Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

Renata Dziedzic,Manjot Kiran,Malini Rajagopalan,Malgorzata Ziolkiewicz,Indumati S Vadrevu,Murty Madiraju,Jaroslaw Dziadek,Meredith Moomey,Anna Brzostek,Przemyslaw Plocinski,David M Ojcius

doi:10.1371/journal.pone.0011058

Abstract

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.

Highlights

Mycobacterium tuberculosis, the causative agent of tuberculosis, has spread dramatically worldwide, and recent years have seen an increase in the number of multidrug-resistant strains and the emergence of extensively drug-resistant M. tuberculosis [1,2]
Since M. tuberculosis cells growing in macrophages and those exposed to cephalexin antibiotic are deficient in FtsZ assembly [5,26], we examined if ClpX is involved in regulating FtsZ assembly and cell division in M. tuberculosis
The results presented in this study show that M. tuberculosis ClpX interacts with FtsZ and a possible consequence of these interactions is modulation of Z-ring assembly at midcell sites in vivo and interference with GTP-dependent FtsZ polymerization activity in vitro (Figs. 7, 8 and 2)

Summary

Introduction

Mycobacterium tuberculosis, the causative agent of tuberculosis, has spread dramatically worldwide, and recent years have seen an increase in the number of multidrug-resistant strains and the emergence of extensively drug-resistant M. tuberculosis [1,2]. Eradication of M. tuberculosis infection necessitates the development of novel drugs targeted to hitherto unidentified metabolic processes and pathways of the pathogen, and FtsZ catalyzed cell division is one such process. M. tuberculosis FtsZ, like its bacterial counterparts, exhibits GTP binding and hydrolysis activities and localizes to the midcell division site in the form of a Z-ring [5,6]. FtsZ-ring assembly in M. tuberculosis is delayed under several conditions relevant to its growth; two of these are growth in macrophages and exposure to cephalexin [5]. The identities of the regulators affecting Z-ring assembly and the cell division process in M. tuberculosis, are largely unknown. Identification and characterization of such regulators would improve our understanding of the cell division process in M. tuberculosis

Methods

Results

Conclusion