Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

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.

Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes

“…Transformants were selected in this same medium supplemented with agar containing either Km (10 g/ml), Hyg (50 g/ml), or both. When required, acetamide and/or anhydrotetracycline was supplied in the growth medium at a final concentration of 0.2% acetamide and 10 to 100 ng/ml anhydrotetracycline (19,20). Growth was followed by monitoring absorbance at 600 nm.…”

“…Wild-type (WT) and recombinant M. tuberculosis strains were grown for various periods of time with shaking and were then harvested by centrifugation, washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde, and stored at 4°C until further use. GFP-CrgA, enhanced cyan fluorescent protein (ECFP)-CrgA, GFP-PBPB, and GFP-PBPA localization could not be visualized in M. tuberculosis due to fluorescence bleaching, which was caused by the paraformaldehyde fixation required for this virulent strain (20). Therefore, all the cellular localization experiments were carried out in the nonpathogenic M. smegmatis mc 2 155 strain.…”

“…Therefore, all the cellular localization experiments were carried out in the nonpathogenic M. smegmatis mc 2 155 strain. Cell division proteins in various mycobacterial species have shown a high degree of homology, and M. smegmatis has therefore served as a favorite surrogate host for cell division protein localization studies (6,14,19,20,33). Bocillin-FL, a fluorescent derivative of penicillin V, was used to visualize the overall localization of penicillin-binding proteins in M. smegmatis strains expressing altered CrgA levels.…”

“…(i) BACTH assay. A BACTH system kit (Euromedex) was used to investigate CrgA interactions with other cell division proteins as previously described (20). Escherichia coli BTH101 recombinants containing various combinations of plasmids (Table 1) were selected on LB agar supplemented with 40 g/ml 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside.…”

“…MciZ is a 40-amino-acid (aa) peptide involved in the inhibition of FtsZ ring assembly following sporulation in B. subtilis (29). ClpX, a part of the ClpXP protease complex, inhibits FtsZ polymerization and possibly helps to maintain cytoplasmic pools of unpolymerized FtsZ subunits (5,20,25,44,50). Finally, the Min system spatially regulates cell division by preventing FtsZ assembly at the cell poles (27).…”