title: Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.
abstract: Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.
authors.affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, New York, United States of America.
authors.forename: Catherine
authors.initials: C
authors.lastname: Vilchèze
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS; UMR 5235, Montpellier, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Molle
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS; UMR 5235, Montpellier, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Carrère-Kremer
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS; UMR 5235, Montpellier, France.
authors.forename: Jade
authors.initials: J
authors.lastname: Leiba
authors.affiliation: Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse, France; The Université de Toulouse, Université Paul Sabatier, IPBS, Toulouse, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Mourey
authors.affiliation: Division of Infectious Diseases, Department of Medicine, and the Ruy V. Lourenco Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, New Jersey, United States of America.
authors.forename: Shubhada
authors.initials: S
authors.lastname: Shenai
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS; UMR 5235, Montpellier, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Baronian
authors.affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, New York, United States of America.
authors.forename: Joann
authors.initials: J
authors.lastname: Tufariello
authors.affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, New York, United States of America.
authors.forename: Travis
authors.initials: T
authors.lastname: Hartman
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS; UMR 5235, Montpellier, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Veyron-Churlet
authors.affiliation: Université Lille 1, Unité de Glycobiologie Structurale et Fonctionnelle, UGSF, Villeneuve d'Ascq, France; CNRS, UMR 8576, Villeneuve d'Ascq, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Trivelli
authors.affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, New York, United States of America.
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Tiwari
authors.affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, New York, United States of America.
authors.forename: Brian
authors.initials: B
authors.lastname: Weinrick
authors.affiliation: Division of Infectious Diseases, Department of Medicine, and the Ruy V. Lourenco Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, New Jersey, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Alland
authors.affiliation: Université Lille 1, Unité de Glycobiologie Structurale et Fonctionnelle, UGSF, Villeneuve d'Ascq, France; CNRS, UMR 8576, Villeneuve d'Ascq, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Guérardel
authors.affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, New York, United States of America.
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS; UMR 5235, Montpellier, France; INSERM, DIMNP, Montpellier, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D050356:Lipid Metabolism / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008828:Microbiological Techniques / Q000379:methods; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D009171:Mycolic Acids / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D014376:Tuberculosis / Q000175:diagnosis / Q000378:metabolism / Q000382:microbiology; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D009171:Mycolic Acids; C444656:KasB protein, Mycobacterium tuberculosis; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D017346:Protein Serine-Threonine Kinases