analyses. Proteins that showed changes in expression levels were analyzed in silico using OmicsBox and String analysis to investigate the canonical pathways and functional networks involved in infection. Our results demonstrate that, after cholesterol consumption, M. smegmatis can induce deregulation of protein expression in macrophages. Many of these proteins are related to cytoskeleton remodeling, immune response, the ubiquitination pathway, mRNA processing, and immunometabolism. The identification of these proteins sheds light on the biochemical pathways involved in the mechanisms of action of mycobacteria infection, and may suggest novel protein targets for the development of new and improved treatments.

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

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 (LM13-25KDa) and LAM increased (LAM37-100KDa), when compared these molecules after bacteria culture in complete medium (LM17-25KDa and LAM37-50KDa). 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.

Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil.

Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as Lutzomyia longipalpis sensun latum (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi [suspected vector of Leishmania (Viannia) lindenbergi] are incriminated of transmitting the parasite Leishmania for the vertebrate host. The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG) to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-acetyl-galactosamine (GalNAc) seems to occur in some species of phlebotomines that are classified as permissive. The aim of this study was to characterize the carbohydrate residues that, probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field populations from the Brazilian Amazonian region. We observed the presence of GalNAc, mannose, galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L. i.chagasi to the midguts of different species of phlebotomines was performed. The attachment of both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian Amazonian region.