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Cholesterol-dependent transcriptome remodeling reveals new insight into the contribution of cholesterol to Mycobacterium tuberculosis pathogenesis.

ABSTRACT: Mycobacterium tuberculosis (Mtb) is an obligate human pathogen that can adapt to the various nutrients available during its life cycle. However, in the nutritionally stringent environment of the macrophage phagolysosome, Mtb relies mainly on cholesterol. In previous studies, we demonstrated that Mtb can accumulate and utilize cholesterol as the sole carbon source. However, a growing body of evidence suggests that a lipid-rich environment may have a much broader impact on the pathogenesis of Mtb infection than previously thought. Therefore, we applied high-resolution transcriptome profiling and the construction of various mutants to explore in detail the global effect of cholesterol on the tubercle bacillus metabolism. The results allow re-establishing the complete list of genes potentially involved in cholesterol breakdown. Moreover, we identified the modulatory effect of vitamin B₁₂ on Mtb transcriptome and the novel function of cobalamin in cholesterol metabolite dissipation which explains the probable role of B₁₂ in Mtb virulence. Finally, we demonstrate that a key role of cholesterol in mycobacterial metabolism is not only providing carbon and energy but involves also a transcriptome remodeling program that helps in developing tolerance to the unfavorable host cell environment far before specific stress-inducing phagosomal signals occur.

SUBMITTER: Pawelczyk J

PROVIDER: S-EPMC8196197 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Cholesterol-dependent transcriptome remodeling reveals new insight into the contribution of cholesterol to Mycobacterium tuberculosis pathogenesis.

Pawełczyk Jakub J Brzostek Anna A Minias Alina A Płociński Przemysław P Rumijowska-Galewicz Anna A Strapagiel Dominik D Zakrzewska-Czerwińska Jolanta J Dziadek Jarosław J

Scientific reports 20210611 1

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen that can adapt to the various nutrients available during its life cycle. However, in the nutritionally stringent environment of the macrophage phagolysosome, Mtb relies mainly on cholesterol. In previous studies, we demonstrated that Mtb can accumulate and utilize cholesterol as the sole carbon source. However, a growing body of evidence suggests that a lipid-rich environment may have a much broader impact on the pathogenesis of Mtb ...[more]

PMID: 34117327

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Project description:Tuberculosis is the leading killer among infectious diseases worldwide. Increasing multidrug resistance has prompted new approaches for tuberculosis drug development, including targeted inhibition of virulence determinants and of signaling cascades that control many downstream pathways. We used a multisystem approach to determine the effects of a potent small-molecule inhibitor of the essential Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB. We observed differential levels of phosphorylation of many proteins and extensive changes in levels of gene expression, protein abundance, cell wall lipids, and intracellular metabolites. The patterns of these changes indicate regulation by PknA and PknB of several pathways required for cell growth, including ATP synthesis, DNA synthesis, and translation. These data also highlight effects on pathways for remodeling of the mycobacterial cell envelope via control of peptidoglycan turnover, lipid content, a SigE-mediated envelope stress response, transmembrane transport systems, and protein secretion systems. Integrated analysis of phosphoproteins, transcripts, proteins, and lipids identified an unexpected pathway whereby threonine phosphorylation of the essential response regulator MtrA decreases its DNA binding activity. Inhibition of this phosphorylation is linked to decreased expression of genes for peptidoglycan turnover, and of genes for mycolyl transferases, with concomitant changes in mycolates and glycolipids in the cell envelope. These findings reveal novel roles for PknA and PknB in regulating multiple essential cell functions and confirm that these kinases are potentially valuable targets for new antituberculosis drugs. In addition, the data from these linked multisystems provide a valuable resource for future targeted investigations into the pathways regulated by these kinases in the M. tuberculosis cell.IMPORTANCE Tuberculosis is the leading killer among infectious diseases worldwide. Increasing drug resistance threatens efforts to control this epidemic; thus, new antitubercular drugs are urgently needed. We performed an integrated, multisystem analysis of Mycobacterium tuberculosis responses to inhibition of its two essential serine/threonine protein kinases. These kinases allow the bacterium to adapt to its environment by phosphorylating cellular proteins in response to extracellular signals. We identified differentially phosphorylated proteins, downstream changes in levels of specific mRNA and protein abundance, and alterations in the metabolite and lipid content of the cell. These results include changes previously linked to growth arrest and also reveal new roles for these kinases in regulating essential processes, including growth, stress responses, transport of proteins and other molecules, and the structure of the mycobacterial cell envelope. Our multisystem data identify PknA and PknB as promising targets for drug development and provide a valuable resource for future investigation of their functions.

Project description:Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k(cat)/K(m) = 1000 +/- 100 M(-1) s(-1) versus 700 +/- 100 M(-1) s(-1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k(cat)/K(m) = 80 +/- 40 M(-1) s(-1)). In the presence of 3-HSA the K(m)(app) for O(2) was 100 +/- 10 microM. The crystal structure of HsaA to 2.5-A resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val(367)-Val(394)) could adopt two conformations differing by a rigid body rotation of 25 degrees around Arg(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.

Dataset Information

Cholesterol-dependent transcriptome remodeling reveals new insight into the contribution of cholesterol to Mycobacterium tuberculosis pathogenesis.

Publications

Cholesterol-dependent transcriptome remodeling reveals new insight into the contribution of cholesterol to Mycobacterium tuberculosis pathogenesis.

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