The protein kinase PknB negatively regulates biosynthesis and trafficking of mycolic acids in mycobacteria

Mycobacterium tuberculosis is the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug-development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we used genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium. The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates the export of MAs and promotes the remodeling of the mycobacterial cell envelope. In particular, we identified the essential MmpL3 as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way for novel antimycobacterial strategies. Mycobacterium tuberculosis is the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug-development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we used genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium. The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates the export of MAs and promotes the remodeling of the mycobacterial cell envelope. In particular, we identified the essential MmpL3 as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way for novel antimycobacterial strategies. Mycobacterium tuberculosis (Mtu), a causative agent of tuberculosis (TB), remains one of the most widespread and deadliest pathogens in the world. Currently available treatment regimens for patients with active TB are significantly more complex than other bacterial infections and require the use of multiple antibiotics taken over the course of 6 to 9 months. The length, complexity, and side effects of this chemotherapy may have contributed to the emergence of multi drug-resistant and extensively drug-resistant Mtu strains due to low compliance. This highlights the need for the continued development of additional TB treatment options as well as further research into Mtu biological features that set it apart from other bacterial pathogens. One such distinguishing feature of mycobacteria is the one-of-a-kind architecture of their cell wall, which provides a protective layer and renders the pathogen recalcitrant to chemical damage, dehydration, and antibiotic treatment. This cell wall contains various species-specific lipids, most notably mycolic acids (MAs), that are unique to mycobacteria and play key roles in mycobacterial physiology and fitness (1Jamet S. Slama N. Domingues J. Laval F. Texier P. Eynard N. Quémard A. Peixoto A. Lemassu A. Daffé M. et al.The non-essential mycolic acid biosynthesis genes hadA and hadC contribute to the physiology and fitness of Mycobacterium smegmatis.PLoS One. 2015; 10: e0145883Crossref PubMed Scopus (18) Google Scholar) as well as in Mtu virulence and persistence within infected cells (2Verschoor J.A. Baird M.S. Grooten J. Towards understanding the functional diversity of cell wall mycolic acids of Mycobacterium tuberculosis.Prog. Lipid Res. 2012; 51: 325-339Crossref PubMed Scopus (66) Google Scholar). MAs are very long-chain α-branched, β-hydroxylated FAs ranging from C70 to C90 (3Daffé M.Q.A. Marrakchi H. Mycolic acids: from chemistry to biology.in: Geiger O. Biogenesis of Fatty Acids, Lipids and Membranes. Springer, Cham, Switzerland2017Crossref Google Scholar). MAs represent the main constituents of the cell-wall outer membrane (or mycomembrane), wherein they are anchored. MAs are transported across the plasma (inner) membrane as trehalose monomycolates (TMMs) and then are either covalently linked to the arabinogalactan-peptidoglycan to form the mycoloyl arabinogalactan-peptidoglycan complex or incorporated into trehalose dimycolates (TDMs), which are found in the outermost leaflet of the mycomembrane. The outer leaflet also contains other noncovalently associated lipids, such as phthiocerol dimycocerosates and sulfolipids (4Daffé M. Marrakchi H. Unraveling the structure of the mycobacterial envelope.Microbiol. Spectr. 2019; 7 (10.1128/microbiolspec.GPP3-0027-2018)Crossref PubMed Scopus (46) Google Scholar). MAs are synthesized in the cytoplasm via a highly conserved and well-characterized mixed FAS/polyketide synthase biosynthetic pathway that has been extensively studied (5Marrakchi H. Lanéelle M-A.A. Daffé M. Mycolic acids: structures, biosynthesis, and beyond.Chem. 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MmpL3 is the flippase for mycolic acids in mycobacteria.Proc. Natl. Acad. Sci. USA. 2017; 114: 7993-7998Crossref PubMed Scopus (118) Google Scholar). However, the intracellular regulation of cell-wall biosynthesis still remains poorly understood. Recently, phosphorylation mediated by Ser/Thr protein kinases (STPKs) has begun to emerge as a major regulation mechanism for many biological processes that control mycobacterial infection and persistence (10Wehenkel A. Bellinzoni M. Graña M. Duran R. Villarino A. Fernandez P. Andre-Leroux G. England P. Takiff H. Cerveñansky C. et al.Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential.Biochim. Biophys. Acta. 2008; 1784: 193-202Crossref PubMed Scopus (144) Google Scholar, 11Richard-Greenblatt M. Av-Gay Y. Epigenetic phosphorylation control of mycobacterium tuberculosis infection and persistence.Microbiol. 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Bellinzoni M. et phosphorylation regulates the fatty activity of essential in mycolic acid Biol. Chem. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). PknB is to one of the of Ser/Thr in Mtu and in the Ser/Thr protein kinase of Mycobacterium Biol. Chem. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar, P. M. Y. S. K. et with of Mycobacterium tuberculosis PknB Commun. 2019; 10: PubMed Scopus Google Scholar). is essential and regulates the activity of a of notably involved in cell-wall The of has been to cellular and Y. S. S. F. kinase of Mycobacterium tuberculosis is essential for growth of the pathogen in as well as for within the Biol. Chem. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar, The Mycobacterium tuberculosis kinases and substrate and regulation of cell 19: PubMed Scopus Google Scholar), which suggests that the and activity of this kinase the is in other essential genes involved in Ser/Thr and is the of Mtu to a Ser/Thr that a functional with PknB to control mycobacterial cell growth via the of phosphorylation/dephosphorylation in substrate proteins P. M. Y. S. K. et with of Mycobacterium tuberculosis PknB Commun. 2019; 10: PubMed Scopus Google Scholar, M. R. F. Cerveñansky C. PknB kinase activity is regulated by phosphorylation in and by the in Mycobacterium Microbiol. PubMed Scopus Google Scholar). The regulation has been reported for a Mtu such as the activity of involved in the MA pathway Molle V. Eynard N. M. A. F. S. V. G. Bellinzoni M. et phosphorylation regulates the fatty activity of essential in mycolic acid Biol. Chem. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). the of remains and the and intracellular phosphorylation/dephosphorylation and cell-wall biogenesis is well understood. to this we a lipidomics and phosphoproteomics analysis of M. strains to Ser/Thr phosphorylation genetic to Ser/Thr phosphorylation and combined with of PknB and to cell-wall and in lipid identified MmpL3 as the substrate of PknB and this phosphorylation as a major in MA and cell-wall PknB in the into mycobacterial Molle V. Eynard N. M. A. F. S. V. G. Bellinzoni M. et phosphorylation regulates the fatty activity of essential in mycolic acid Biol. Chem. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). 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D. Singh A. A. Molle V. Singh Y. protein of Mycobacterium tuberculosis is for cell and of Biol. Chem. 2016; Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The by the of bacterial cells and lipid the analysis of the and MA in their the and strains However, bacterial lipids to the we a of the of in the with are the mycoloyl of the major mycolate-containing lipids and mycoloyl of the mycomembrane. their notably the with a in the by results that is responsible for a of in This that the transport, or of may regulated by that MA biosynthesis is regulated by kinase activities and the essential and of PknB, we that PknB play a major role in MA metabolism, in cell-wall to this in a more system by PknB. the role of phosphorylation the cell-wall PknB from Mtu with in a we also the Mtu which is to Y. S. S. F. kinase of Mycobacterium tuberculosis is essential for growth of the pathogen in as well as for within the Biol. Chem. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). This that the the strains or to PknB kinase activity and due to in protein The of Mtu PknB the growth with or Mtu which is in with The Mycobacterium tuberculosis kinases and substrate and regulation of cell 19: PubMed Scopus Google Scholar). thus to of the drug this the strains have a growth and which to the and of analysis of the protein from the strains the of PknB and and their by that they in In to the of PknB kinase activity the we the of and that the cells by the lipid of the and and that via proteins H. The of PubMed Scopus Google Scholar, C. S. I. S. H. M. provides the main pathway the cell wall of Mycobacterium Microbiol. PubMed Scopus Google Scholar) the strains or the Mtu more to and with the control and the This by a of the which a of the the other in the to the and in strains PknB results a of the cell-wall from the of PknB kinase that the of to and the of PknB kinase activity from of the investigate the of PknB kinase activity and MA bacteria the of the In to MA production and the of in metabolism, cells and with for The of in the to MAs of bacterial and in the MAs are as trehalose and cells either and the lipids and or with to noncovalently linked lipids lipids by and The of into FAs and MAs in Mtu PknB and with the control the and the the MA production the of PknB kinase In in the noncovalently linked lipids, in the form of notably significantly in the This of in of the inhibition of the either H. V.A. M.S. V. V. J. et of mycolic acid transport across the Mycobacterium tuberculosis plasma Chem. Biol. 2012; 8: PubMed Scopus Google Scholar, K. R. K. V. 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M. of the Mycobacterium tuberculosis mycolic acid 2016; PubMed Scopus Google Scholar). in the of the strains the of into of and MA also Taken together, our that PknB kinase activity and MA also the transport of MAs to the cell wall, that has been the that PknB affects MA transport via phosphorylation of involved in this process, we a analysis of the of with and proteins and then and by to the of protein and phosphorylation across then by a of the to the proteins and phosphorylation regulated the of PknB kinase identified as many phosphorylation Mtu PknB with The phosphorylation in control as The kinase activity of Mtu PknB the of and their in as the of Mtu PknB more than in of the control phosphoproteomics a in phosphorylation the of PknB than of the significantly the of Mtu PknB with the set This suggests that the the strains are more due to the phosphorylation of protein than to in protein the and of significantly regulated phosphorylation the of over and which is in with the reported of mycobacterial S. S. D. G. H. phosphorylation with by Mycobacterium tuberculosis protein Natl. Acad. Sci. USA. PubMed Scopus Google Scholar). analysis identified phosphorylation proteins set which phosphorylation differentially regulated the strains PknB kinase activity with the regulated identified proteins to regulated by PknB, such as PknB and essential protein cell-wall biosynthesis the cellular M. Av-Gay Y. Epigenetic phosphorylation control of mycobacterium tuberculosis infection and persistence.Microbiol. Spectr. 2017; 5 (10.1128/microbiolspec.TBTB2-0005-2015)Crossref PubMed Scopus (16) Google Scholar) In to results from S. S. D. G. H. phosphorylation with by Mycobacterium tuberculosis protein Natl. Acad. Sci. USA. PubMed Scopus Google Scholar, S. 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PubMed Scopus Google Scholar). we also that to to phosphorylation such as the highly regulated phosphorylation we identified further the role of PknB activity in the regulation of MA our and reveal that PknB a role in the production of MAs also their export to the thus in the remodeling of the cell STPKs have emerged as of and for by substrate proteins involved in MA biosynthesis to for Mtu cell-wall a and of Ser/Thr In Mtu and the PknB is one of the characterized kinases that are for the regulation of mycobacterial additional kinases are as well that Mtu genome for 11 is the identified to Therefore, the PknB and activities is to for mycobacterial we this that this a role in cell A.K. D. Singh A. A. Molle V. Singh Y. protein of Mycobacterium tuberculosis is for cell and of Biol. Chem. 2016; Full Text Full Text PDF PubMed Scopus (32) Google Scholar, N. P. O. regulates phosphorylation and cell 2019; PubMed Scopus Google Scholar), as well as cell-wall biosynthesis and phosphorylation N. P. O. regulates phosphorylation and cell 2019; PubMed Scopus Google Scholar). Here, we more evidence that role in MA biosynthesis the that significantly the of a in MA further investigate this we the and essential protein kinase PknB from Mtu and in the of Mtu PknB the it a major in the of the cells to the and remodeling of the envelope. The Mycobacterium tuberculosis kinases and substrate and regulation of cell 19: PubMed Scopus Google Scholar), a of the growth of of the of bacterial cells with a of MA and a and in the of trehalose in the of with a of the of the of These the kinase activity of PknB in the and to the inhibition of trehalose transport from the to the cell wall by A analysis of the of mycobacterial strains the phosphorylation of proteins involved in the and transport of MmpL3 In the phosphorylation of MmpL3 the mycobacterial of this protein and the inhibition of the cell-wall lipid export by MmpL3 H. V.A. M.S. V. V. 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In this the of phosphoproteomics with lipidomics to cell-wall remodeling Ser/Thr phosphorylation proteins of the MA and transport, including essential and the of many antimycobacterial development H. V.A. M.S. V. V. J. et of mycolic acid transport across the Mycobacterium tuberculosis plasma Chem. Biol. 2012; 8: PubMed Scopus Google Scholar, K. R. K. V. D. et a membrane of trehalose involved in mycolic acid to the cell wall of Mycobacterium 2012; PubMed Scopus Google Scholar, A. M. D. M. of the Mycobacterium tuberculosis mycolic acid 2016; PubMed Scopus Google Scholar, A. Y. V. et insights into the mechanism of inhibition of a of multiple in Mycobacterium 2014; PubMed Scopus Google Scholar). These findings are in with a recent to mycobacterial lipid and the phosphorylation of MmpL3 treatment with and PknB J. M. Z. J. S. et analysis of Mycobacterium tuberculosis remodeling of the PubMed Scopus Google Scholar). The functional insights from phosphoproteomics further by them with other such as and to a more of the role of phosphorylation in mycobacterial that in strategies. The have been to the via the J.A. A. N. J. I. G. Y. F. et of the and Res. 2016; PubMed Scopus Google Scholar) with the and the are available a for 2014; Full Text Full Text PDF PubMed Scopus Google Scholar) The to the analysis found The for of the M. with mycolic acid Mycobacterium Mycobacterium tuberculosis Ser/Thr protein kinase tuberculosis trehalose lipid acid trehalose