Abstract

Cholesterol of the host macrophage membrane is vital for mycobacterial infection, replication, and persistence. During chronic infection within host lung tissues, cholesterol facilitates the phagocytosis of mycobacteria into macrophages. Cholesterol degradation leads to increased flux of acetyl-coenzyme A (CoA) and propionyl-CoA, providing energy and building blocks for virulence macromolecules as well as donors for global protein acylation. Potential functions of lysine acylation are gradually revealed in bacterial survival and pathogenesis. However, the mycobacterial proteome and posttranslational modification (PTM) changes involved in the cholesterol catabolism bioprocess remain unclear. Here, we used nonpathogenic Mycobacterium smegmatis as a model and simultaneously monitored mycobacterial proteome and acetylome changes in the presence of glucose and cholesterol. We discovered that cholesterol metabolic enzymes were upregulated with respect to both protein expression levels and lysine acylation levels during the metabolic shift from glucose to cholesterol. After that, adenylating enzymes related to cholesterol metabolism were proven to be precisely regulated at the propionylation level by mycobacterial acyltransferase M. smegmatis Kat (MsKat) in response to cellular propionyl-CoA accumulation. Furthermore, the kinase expression and phosphorylation levels were also changed along with fluctuations in cholesterol levels. Our results expanded current knowledge of acylation regulation in the cholesterol catabolism of mycobacteria and provided references for possible antimycobacterium strategy.IMPORTANCE Cholesterol assimilation is a critical step in mycobacterial chronic infection. However, knowledge from the dynamic characterization of cholesterol metabolism in mycobacteria at the protein expression and PTM levels remains limited. Our study uncovered the landscape of protein expression, lysine acetylation, lysine propionylation, and S/T/Y phosphorylation during the metabolic changes from glucose to cholesterol in mycobacteria. The data showed that cholesterol-induced carbon shift resulted in the elevation of protein expression and lysine acylation in diverse metabolic enzymes involved in cholesterol degradation and that the presence of cholesterol also promoted the perturbations at the phosphorylation level in the kinase system in mycobacteria. This study systematically characterized the regulation of cholesterol catabolism at several different levels, which provided the detailed references in mycobacterial proteome and potential antimycobacterial strategies.

Highlights

  • Cholesterol of the host macrophage membrane is vital for mycobacterial infection, replication, and persistence

  • In this study, using nonpathogenic Mycobacterium smegmatis as a model, we mainly presented the global picture of protein expression, acetylation, propionylation, and phosphorylation changes for M. smegmatis growing in cholesterol compared with its growth in glucose

  • The pathway enrichment analysis of the significantly changed protein indicated that proteins involved in the ribosome process and in biosynthesis of siderophore group nonribosomal peptides were downregulated and that proteins that participated in oxidative phosphorylation, propanoate metabolism, and steroid degradation were upregulated in the presence of cholesterol (Fig. 1C; see Data Set S1C)

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Summary

Introduction

Cholesterol of the host macrophage membrane is vital for mycobacterial infection, replication, and persistence. The data showed that cholesterol-induced carbon shift resulted in the elevation of protein expression and lysine acylation in diverse metabolic enzymes involved in cholesterol degradation and that the presence of cholesterol promoted the perturbations at the phosphorylation level in the kinase system in mycobacteria. Owing to the mass spectrometry (MS)-based proteomic analyses performed in recent years, we and other groups have expanded knowledge of lysine acylomes in various bacteria, including Escherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Thermus thermophilus, Saccharopolyspora erythraea, and Mycobacterium tuberculosis [9,10,11,12] In those studies, lysine acylations were proven to modify virulence factors and to be involved in bacterial pathogenesis [13]. Quantitative proteome and phosphorylome data showed that cholesterol changes influenced levels of protein expression and phosphorylation of protein kinases, including PknA, PknB, and PknK This comprehensive omic profiling functionally connected the specific characterization of cholesterol assimilation with the proteomic and PTM msystems.asm.org 2

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