Mycobacterium tuberculosis ClpX interacts with FtsZ and interferes with FtsZ assembly.
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.
Dziedzic
Renata
R
The University of Texas Health Science Center, Tyler Biomedical Research, Tyler, Texas, United States of America.
Kiran
Manjot
M
Plocinski
Przemyslaw
P
Ziolkiewicz
Malgorzata
M
Brzostek
Anna
A
Moomey
Meredith
M
Vadrevu
Indumati S
IS
Dziadek
Jaroslaw
J
Madiraju
Murty
M
Rajagopalan
Malini
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria