Slow polymerization of Mycobacterium tuberculosis FtsZ

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90 degrees light scattering. The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg(2+) and GTP. The minimum concentration of FtsZ needed for polymerization is 3 microM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.     

Assembly dynamics of Mycobacterium tuberculosis FtsZ

We have investigated the assembly of FtsZ from Mycobacterium tuberculosis (MtbFtsZ). Electron microscopy confirmed the previous observation that MtbFtsZ assembled into long, two-stranded filaments at pH 6.5. However, we found that assembly at pH 7.2 or 7.7 produced predominantly short, one-stranded protofilaments, similar to those of Escherichia coli FtsZ (EcFtsZ). Near pH 7, which is close to the pH of M. tuberculosis cytoplasm, MtbFtsZ formed a mixture of single- and two-stranded filaments. We developed a fluorescence resonance energy transfer assay to measure the kinetics of initial assembly and the dynamic properties at steady state. Assembly of MtbFtsZ reached a plateau after 60-100 s, about 10 times slower than EcFtsZ. The initial assembly kinetics were similar at pH 6.5 and 7.7, despite the striking difference in the polymer structures. Both were fit with a cooperative assembly mechanism involving a weak dimer nucleus, similar to EcFtsZ but with slower kinetics. Subunit turnover and GTPase at steady state were also about 10 times slower for MtbFtsZ than for EcFtsZ. Specifically, the half-time for subunit turnover in vitro at pH 7.7 was 42 s for MtbFtsZ compared with 5.5 s for EcFtsZ. Photobleaching studies in vivo showed a range of turnover half-times with an average of 25 s for MtbFtsZ as compared with 9 s for EcFtsZ.     

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.     

Interaction between cell division proteins FtsE and FtsZ

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.     

Interaction between FtsZ and inhibitors of cell division.

The interaction between inhibitors of cell division and FtsZ were assessed by using the yeast two-hybrid system. An interaction was observed between FtsZ and SulA, a component of the SOS response, and the interacting regions were mapped to their conserved domains. This interaction was reduced by mutations in sulA and by most mutations in ftsZ that make cell refractory to sulA. No interaction was detected between FtsZ and MinCD, an inhibitory component of the site selection system. However, interactions were observed among various members of the Min system, and MinE was found to reduce the interaction between MinC and MinD. The implications of these findings for cell division are discussed.     

Structure of Mycobacterium tuberculosis FtsZ reveals unexpected, G protein-like conformational switches

We report three crystal structures of the Mycobacterium tuberculosis cell division protein FtsZ, as the citrate, GDP, and GTPgammaS complexes, determined at 1.89, 2.60, and 2.08A resolution. MtbFtsZ crystallized as a tight, laterally oriented dimer distinct from the longitudinal polymer observed for alphabeta-tubulin. Mutational data on Escherichia coli FtsZ suggest that this dimer interface is important for proper protofilament and "Z-ring" assembly and function. An alpha-to-beta secondary structure conformational switch at the dimer interface is spatially analogous to, and has many of the hallmarks of, the Switch I conformational changes exhibited by G-proteins upon activation. The presence of a gamma-phosphate in the FtsZ active site modulates the conformation of the "tubulin" loop T3 (spatially analogous to the G-protein Switch II); T3 switching upon gamma-phosphate ligation is directly coupled to the alpha-to-beta switch by steric overlap. The dual conformational switches observed here for the first time in an FtsZ link GTP binding and hydrolysis to FtsZ (and tubulin) lateral assembly and Z-ring contraction, and they are suggestive of an underappreciated functional analogy between FtsZ, tubulin and G-proteins.     

Interaction between FtsW and penicillin-binding protein 3 (PBP3) directs PBP3 to mid-cell, controls cell septation and mediates the formation of a trimeric complex involving FtsZ, FtsW and PBP3 in mycobacteria

In bacteria, biogenesis of cell wall at the division site requires penicillin-binding protein 3 (PBP3) (or Ftsl). Using pull-down, bacterial two-hybrid, and peptide-based interaction assays, we provide evidence that FtsW of Mycobacterium tuberculosis (FtsWMTB) interacts with PBP3 through two extracytoplasmic loops. Pro306 in the larger loop and Pro386 in the smaller loop of FtsW are crucial for these interactions. Fluorescence microscopy shows that conditional silencing of ftsW in Mycobacterium smegmatis prevents cell septation and positioning of PBP3 at mid-cell. Pull-down assays and conditional depletion of FtsW in M. smegmatis provide evidence that FtsZ, FtsW and PBP3 of mycobacteria are capable of forming a ternary complex, with FtsW acting as a bridging molecule. Bacterial three-hybrid analysis suggests that in M. tuberculosis, the interaction (unique to mycobacteria) of FtsZ with the cytosolic C-tail of FtsW strengthens the interaction of FtsW with PBP3. ftsW of M. smegmatis could be replaced by ftsW of M. tuberculosis. FtsWMTB could support formation of the FtsZ-FtsW-PBP3 ternary complex in M. smegmatis. Our findings raise the possibility that in the genus Mycobacterium binding of FtsZ to the C-tail of FtsW may modulate its interactions with PBP3, thereby potentially regulating septal peptidoglycan biogenesis.     

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.     

In vitro polymerization of Mycobacterium leprae FtsZ OR Mycobacterium tuberculosis FtsZ is revived or abolished, respectively, by reciprocal mutation of a single residue

A single residue that dramatically influences polymerization of principal cell division protein FtsZ of Mycobacterium leprae (MlFtsZ) and Mycobacterium tuberculosis (MtFtsZ) has been identified. Soluble, recombinant MlFtsZ did not show polymerization in vitro, in contrast to MtFtsZ, which polymerised. Mutation of the lone non-conserved residue T172 in the N-terminal domain of MlFtsZ to A172, as it exists in MtFtsZ, showed dramatic polymerization of MlFtsZ-T172A in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in MlFtsZ, abolished polymerization of MtFtsZ-A172T in vitro. While T172A mutation enhanced weak GTPase activity of MlFtsZ, reciprocal A172T mutation marginally reduced GTPase activity of MtFtsZ in vitro. These observations demonstrate that the residue at position 172 plays critical role in the polymerization of MlFtsZ and MtFtsZ. A possible evolutionary correlation between the presence of polymerization-adversive or polymerization-favouring residue at position 172 in FtsZ and generation time of the respective bacterium are discussed.     

Cysteine 155 plays an important role in the assembly of Mycobacterium tuberculosis FtsZ

The assembly of FtsZ plays an important role in bacterial cell division. Mycobacterium tuberculosis FtsZ (MtbFtsZ) has a single cysteine residue at position 155. We have investigated the role of the lone cysteine residue in the assembly of MtbFtsZ using different complimentary approaches, namely chemical modification by a thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or a cysteine-chelating agent HgCl(2), and site-directed mutagenesis of the cysteine residue. HgCl(2) strongly reduced the polymerized mass of MtbFtsZ while it had no detectable effect on the polymerization of Escherichia coli FtsZ, which lacks a cysteine residue. HgCl(2) inhibited the protofilamentous assembly of MtbFtsZ and induced the aggregation of the protein. Further, HgCl(2) perturbed the secondary structure of MtbFtsZ and increased the binding of a hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) with MtbFtsZ, indicating that the binding of HgCl(2) altered the conformation of MtbFtsZ. Chemical modification of MtbFtsZ by DTNB also decreased the polymerized mass of MtbFtsZ. Further, the mutagenesis of Cys-155 to alanine caused a strong reduction in the assembly of MtbFtsZ. Under assembly conditions, the mutated protein formed aggregates instead of protofilaments. Far-UV CD spectroscopy and ANS binding suggested that the mutated MtbFtsZ has different conformation than that of the native MtbFtsZ. The effect of the mutation or chemical modification of Cys-155 on the MtbFtsZ assembly has been explained considering its location in the MtbFtsZ crystal structure. The results together suggest that the cysteine residue (Cys-155) of MtbFtsZ plays an important role in the assembly of MtbFtsZ into protofilaments.     

牛分支杆菌与肺结核分支杆菌基因组的比较

通过比较基因组学的方法研究发现,牛分支杆菌与肺结核杆菌基因组的同源性为99．95％,但在牛分枝杆菌基因组中有11个缺失区,大小从1kb到12．7kb,遗传信息的缺失引起牛分枝杆菌的基因组减小;牛分枝杆菌与肺结核分枝杆菌H37Rv间存在着2437个单核苷酸多态性（SNPs）,与肺结核分枝杆菌CDC1551间存在着2423个单核苷酸多态性（SNPs）,牛分支杆菌与肺结核分枝杆菌在编码细胞壁和分泌蛋白上变异程度也是巨大的。研究结果揭示了牛分支杆菌与肺结核分枝杆菌的遗传关系,为研究分支杆菌疫苗和诊断试剂提供理论依据,对牛肺结核病的防治有着非常重要的意义。     

结核分枝杆菌与巨噬细胞相互作用的研究进展

结核分枝杆菌是一种胞内感染菌,巨噬细胞是其寄生场所。结核分枝杆菌通过阻止吞噬溶酶体的融合、减少巨噬细胞凋亡、降低巨噬细胞对刺激应答的敏感性等途径逃避巨噬细胞的免疫监视和攻击,并在细胞内存活、增殖;而巨噬细胞又是抗菌免疫的主要效应细胞,通过直接杀伤和分泌多种细胞因子,对结核分枝杆菌具有免疫调节、呈递抗原等作用。深入研究结核分枝杆菌对巨噬细胞的免疫逃逸机制及巨噬细胞抗结核免疫作用,对研究宿主抗结核免疫机制及设计新型结核病疫苗有重要意义。     

Interaction of Mycobacterium tuberculosis CYP130 with Heterocyclic Arylamines

The Mycobacterium tuberculosis P450 enzymes are of interest for their pharmacological development potential, as evidenced by their susceptibility to inhibition by antifungal azole drugs that normally target sterol 14α-demethylase (CYP51). Although antifungal azoles show promise, direct screening of compounds against M. tuberculosis P450 enzymes may identify novel, more potent, and selective inhibitory scaffolds. Here we report that CYP130 from M. tuberculosis has a natural propensity to bind primary arylamines with particular chemical architectures. These compounds were identified via a high throughput screen of CYP130 with a library of synthetic organic molecules. As revealed by subsequent x-ray structure analysis, selected compounds bind in the active site by Fe-coordination and hydrogen bonding of the arylamine group to the carbonyl oxygen of Gly²⁴³. As evidenced by the binding of structural analogs, the primary arylamine group is indispensable, but synergism due to hydrophobic contacts between the rest of the molecule and protein amino acid residues is responsible for a binding affinity comparable with that of the antifungal azole drugs. The topology of the CYP130 active site favors angular coordination of the arylamine group over the orthogonal coordination of azoles. Upon substitution of Gly²⁴³ by an alanine, the binding mode of azoles and some arylamines reverted from type II to type I because of hydrophobic and steric interactions with the alanine side chain. We suggest a role for the conserved Ala(Gly)²⁴³-Gly²⁴⁴ motif in the I-helix in modulating both the binding affinity of the axial water ligand and the ligand selectivity of cytochrome P450 enzymes.CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 (P450, CYP)² enzymes and is one of three (CYP51, CYP121, and CYP130) that have been studied as individually expressed proteins at the structural level. Evidence has accumulated for the importance of M. tuberculosis P450 enzymes in virulence (CYP132) (1), host infection (CYP125) (2), and pathogen viability (CYP128, CYP121) (3, 4), although neither their exact biological functions nor any of the endogenous substrates upon which these enzymes operate have yet been established. However, it has recently been shown in vitro that CYP121 catalyzes a C–C coupling reaction between two tyrosine groups (5). CYP130 is absent from the genome of Mycobacterium bovis, suggesting that it might play specific role(s) in the infection of the human host and thus constitute a potential therapeutic target.The potential of M. tuberculosis P450 enzymes for pharmacologic development was initially suggested by their susceptibility to inhibition by antifungal azole drugs such as fluconazole, econazole, and clotrimazole. These drugs block sterol 14α-demethylase CYP51 in fungi (6), tightly bind to M. tuberculosis P450 proteins (7, 8), and display inhibitory potential against latent and multidrug-resistant forms of tuberculosis both in vitro and in tuberculosis-infected mice (9–14).The substantial differences between fungal CYP51 and the potential P450 targets in microbial pathogens, including M. tuberculosis, suggest that the direct screening of compounds against M. tuberculosis CYP enzymes could identify novel inhibitory scaffolds that are more potent and selective than antifungal drugs. Structurally characterized screening targets are advantageous, as the already defined purification and crystallization protocols can be applied to obtain co-crystal structures and to elucidate the binding modes of screening hits. This approach has been successfully applied to CYP51, resulting in identification of novel inhibitory scaffolds for CYP51 therapeutic targets (15, 16).Toward this goal, the property of P450 enzymes to shift the ferric heme iron Soret band on ligand binding (17) provides an experimental platform for high throughput screening of compound libraries to select chemotypes with high binding affinities for the target. Expulsion of the heme iron axial water ligand from the Fe-coordination sphere by the incoming substrate followed by transition of the ferric heme from the low-spin hexacoordinated to the high-spin pentacoordinated state characterize type I spectral shifts and are a prerequisite for P450 catalytic activity. Replacement of a weak axial ligand, the water molecule, with a stronger one possessing a nitrogen-containing aliphatic or aromatic group coordinating to the heme iron characterizes type II spectral shifts.To find new high affinity ligands of CYP130, a commercial library of 20,000 small organic molecules comprising a large selection of molecular scaffolds was screened against the enzyme. In contrast to the results with CYP51, no type I binding hits were identified. Screening produced about a dozen structurally diverse type II hits that were unexpectedly devoid of the usual aromatic nitrogen atoms readily accessible for axial coordination of the heme iron, suggesting an alternative coordination mode. High resolution x-ray structure analysis determined that two compounds coordinated to the heme iron via a primary arylamine group, providing the first structural evidence on P450-heterocyclic arylamine interactions.     

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non‐synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species‐specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia‐related genes between M. bovis and M. tuberculosis.     

Novel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division

The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, 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. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish 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 showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress.     

结核分枝杆菌与巨噬细胞相互作用的研究进展

结核分枝杆菌（Mycobacterium tuberculosis,MTB）是一种典型的胞内致病菌,巨噬细胞是MTB在体内的主要宿主细胞。巨噬细胞具有强大的吞噬功能,在机体固有免疫和适应性免疫中均发挥着重要作用,可有效保护宿主免受结核分枝杆菌的感染。MTB在与宿主巨噬细胞的长期相互作用过程中,逐渐形成多种逃避杀灭的有效策略,得以在宿主体内存活并增殖。该文从巨噬细胞抗MTB感染及MTB逃避巨噬细胞杀灭两个方面综述国内外的研究进展。     

结核分枝杆菌FtsZ的异源表达及酶学性质研究

作为细菌分裂所必需的微管蛋白类似物,丝状温度敏感蛋白Z(filamentous temperature-sensitive protein Z,FtsZ)被认为是一个具有潜力的药物作用新靶点。为了构建高纯度的FtsZ重组蛋白分离纯化体系,探讨其酶学性质,该研究利用大肠杆菌BL21异源表达结核分枝杆菌FtsZ重组蛋白,通过Ni亲和层析柱和G-50层析柱纯化目的蛋白,采用孔雀石绿法和90°光散射法测定FtsZ重组蛋白的GTP酶活和蛋白聚集。研究结果表明:成功获得具有生物学活性的结核分枝杆菌FtsZ重组蛋白,其相对分子质量约为49kD;该酶最适反应温度为37℃,最适pH为6.8,金属离子Mg~(2+)和K~+对FtsZ重组蛋白酶活具有促进作用,有机溶剂DMSO和TritonX-100的体积分数分别高于0.1%和0.005%时对酶活有显著抑制作用(P0.05)。此外,FtsZ重组蛋白在加入底物GTP诱导后,快速聚集。本实验利用基因工程技术成功获得具有生物活性的FtsZ重组蛋白,并明确了该蛋白质的酶学性质,为其进一步的研究和应用奠定了基础。