International Symposium on Emerging Trends In Infectious Diseases: TB & GI infections

11

Novel players in mycobacterial cell division under oxidative stress

Joyoti Basu
Bose Institute, Kolkata, India

The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Wag31 of Mycobacterium tuberculosis belongs to the DivIVA family of proteins known to regulate cell morphology in Gram-positive bacteria. We demonstrate that penicillinbinding protein 3 (PBP3), a septal transpeptidase is a target of the intramembrane metalloprotease Rv2869c in oxidatively stressed cells. Amino acids 102A and 103A of PBP3 are essential for Rv2869c-mediated cleavage. Wag31 interacts with PBP3 through amino acid residues 46NSD48 and protects it from oxidative stress-induced cleavage. M. smegmatis (strain PM2) harboring mutant wag31 (D46NSD48), and wag31-depleted M. tuberculosis are compromised in their ability to withstand oxidative stress. PBP3 fails to localize to the septum in oxidatively stressed PM2 cells underscoring the importance of the Wag31-PBP3 interaction. Wag31-depleted M. tuberculosis is less competent in survival within macrophages and more susceptible towards the drugs isoniazid and ofloxacin, drugs which generate reactive oxygen species (ROS) associated with the proteolytic cleavage of PBP3 in wag31depleted mycobacteria. We have demonstrated interrelationship between the products of the M. tuberculosis gene cluster Rv0014cRv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. We have identified FipA as an interacting partner of FtsZ and FtsQ and established that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and shows elongation of cells due to a defect in septum formation. Merodiploid strains of M. smegmatis expressing the FtsZ (T343A) show inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA is associated with elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. We conclude that phosphorylation-dependent proteinprotein interactions involving FipA and FtsZ, sustain mycobacterial cell division under oxidative stress. References: Mukherjee et al. (2009) Mol. Microbiol. 73, 113-119. Sureka et al. (2009) PLoS One in press