Murty V. V. S. Madiraju

Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulator

Paired two‐component regulatory systems consisting of a sensor kinase and a response regulator are the major means by which bacteria sense and respond to different stimuli. The role of essential response regulator, MtrA, in Mycobacterium tuberculosis proliferation is unknown. We showed that elevating the intracellular levels of MtrA prevented M. tuberculosis from multiplying in macrophages, mice lungs and spleens, but did not affect its growth in broth. Intracellular trafficking analysis revealed that a vast majority of MtrA overproducing merodiploids were associated with lysosomal associated membrane protein (LAMP‐1) positive vacuoles, indicating that intracellular growth attenuation is, in part, due to an impaired ability to block phagosome–lysosome fusion. A merodiploid strain producing elevated levels of phosphorylation‐defective MtrA (MtrAD53N) was partially replicative in macrophages, but was attenuated in mice. Quantitative real‐time PCR analyses revealed that expression of dnaA, an essential replication gene, was sharply upregulated during intramacrophage growth in the MtrA overproducer in a phosphorylation‐dependent manner. Chromatin immunoprecipitation using anti‐MtrA antibodies provided direct evidence that MtrA regulator binds to dnaA promoter in vivo indicating that dnaA promoter is a MtrA target. Simultaneous overexpression of mtrA regulator and its cognate mtrB kinase neither inhibited growth nor sharply increased the expression levels of dnaA in macrophages. We propose that proliferation of M. tuberculosis in vivo depends, in part, on the optimal ratio of phosphorylated to non‐phosphorylated MtrA response regulator.

Mycobacterium tuberculosis Cells Growing in Macrophages Are Filamentous and Deficient in FtsZ Rings

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.

Interference of Mycobacterium tuberculosis cell division by Rv2719c, a cell wall hydrolase

The genetic factors responsible for the regulation of cell division in Mycobacterium tuberculosis are largely unknown. We showed that exposure of M. tuberculosis to DNA damaging agents, or to cephalexin, or growth of M. tuberculosis in macrophages increased cell length and sharply elevated the expression of Rv2719c, a LexA‐controlled gene. Overexpression of Rv2719c in the absence of DNA damage or of antibiotic treatment also led to filamentation and reduction in viability both in broth and in macrophages indicating a correlation between Rv2719c levels and cell division. Overproduction of Rv2719c compromised midcell localization of FtsZ rings, but had no effect on the intracellular levels of FtsZ. In vitro, the Rv2719c protein did not interfere with the GTP‐dependent polymerization activity of FtsZ indicating that the effects of Rv2719c on Z‐ring assembly are indirect. Rv2719c protein exhibited mycobacterial murein hydrolase activity that was localized to the N‐terminal 110 amino acids. Visualization of nascent peptidoglycan (PG) synthesis zones by probing with fluoresceinated vancomycin (Van‐FL) and localization of green fluorescent protein‐Rv2719c fusion suggested that the Rv2719c activity is targeted to potential PG synthesis zones. We propose that Rv2719c is a potential regulator of M. tuberculosis cell division and that its levels, and possibly activities, are modulated under a variety of growth conditions including growth in vivo and during DNA damage, so that the assembly of FtsZ‐rings, and therefore the cell division, can proceed in a regulated manner.

The Two-Domain LysX Protein of Mycobacterium tuberculosis Is Required for Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides

The well-recognized phospholipids (PLs) of Mycobacterium tuberculosis (Mtb) include several acidic species such as phosphatidylglycerol (PG), cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG), is a component of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF)-lysyl-tRNA synthetase (lysU) protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows increased association with lysosome-associated membrane protein–positive vesicles, and it exhibits altered membrane potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and for survival of the pathogen upon infection.

Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts

The ftsZ gene of Mycobacterium tuberculosis H37Rv has been characterized as the first step in determining the molecular events involved in the cell division process in mycobacteria. Western analysis revealed that intracellular levels of FtsZ are growth phase dependent in both M. tuberculosis and Mycobacterium smegmatis. Unregulated expression of M. tuberculosis ftsZ from constitutive hsp60 and dnaA promoters in M. tuberculosis hosts resulted in lethality whereas expression from only the hsp60 promoter was toxic in M. smegmatis hosts. Expression of ftsZ from the dnaA promoter in M. smegmatis resulted in approximately sixfold overproduction and the merodiploids exhibited slow growth, an increased tendency to clump and filament, and in some cases produced buds and branches. Many of the cells also contained abnormal and multiple septa. Expression of ftsZ from the chemically inducible acetamidase promoter in M. smegmatis hosts resulted in approximately 22-fold overproduction of FtsZ and produced filamentous cells, many of which lacked any visible septa. Visualization of the M. tuberculosis FtsZ tagged with green fluorescent protein in M. smegmatis by fluorescence microscopy revealed multiple fluorescent FtsZ foci, suggesting that steps subsequent to the formation of organized FtsZ structures but prior to septum formation are blocked in FtsZ-overproducing cells. Together these results suggest that the intracellular concentration of FtsZ protein is critical for productive septum formation in mycobacteria.

Conditional expression of Mycobacterium smegmatis dnaA, an essential DNA replication gene

To begin to understand the role of Mycobacterium smegmatis dnaA in DNA replication, the dnaA gene was characterized at the genetic level. Western analyses revealed that DnaA accounts for approximately 0.18% of the total cellular protein during both the active and stationary growth periods. Expression of antisense dnaA RNA reduced viability, indicating that dnaA is an essential gene in replication. To further understand the role(s) of dnaA in replication, a conditionally expressing strain was constructed in which expression of dnaA was controlled by acetamide. Growth in the presence of 0.2% acetamide elevated the intracellular levels of DnaA and increased cell length, but did not affect viability. Visualization of DNA by fluorescence microscopy revealed that DnaA-overproducing cells were multinucleoidal, indicating a loss of synchrony between the replication and cell-division cycles. Withdrawal of acetamide resulted in the depletion of the intracellular levels of DnaA, reduced viability and gradually blocked DNA synthesis. Acetamide-starved cells were very filamentous, several times the size of the parent cells and showed either abnormal or multi-nucleoid morphology, indicating a blockage in cell-division events. The addition of acetamide to the starved cells restored their viability and shortened the lengths of their filaments back to the size of the parent cells. Thus, both increasing and decreasing the levels of DnaA have an effect on the cells, indicating that the level of DnaA is critical to the maintenance of coordination between DNA replication and cell division. It is concluded that DNA replication and cell-division processes in M. smegmatis are linked, and it is proposed that DnaA has a role in both of these processes.

recO and recR mutations delay induction of the SOS response in Escherichia coli

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage; therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of β-galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.

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.

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

The role(s) in cell division of the Mycobacterium tuberculosis Rv0011c gene product, a homolog of the Streptomyces CrgA protein that is responsible for coordinating growth and cytokinesis in sporogenic aerial hyphae, is largely unknown. We show that an enhanced cyan fluorescent protein-M. tuberculosis CrgA (ECFP-CrgA(MT)) fusion protein is localized to the cell membrane, midcell, and cell pole regions in Mycobacterium smegmatis. Furthermore, the ECFP-CrgA(MT) fusion protein colocalized with FtsZ-enhanced yellow fluorescent protein (EYFP) in M. smegmatis. Bacterial two-hybrid assays indicated strong interactions of M. tuberculosis CrgA with FtsZ, FtsQ, and the class B penicillin-binding proteins, FtsI (PBPB) and PBPA. The midcell localization of CrgA(MT) was severely compromised under conditions of FtsZ depletion, which indicated that CrgA localizes to the midcell region after assembly of the FtsZ ring. M. tuberculosis cells with reduced CrgA levels were elongated and grew more slowly than wild-type cells, which indicated defects in cell division, whereas CrgA overproduction did not show growth defects. A M. smegmatis ΔcrgA strain exhibited a bulged cell morphology, elongated cells with a chain-like phenotype, cells with polar bulbous structures, and a modest growth defect. FtsZ and FtsI levels were not affected in cells producing altered levels of CrgA. Septal and membrane localization of GFP-FtsI was enhanced by CrgA overproduction and was diminished in a ΔcrgA strain, which indicates that one role of CrgA is to promote and/or stabilize FtsI localization. Overall, these data indicate that CrgA is a novel member of the cell division complex in mycobacteria and possibly facilitates septum formation.

Mycobacterium tuberculosis CwsA interacts with CrgA and Wag31, and the CrgA-CwsA complex is involved in peptidoglycan synthesis and cell shape determination.

Bacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein from Mycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog in M. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [(14)C]N-acetylglucosamine cell wall precursor. The M. smegmatis MSMEG_0023 crgA double mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA for cell wall synthesis and cell shape protein A, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.