title: Phosphorylation of mycobacterial PcaA inhibits mycolic acid cyclopropanation: consequences for intracellular survival and for phagosome maturation block.
abstract: Pathogenic mycobacteria survive within macrophages by residing in phagosomes, which they prevent from maturing and fusing with lysosomes. Although several bacterial components were seen to modulate phagosome processing, the molecular regulatory mechanisms taking part in this process remain elusive. We investigated whether the phagosome maturation block (PMB) could be modulated by signaling through Ser/Thr phosphorylation. Here, we demonstrated that mycolic acid cyclopropane synthase PcaA, but not MmaA2, was phosphorylated by mycobacterial Ser/Thr kinases at Thr-168 and Thr-183 both in vitro and in mycobacteria. Phosphorylation of PcaA was associated with a significant decrease in the methyltransferase activity, in agreement with the strategic structural localization of these two phosphoacceptors. Using a BCG ΔpcaA mutant, we showed that PcaA was required for intracellular survival and prevention of phagosome maturation in human monocyte-derived macrophages. The physiological relevance of PcaA phosphorylation was further assessed by generating PcaA phosphoablative (T168A/T183A) or phosphomimetic (T168D/T183D) mutants. In contrast to the wild-type and phosphoablative pcaA alleles, introduction of the phosphomimetic pcaA allele in the ΔpcaA mutant failed to restore the parental mycolic acid profile and cording morphotype. Importantly, the PcaA phosphomimetic strain, as the ΔpcaA mutant, exhibited reduced survival in human macrophages and was unable to prevent phagosome maturation. Our results add new insight into the importance of mycolic acid cyclopropane rings in the PMB and provide the first evidence of a Ser/Thr kinase-dependent mechanism for modulating mycolic acid composition and PMB.
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS, UMR 5235, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Rosa Milagros
authors.initials: RM
authors.lastname: Corrales
authors.forename: Virginie
authors.initials: V
authors.lastname: Molle
authors.forename: Jade
authors.initials: J
authors.lastname: Leiba
authors.forename: Lionel
authors.initials: L
authors.lastname: Mourey
authors.forename: Chantal
authors.initials: C
authors.lastname: de Chastellier
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D003521:Cyclopropanes / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009163:Mycobacterium bovis / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009171:Mycolic Acids / Q000378:metabolism*; D010588:Phagosomes; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003521:Cyclopropanes; D009171:Mycolic Acids; D008780:Methyltransferases; C019964:cyclopropane synthetase; D017346:Protein Serine-Threonine Kinases