Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Catherine Vilchèze (1) , Virginie Molle (2) , Severine Carrere-Kremer (2) , Jade Leiba (2) , Lionel Mourey (3) , Shubhada Shenai (4) , Gregory Baronian (2) , Joann Tufariello (1) , Travis Hartman (1) , Romain Veyron-Churlet (2) , Xavier Trivelli (5) , Sangeeta Tiwari (1) , Brian Weinrick (1) , David Alland (6) , Yann Guérardel (5) , William Jacobs (1) , Laurent Kremer (2)

1 HHMI - Howard Hughes Medical Institute [New York]
2 DIMNP - Dynamique des interactions membranaires normales et pathologiques
3 IPBS - Institut de pharmacologie et de biologie structurale
4 NJMS - Rutgers New Jersey Medical School
5 UGSF - Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576
6 Rutgers University [Newark]

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.

Mots clés: Cell walls; Mycobacterium tuberculosis; Phosphorylation; Acid-fast stain; Macrophages; Mutant strains; Thin-layer chromatography; Polymerase chain reaction

Domaines: Bactériologie; Biochimie, Biologie Moléculaire

Catherine Vilchèze, Virginie Molle, Severine Carrere-Kremer, Jade Leiba, Lionel Mourey, et al.. Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis. PLoS Pathogens, 2014, 10 (5), pp.e1004115. ⟨10.1371/journal.ppat.1004115⟩. ⟨hal-02282645⟩