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Arabinogalactan enhanced Mycobacterium virulence through reprogramming phagocyte oxidative metabolism and inducing cell death

ABSTRACT: Arabinogalactan (AG) participates in forming the cell wall core of mycobacteria which is known as mAGP complex. Few studies on effects of AG on real-world virulence revealed its interaction with the host immune system, especially when taking the whole mycobacterial conditions into account. By CRISPRi, conditional mutant strains were constructed with low-expression of embA or glfT2 which involved in AG biosynthesis. HPGPC and HPLC confirmed EmbA_KD strain had a remarkable decrease in AG content with fragmentary arabinose chains, and GlfT2_KD strain displayed a modest reduction in content with dramatically cut-down galactose chains. Loose and thickened cell wall structure occurred with AG loss, and extracellular leakage happened from decreased arabinose under TEM and SEM. Phenotypes of EmbA_KD strain including spreading motility, biofilm formation and ethambutol resistance altered. When murine Tr4A.1 macrophages and human Gal9 KO cells were infected, EmbA_KD/GlfT2_KD strains displayed limited proliferation. Through transcriptome profiling, phagocytes infected by EmbA_KD/GlfT2_KD strains showed enhanced oxidative metabolism, and macrophages trended to survival when exposed to EmbA_KD strain. Secretion of proinflammatory cytokines such as IL-6/MCP-1/MIP-1α/MIP-1β were induced by EmbA_KD strain via the TLR2 - C/EBPβ signaling pathway, which benefited the bactericidal effect. The onset progressions of knock-down strains in larvae zebrafish and murine tail infection models significantly delayed with attenuate inflammatory infiltrate in murine tail, further confirmed that AG was an important virulence factor at the whole bacterial level. In adult zebrafish, EmbA_KD showed decreased CFU/granulomas counts and restricted formation and distribution of granulomas. In conclusion, AG restrained host innate immune responses to enhance proliferation through reprogramming oxidative metabolism and cell death, and inhibiting phagocytes migration possibly. Arabinose chains contribute more to AG virulence, and cause easier recognition and removal of Mycobacterium by the host immune cells, which mimics the ethambutol inhibition process.

ORGANISM(S): Mus musculus

PROVIDER: GSE197488 | GEO | 2022/03/04

REPOSITORIES: GEO

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Project description:L-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Especially at low concentrations of L-arabinose, its uptake via the Gal2 galactose transporter is an important rate-controlling step in the complete conversion of these feedstocks by engineered, pentose-metabolizing Saccharomyces cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least three-fold higher in L-arabinose-limited cultures than in glucose-limited and ethanol-limited cultures. Of five genes that showed an over 30-fold higher transcript level in arabinose-grown cultures, only one (Pc20g01790) restored growth on L-arabinose upon expression in an engineered L-arabinose-fermenting S. cerevisiae strain in which GAL2 had been deleted. Sugar-transport assays indicated that Pc20g01790this transporter, designated as PcAraT, encodes functions as a high-affinity (Km = 0.13 mM) L-arabinose-proton symporter that does not transport xylose or glucose. An L-arabinose-metabolizing S. cerevisiae strain co-expressing Pc20g01790PcAraT and GAL2 showed lower residual substrate concentrations in L-arabinose-limited chemostat cultures (4 mg L-1) than a congenic strain in which L-arabinose import exclusively depended on Gal2 (1.8 g L-1). Inhibition of L-arabinose transport by these sugars was less pronounced than observed with Gal2. A hexose-phosphorylation-deficient, L-arabinose-metabolizing S. cerevisiae strain expressing PcAraT Pc20g0190 grew on 20 g L-1 L-arabinose in the presence of 20 g L-1 glucose, which completely inhibited growth on L-arabinose of a congenic strain dependent on L-arabinose transport via Gal2. Its high affinity and specificity for L-L-arabinose, combined with limited sensitivity to inhibition by glucose and D-D-xylose make PcAraT/ Pc20g01790 a valuable transporter gene for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

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Arabinogalactan enhanced Mycobacterium virulence through reprogramming phagocyte oxidative metabolism and inducing cell death

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