Date Published: May 8, 2014
Publisher: Public Library of Science
Author(s): Catherine Vilchèze, Virginie Molle, Séverine Carrère-Kremer, Jade Leiba, Lionel Mourey, Shubhada Shenai, Grégory Baronian, Joann Tufariello, Travis Hartman, Romain Veyron-Churlet, Xavier Trivelli, Sangeeta Tiwari, Brian Weinrick, David Alland, Yann Guérardel, William R. Jacobs, Laurent Kremer, Christopher M. Sassetti.
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.
Mycobacterium tuberculosis (Mtb) is an extraordinarily versatile pathogen that can exist in two distinct states in the host, leading to asymptomatic latent infection in which bacilli are present in a non-replicating dormant form, or to active tuberculosis (TB), characterized by actively replicating organisms. Establishment of these different (patho)physiological states requires mechanisms to sense a wide range of environmental signals and to coordinately regulate multiple metabolic and cellular processes. Many of the stimuli encountered by Mtb are transduced via transmembrane sensor kinases, allowing the pathogen to adapt to survive in hostile environments. In addition to the 12 classical two-component systems , Mtb contains 11 eukaryotic-like Ser/Thr protein kinases (STPK) , , suggesting that these two phospho-based signaling systems are of comparable importance in this microorganism. Knowledge of the substrates of each of the Mtb STPK is essential for understanding their function. Several kinase-substrates pairs have been identified and characterized during the last decade. In addition, a recent comprehensive understanding of in vivo phosphorylation event in Mtb was gained using a mass spectrometry-based approach to identify phosphorylation sites in Mtb proteins . This provided insights into the range of functions regulated by Ser/Thr phosphorylation, underpinning the involvement of many STPK in regulating metabolic processes, transport of metabolites, cell division or virulence , , .
Recent studies have provided clear insights into the vast range of pathways regulated by STPK in Mtb. These include multiple metabolic processes, transport of cell wall components as well as cell division or virulence functions , , . Several STPK, such as PknH or PknG, have been reported to play a crucial role in modulating Mtb virulence , , , , . However, little is known regarding the specific substrates contributing to mycobacterial virulence and regulated by these kinases. Here, we report the critical role of STPK-dependent phosphorylation of KasB, which is directly linked to Mtb virulence. Through the design of a KasB phosphomimetic Mtb mutant, we demonstrate that, in vivo, the replacement of the two Thr by Asp residues was characterized by highly pronounced phenotypes corresponding to i) loss of acid-fastness, ii) production of shorter mycolic acids with defects in trans-cyclopropanation, iii) defect in macrophage invasion, iv) incapacity to grow and establish a persistent infection in both immune-compromised and immune-deficient mice, and v) absence of pathology in infected animals (Fig. 9). The long-term persistence of the KasB phosphomimetic strain without causing disease or mortality makes it an attractive model for studying latent Mtb infections and suggests that this attenuated strain may represent a valuable vaccine candidate against TB.