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.

Insights into the smooth‐to‐rough transitioning in Mycobacterium bolletii unravels a functional Tyr residue conserved in all mycobacterial MmpL family members

“…The mmpL4a open reading frame (ORF) was PCR amplified using purified genomic DNA of M. bolletii (extracted from the progenitor strain of the M. bolletii MmpL4a_Y842H mutant), Phusion DNA polymerase (Finnzymes, Finland) and the primers mmpL4af and mmpL4ar. The amplicon was digested with NdeI and HindIII and used to replace the eGFP ORF flanked by NdeI and HindIII recognition sequences in the replicative plasmid pVV16_ eGFP (Vilchèze et al ., ). The resulting construct, pVV16_ mmpL4a , contained the mmpL4a ORF under control of the constitutive hsp60 promoter.…”

Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids

“…For example, phosphorylation of the fatty acid synthase II (FASII) enoyl-ACP reductase InhA reduces activity by lowering the affinity for the NADH cofactor (34). Inhibition of β-ketoacyl acyl carrier protein synthase KasB upon phosphorylation is attributed to the proximity of the modified Thr residues to the catalytic triad (36). Since PDIM levels are modulated by growth conditions, infection or the presence of PknH, we hypothesized that Mtb Ser/Thr kinases may also regulate PDIM production by modifying PapA5.…”

“…Phosphorylated peptides corresponding to Mas and Pks1/15 have also been detected in Mtb cell lysates (32, 33). We hypothesized that phosphorylation modulates PDIM biosynthesis in part via a direct effect on the catalytic activity of PapA5, as has been shown for other lipid biosynthetic enzymes in Mtb (34–36). …”

A unique PE_PGRS protein inhibiting host cell cytosolic defenses and sustaining full virulence ofMycobacterium marinumin multiple hosts

“…The 200 bp promoter region of MMAR_0242 was amplified from Mma E11 genomic DNA using the 02260_Prom Forward (XbaI) and 02260_Prom Reverse (NdeI) primers (Table S2). The amplified promoter region was restricted with XbaI/NdeI and ligated into the XbaI/NdeI‐digested pVV16‐EGFP (Vilchèze et al ., ). The resulting plasmid, pVV16‐Prom 0242 _EGFP was electroporated into Mma E11 wild‐type.…”