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Background: Tannins are the polyphenolic group of plant compounds having strong antimicrobial potential. Research on human pathogens using silver nanoparticles for antimicrobial purposes has opened up new possibilities in nanomedicine. Objective: The present study was concerned with combining the effectiveness of the two by the green synthesis of silver nanoparticles with plant tannin. Methods: Synthesis of silver nanoparticles was done based on the tannin content of the selected plants. Characterization of the synthesized nanoparticles has been performed through UV–VIS, FTIR spectroscopy, Zeta potential, XRD analysis and FEGSEM imaging. The antibacterial potentiality of the nanoparticles was checked against two most susceptible bacteria Staphylococcus epidermidis and Salmonella typhi. Results: Among the studied plants, Phyllanthus emblica showed highest tannin content and best bactericidal properties. The nanoparticles synthesized with P. emblica showed the highest zone of inhibition against the studied bacteria. An in-silico comparative molecular docking study of the bioactive compounds from the selected plants was performed against the FtsZ protein of S. epidermidis and YfdX protein of S. typhi. Three compounds namely Isocorilagin,1(β), 6-di-o-galloylglucose and Hamamelitannin appeared as the best inhibitors of the said proteins and among them, Isocorilagin, a natural tannin showed the best docking score of -10.2 Kcal/mol with FtsZ protein. Further molecular dynamics simulation studies of the FtsZ protein-Isocorilagin complex support its stability indicating Isocorilagin as the natural inhibitor of the pathogenic bacterial protein FtsZ. Conclusion: These results concluded that synthesis of nanoparticles with plant tannin is a cost effective green approach and the synthesised nanoparticles appear as efficient antimicrobial agent.

Microbial morphology engineering is a novel approach for cell factory to improve the titer of target product in bio-manufacture. Hyaluronic acid (HA), a valuable glycosaminoglycan polymerized by HA synthase (HAS), a membrane protein, is particularly selected as the model product to improve its single-cell HA-producing capacity via morphology engineering. DivIVA and FtsZ, the cell-elongation and cell division related protein, respectively, were both down/up dual regulated in C. glutamicum via weak promoter substitution or plasmid overexpression. Different from the natural short-rod shape, varied morphologies of engineered cells, i.e. small-ellipsoid-like (DivIVA-reduced), bulb-like (DivIVA-enhanced), long-rod (FtsZ-reduced) and dumbbell-like (FtsZ-enhanced), were observed. Applying these morphology-changed cells as hosts for HA production, the reduced expression of both DivIVA and FtsZ seriously inhibited normal cell growth; meanwhile, overexpression of DivIVA didn't show morphology changes, but overexpression of FtsZ surprisingly change the cell-shape into long and thick rod with remarkably enlarged single-cell surface area (more than 5.2-fold-increase). And finally, the single-cell HA-producing capacity of the FtsZ-overexpressed C. glutamicum was immensely improved by 13.5-folds. Flow cytometry analyses verified that the single-cell HAS amount on membrane was enhanced by 2.1 folds. This work is pretty valuable for high titer synthesis of diverse metabolic products with microbial cell factory.

The appearance of drug-resistant strains of Mycobacterium tuberculosis and the dramatic increase in infection rates worldwide evidences the urgency of developing new and effective compounds for treating tuberculosis. Benzimidazoles represent one possible source of new compounds given that antimycobacterial activity has already been documented for some derivatives, such as those bearing electron-withdrawing groups. The aim of this study was to synthesize two series of benzimidazoles, di- and trisubstituted derivatives, and evaluate their antimycobacterial activity. Accordingly, 5a and 5b were synthesized from hydroxymoyl halides 3a and 3b, and nitro-substituted o-phenylenediamine 4. Compound 11 was synthesized from an aromatic nitro compound, 4-chloro-1,2-phenylenediamine 9, mixed with 3-nitrobenzaldehyde 10, and bentonite clay. Although the synthesis of 11 has already been reported, its antimycobacterial activity is herein examined for the first time. 1,2,5-trisubstituted benzimidazoles 7a, 7b, and 12 were obtained from N-alkylation of 5a, 5b, and 11. All benzimidazole derivatives were characterized by FT-IR, NMR, and HR-MS, and then screened for their in vitro antimycobacterial effect against the M. tuberculosis H37Rv strain. The N-alkylated molecules (7a, 7b, and 12) generated very limited in vitro inhibition of mycobacterial growth. The benzimidazoles (5a, 5b, and 11) showed in vitro potency against mycobacteria, reflected in minimal inhibitory concentration (MIC) values in the range of 6.25–25 μg/mL. Consequently, only the 2,5-disubstituted benzimidazoles were assessed for biological activity on mouse macrophages infected with M. tuberculosis. A good effect was found for the three compounds. The cytotoxicity assay revealed very low toxicity for all the test compounds against the macrophage cell line. According to the docking study, 2,5-disubstituted benzimidazoles exhibit high affinity for an interdomain cleft that plays a key role in the GTP-dependent polymerization of the filamentous temperature-sensitive Z (FtsZ) protein. The ability of different benzimidazoles to impede FtsZ polymerization is reportedly related to their antimycobacterial activity. On the other hand, the 1,2,5-trisubstituted benzimidazoles docked to the N-terminal of the protein, close to the GTP binding domain, and did not show strong binding energies. Overall, 5a, 5b, and 11 proved to be good candidates for in vivo testing to determine their potential for treating tuberculosis.

The overuse of antibiotics in recent years has resulted in a development of antibiotic-resistant bacteria. There are two potential solutions to overcome this problem. New drugs can be developed, but this takes considerable time and money. Old drugs can also be remade and reused, and bacteria may have a harder time becoming resistant. The energy of interaction between protein FtsZ (found in Caulobacter crescentus bacteria) and 7,409 Food and Drug Administration (FDA) approved drugs was calculated using a Python code, where negative values represented high energy of interaction and positive values, low energy of interaction. The drugs that had the highest energy of interaction are recommended for further experimentation. [ABSTRACT FROM AUTHOR]

As one of the key elements in bacterial cell division, the cytoskeletal protein FtsZ appears to be highly involved in circumferential treadmilling along the inner membrane, yielding circular vortices when transferred to flat membranes. However, it remains unclear how a membrane-targeted protein can produce these dynamics. Here, we dissect the roles of membrane binding, GTPase activity, and the unstructured C-terminal linker on the treadmilling of a chimera FtsZ protein through in vitro reconstitution of different FtsZ-YFP-mts variants on supported membranes. In summary, our results suggest substantial robustness of dynamic vortex formation, where only significant mutations, resulting in abolished membrane binding or compromised lateral interactions, are detrimental for the generation of treadmilling rings. In addition to GTPase activity, which directly affects treadmilling dynamics, we found a striking correlation of membrane binding with treadmilling speed as a result of changing the MTS on our chimera proteins. This discovery leads to the hypothesis that the in vivo existence of two alternative tether proteins for FtsZ could be a mechanism for controlling FtsZ treadmilling. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a well-known pathogen for its rapid development of multi-drug antibiotic resistance. This pathogen is responsible for numerous human diseases, particularly affecting immunocompromised and elderly patients. Hence, discovering novel therapeutics has become necessary in the fight against antimicrobial resistance. This study is focused on evaluating the potential inhibitory activity of eleven phytocompounds from Azadirachta indica against the nucleotide-binding site of the FtsZ protein of P. aeruginosa through a cheminformatics approach. FtsZ is an indispensable and highly conserved protein in prokaryotic cell division. Docking studies revealed favourable binding energies (ΔG= - 8.3 to - 5.4 kcal/mol) for all selected phytoconstituents. Finally, we selected Nimbiol (CID 11119228), as a lead compound, exhibiting a binding energy (ΔG= -7.8 kcal/mol) for the target. Based on our findings, Nimbiol shows potential as an anti-FtsZ compound, making it a promising candidate for further in vitro and in vivo investigations to assess its antimicrobial activity.

Addressing the growing problem of antibiotic resistance requires the development of new drugs with novel antibacterial targets. FtsZ has been identified as an appealing new target for antibacterial agents. Here, we describe the structure-guided design of a new fluorescent probe (BOFP) in which a BODIPY fluorophore has been conjugated to an oxazole-benzamide FtsZ inhibitor. Crystallographic studies have enabled us to identify the optimal position for tethering the fluorophore that facilitates the high-affinity FtsZ binding of BOFP. Fluorescence anisotropy studies demonstrate that BOFP binds the FtsZ proteins from the Gram-positive pathogens Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae with Kd values of 0.6–4.6 µM. Significantly, BOFP binds the FtsZ proteins from the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii with an even higher affinity (Kd = 0.2–0.8 µM). Fluorescence microscopy studies reveal that BOFP can effectively label FtsZ in all the above Gram-positive and Gram-negative pathogens. In addition, BOFP is effective at monitoring the impact of non-fluorescent inhibitors on FtsZ localization in these target pathogens. Viewed as a whole, our results highlight the utility of BOFP as a powerful tool for identifying new broad-spectrum FtsZ inhibitors and understanding their mechanisms of action. [ABSTRACT FROM AUTHOR]

During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner. The Z-ring, constituted of the tubulin homolog FtsZ protein, plays an essential role for bacterial cell division. Here the authors use an in vitro reconstitution approach to determine how the regulatory protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A new 8,8ˈ-binaphthopyranone (mycopyranone, 1) was discovered and elucidated. • Absolute configuration was elucidated via NOESY, Mosher's esters, and ECD data. • Active in vitro vs a clinically-relevant methicillin-resistant S. aureus strain. • Molecular docking studies suggests binding to the FtsZ (tubulin-like) protein. Abstract A new 8,8ˈ-binaphthopyranone (mycopyranone, 1) was isolated from a solid fermentation of Phialemoniopsis sp. (fungal strain MSX61662), and the structure was elucidated via analysis of the NMR and HRESIMS data. The axial chirality of 1 was determined to be M by ECD. The central chirality at C-4/C-4ˈ was assigned through a modified Mosher's method, while the absolute configuration at C-3/C-3ˈ was deduced based on analysis of the 3 J H-3-H-4 values and NOESY correlations. Compound 1 was evaluated for its antimicrobial properties against Staphylococcus aureus SA1199 and a clinically relevant methicillin-resistant S. aureus strain (MRSA USA300 LAC strain AH1263). Compound 1 inhibited the growth of both strains in a concentration dependent manner with IC 50 values in the low μM range. Molecular docking indicated that compound 1 binds to the FtsZ (tubulin-like) protein in the same pocket as viriditoxin (2), suggesting that 1 targets bacterial cell division. [ABSTRACT FROM AUTHOR]

Abstract The development of multi-drug and extensively-drug resistant strains of Mycobacterium tuberculosis (Mtb) have encouraged to develop new anti-TB agents with a unique mechanism of action. FtsZ , an essential protein of bacterial cytokinesis, has emerged as a validated target for antibacterial therapy. This study describes the utility of benzimidazoles (BI) derivatives as potential FtsZ inhibitors. Through 2/3D QSAR (MLR) molecular modeling, new BI-inhibitors with improved activity have been designed. Statistically significant models from 2D-QSAR (r2 = 0.90, q2 = 85 and pred_r2 = 0.60) indicated that the descriptors such as SaaOcount, –ve Potential Surface Area, and SdsCHE-index modulate the activity of a molecule. Similarly, 3D-QSAR (r2 = 0.78, q2 = 0.69 and pred_r2 = 0.59) results indicated both electrostatic and steric descriptors play a pivotal role. These indications prompted us to design 223 new BI-derivatives, out of which three BI-derivatives (D-83 , D-116 and D-223) have shown some promising results. [ABSTRACT FROM AUTHOR]

The emergence of multidrug-resistant bacteria and the slow development of new antibacterial agents have led to a growing global health crisis. Here, we identified an antibacterial agent possessing 1-methyl-2,5-diphenylpyridin-1-ium core, MA220607 , with a dual-targeting mechanism of action (MOA), which exhibited effective killing activity against both Gram-positive (MIC = 0.062–2 μg/mL) and Gram-negative bacteria (MIC = 0.5–4 μg/mL). Moreover, our study revealed that MA220607 could block the formation of bacterial biofilm, which might be the reason for low frequency of resistance. MOA studies showed that MA220607 not only promoted FtsZ protein polymerization, but also increased the permeability of bacterial membranes and altered their proton gradients. In addition, MA220607 had low hemolytic toxicity and could significantly inhibit the growth of bacteria in mice. Molecular dynamics simulations demonstrated that MA220607 could block the transition from the tense (T) to relaxed (R) state of FtsZ protein, thereby perturbing the stepping mechanism of FtsZ protein. Overall, our findings suggest that integrating the dual mechanisms targeting FtsZ protein and cell membranes of bacteria into a single scaffold represents a promising direction for the development of new antibacterial agents. [Display omitted] • 1-Methyl-2,5-diphenylpyridin-1-ium derivatives were designed and synthesized. • MA220607 is a potential antibacterial agent with a dual-targeting mechanism of action. • MA220607 has broad spectrum antibacterial effect. [ABSTRACT FROM AUTHOR]

The biological activities of berberine, a natural plant molecule, are known to be affected by structural modifications, mostly at position 9 and/or 13. A series of new 13-substituted berberine derivatives were synthesized and evaluated in term of antimicrobial activity using various microorganisms associated to human diseases. Contrarily to the original molecule berberine, several derivatives were found strongly active in microbial sensitivity tests against Mycobacterium, Candida albicans and Gram-positive bacteria, including naïve or resistant Bacillus cereus, Staphylococcus aureus and Streptococcus pyogenes with minimal inhibitory concentration (MIC) of 3.12 to 6.25 µM. Among the various Gram-negative strains tested, berberine’s derivatives were only found active on Helicobacter pylori and Vibrio alginolyticus (MIC values of 1.5–3.12 µM). Cytotoxicity assays performed on human cells showed that the antimicrobial berberine derivatives caused low toxicity resulting in good therapeutic index values. In addition, a mechanistic approach demonstrated that, contrarily to already known berberine derivatives causing either membrane permeabilization, DNA fragmentation or interacting with FtsZ protein, active derivatives described in this study act through inhibition of the synthesis of peptidoglycan or RNA. Overall, this study shows that these new berberine derivatives can be considered as potent and safe anti-bacterial agents active on human pathogenic microorganisms, including ones resistant to conventional antibiotics.

The bacterial cytoskeletal protein FtsZ binds and hydrolyzes GTP, self-aggregates into dynamic filaments and guides the assembly of the septal ring on the inner side of the membrane at midcell. This ring constricts the cell during division and is present in most bacteria. Despite exhaustive studies undertaken in the last 25 years after its discovery, we do not yet know the mechanism by which this GTP-dependent self-aggregating protein exerts force on the underlying membrane. This paper reviews recent experiments and theoretical models proposed to explain FtsZ filament dynamic assembly and force generation. It highlights how recent observations of single filaments on reconstituted model systems and computational modeling are contributing to develop new multiscale models that stress the importance of previously overlooked elements as monomer internal flexibility, filament twist and flexible anchoring to the cell membrane. These elements contribute to understand the rich behavior of these GTP consuming dynamic filaments on surfaces. The aim of this review is 2-fold: (1) to summarize recent multiscale models and their implications to understand the molecular mechanism of FtsZ assembly and force generation and (2) to update theoreticians with recent experimental results. [ABSTRACT FROM AUTHOR]

Summary: FtsZ is the key regulator of bacterial cell division. It initiates division by forming a dynamic ring‐like structure, the Z‐ring, at the mid‐cell. What triggers the formation of the Z‐ring during the cell cycle is poorly understood. In Escherichia coli, the common view is that FtsZ concentration is constant throughout its doubling time and therefore regulation of assembly is controlled by some yet‐to‐be‐identified protein‐protein interactions. Using a newly developed functional, fluorescent FtsZ reporter, we performed a quantitative analysis of the FtsZ concentration throughout the cell cycle under slow growth conditions. In contrast to the common expectation, we show that FtsZ concentrations vary in a cell cycle‐dependent manner, and that upregulation of FtsZ synthesis correlates with the formation of the Z‐ring. The first half of the cell cycle shows an approximately fourfold upregulation of FtsZ synthesis, followed by its rapid degradation by ClpXP protease in the last 10% of the cell cycle. The initiation of rapid degradation coincides with the dissociation of FtsZ from the septum. Altogether, our data suggest that the Z‐ring formation in slow growth conditions in E. coli is partially controlled by a regulatory sequence wherein upregulation of an essential cell cycle factor is followed by its degradation. We find that there is a cell cycle‐dependent regulation of FtsZ numbers in slow‐growing E. coli. There is an upregulation of FtsZ synthesis in the first half of the cell cycle, followed by its rapid degradation by the ClpXP protease in the last 10% of the cell cycle. The upregulation of FtsZ synthesis correlates with the formation of the Z‐ring and rapid degradation coincides with dissociation of FtsZ from the constriction. [ABSTRACT FROM AUTHOR]

Abstract The dominance of multi drug resistance in the clinically significant bacteria led to urgency in the development of new antibiotics with novel mechanism of action. Among the biochemical targets explored for selective toxicity, molecular mechanisms involving cell division remained focal point for novel antimicrobial drug discovery. For this purpose we have performed in-silico studies of FtsZ protein and obtained benzimidazolium compounds as potential hits. These molecules obtained in the dock results were synthesized via reacting benzimidazoles with appropriate benzyl halides. The structures of the synthesized compounds were confirmed by their 1H NMR, 13C NMR, IR and mass spectral data. These were evaluated for anti-microbial activity. Among the tested compounds B14, B15 and B20 have shown highest activity (MIC 5 μg/mL) against Staphylococcus aureus , Macrococcus caseolyticus, Escherichia coli and Pseudomonas aeruginosa.. Microscopic examination of drug-treated cultures of Staphylococcus aureus and Pseudomonas aeruginosa showed rod-shaped filamentous growth of the dividing cells, which is a characteristic feature of FtsZ inhibition. Graphical abstract Image 1 Highlights • Novel FtsZ enzyme inhibitors with benzimidazole nucleus were designed and then docking analysis was carried out. • Hit molecules were synthesized and structures were confirmed by IR, NMR and Mass spectra. • They are evaluated for antimicrobial activity and anti fungal activity. • Cell divison inhibitory assay was performed as a proof of concept that it acts on FtsZ protein. [ABSTRACT FROM AUTHOR]

New generation of antibacterial agents are urgently needed in order to fight the emergence of multidrug-resistant bacteria. FtsZ is currently identified as a promising target for new types of antimicrobial compounds development because of its conservative characteristics and its essential role played in bacterial cell division. In the present study, the antibacterial activity of a series of benzofuroquinolinium derivatives was investigated. The results show that the compounds possess potent antibacterial activity against drug resistant pathogens including MRSA, VREF and NDM-1 Escherichia coli. Biological studies reveal that the compound is an effective inhibitor that is able to suppress FtsZ polymerization and GTPase activity and thus stopping the cell division and causing cell death. More importantly, this series of compounds shows low cytotoxicity on mammalian cells and therefore they could be new chemotypes for the development of new antibacterial agents targeting the cell-division protein FtsZ. [ABSTRACT FROM AUTHOR]

FtsZ, the primary protein of the bacterial Z ring guiding cell division, has been recently shown to engage in intriguing treadmilling dynamics along the circumference of the division plane. When coreconstituted in vitro with FtsA, one of its natural membrane anchors, on flat supported membranes, these proteins assemble into dynamic chiral vortices compatible with treadmilling of curved polar filaments. Replacing FtsA by a membrane-targeting sequence (mts) to FtsZ, we have discovered conditions for the formation of dynamic rings, showing that the phenomenon is intrinsic to FtsZ. Ring formation is only observed for a narrow range of protein concentrations at the bilayer, which is highly modulated by free Mg2+ and depends upon guanosine triphosphate (GTP) hydrolysis. Interestingly, the direction of rotation can be reversed by switching the mts from the C-terminus to the N-terminus of the protein, implying that the filament attachment must have a perpendicular component to both curvature and polarity. Remarkably, this chirality switch concurs with previously shown inward or outward membrane deformations by the respective FtsZ mutants. Our results lead us to suggest an intrinsic helicity of FtsZ filaments with more than one direction of curvature, supporting earlier hypotheses and experimental evidence. [ABSTRACT FROM AUTHOR]

Bacterial cytokinesis begins with the assembly of FtsZ into a Z ring at the center of the cell. The Z-ring constriction in Gram-negative bacteria may occur in an environment where the periplasm and the cytoplasm are isoosmotic, but in Gram-positive bacteria the constriction may have to overcome a substantial turgor pressure. We address three potential sources of invagination force. (1) FtsZ itself may generate force by curved protofilaments bending the attached membrane. This is sufficient to constrict liposomes in vitro. However, this force is on the order of a few pN, and would not be enough to overcome turgor. (2) Cell wall (CW) synthesis may generate force by pushing the plasma membrane from the outside. However, this would probably require some kind of Brownian ratchet to separate the CW and membrane sufficiently to allow a glycan strand to slip in. The elastic element is not obvious. (3) Excess membrane production has the potential to contribute significantly to the invagination force. If the excess membrane is produced under the CW, it would force the membrane to bleb inward. We propose here that a combination of FtsZ pulling from the inside, and excess membrane pushing membrane inward may generate a substantial constriction force at the division site. This combined force generation mechanism may be sufficient to overcome turgor pressure. This would abolish the need for a Brownian ratchet for CW growth, and would permit CW to operate by reinforcing the constrictions generated by FtsZ and excess membrane. [ABSTRACT FROM AUTHOR]

Filamentous temperature-sensitive Z (FtsZ) protein plays essential role in bacteria cell division, and its inhibition prevents Mycobacteria reproduction. Here we adopted curcumin derivatives as candidates of novel inhibitors and investigated their specific interactions with FtsZ, using ab initio molecular simulations based on protein–ligand docking, classical molecular mechanics and ab initio fragment molecular orbital (FMO) calculations. Based on FMO calculations, we specified the most preferable site of curcumin binding to FtsZ and highlighted the key amino acid residues for curcumin binding at an electronic level. The result will be useful for proposing novel inhibitors against FtsZ based on curcumin derivatives. [ABSTRACT FROM AUTHOR]

FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples. [ABSTRACT FROM AUTHOR]

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100°C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1–2 μM) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization. [ABSTRACT FROM AUTHOR]

The cytoskeletal GTPase FtsZ assembles at midcell, recruits the division machinery and directs envelope invagination for bacterial cytokinesis. ZapA, a conserved FtsZ-binding protein, promotes Z-ring stability and efficient division through a mechanism that is not fully understood. Here, we investigated the function of ZapA in Caulobacter crescentus. We found that ZapA is encoded in an operon with a small coiled-coil protein we named ZauP. ZapA and ZauP co-localized at the division site and were each required for efficient division. ZapA interacted directly with both FtsZ and ZauP. Neither ZapA nor ZauP influenced FtsZ dynamics or bundling, in vitro, however. Z-rings were diffuse in cells lacking zapA or zauP and, conversely, FtsZ was enriched at midcell in cells overproducing ZapA and ZauP. Additionally, FtsZ persisted at the poles longer when ZapA and ZauP were overproduced, and frequently colocalized with MipZ, a negative regulator of FtsZ polymerization. We propose that ZapA and ZauP promote efficient cytokinesis by stabilizing the midcell Z-ring through a bundling-independent mechanism. The zauPzapA operon is present in diverse Gram-negative bacteria, indicating a common mechanism for Z-ring assembly. [ABSTRACT FROM AUTHOR]

FtsZ, a homolog of tubulin, is found in almost all bacteria and archaea where it has a primary role in cytokinesis. Evidence for structural homology between FtsZ and tubulin came from their crystal structures and identification of the GTP box. Tubulin and FtsZ constitute a distinct family of GTPases and show striking similarities in many of their polymerization properties. The differences between them, more so, the complexities of microtubule dynamic behavior in comparison to that of FtsZ, indicate that the evolution to tubulin is attributable to the incorporation of the complex functionalities in higher organisms. FtsZ and microtubules function as polymers in cell division but their roles differ in the division process. The structural and partial functional homology has made the study of their dynamic properties more interesting. In this review, we focus on the application of the information derived from studies on FtsZ dynamics to study microtubule dynamics and vice versa. The structural and functional aspects that led to the establishment of the homology between the two proteins are explained to emphasize the network of FtsZ and microtubule studies and how they are connected. [ABSTRACT FROM AUTHOR]

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram-negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ-like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ-driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction. [ABSTRACT FROM AUTHOR]

Virtual screenings based on molecular docking play a major role in medicinal chemistry for the identification of new bioactive molecules. For this purpose, several docking methods can be used. Here, using Arguslab as software and a Gold Platinum subset library of commercially available compounds from Asinex, two docking methods associated to the scoring function Ascore were employed to investigate virtual screenings. One method is based on a genetic algorithm and the other based on a shape-based method. As case studies, both docking techniques were explored by targeting the PC190723 binding site of FtsZ protein fromStaphylococcus aureusand the active site of N8 neuraminidase from Influenza virus. Following four docking sequences for each docking engine, the genetic algorithm led to multiple docking results, whereas the shape-based method gave reproducible results. The present study shows that the stochastic nature of the genetic algorithm will require the biological evaluation of more compounds than the shape-based method. This study showed that both methods are complementary and also led to the identification of neuraminidase and FtsZ potential inhibitors. [ABSTRACT FROM PUBLISHER]

The emergence and spread of multidrug-resistant strains of the human pathological bacteria are generating a threat to public health worldwide. In the current study, a series of PC190723 derivatives was synthesized and investigated for their antimicrobial activity. The compounds exhibited good activity against several Gram-positive bacteria as determined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics. Compound 9 with a fluorine substitution on the phenyl ring showed the best antibacterial activity in the series against M. smegmatis with the zone ratio of 0.62, and against S. aureus with the zone ratio of 0.44. The results from this study indicate that based on the unique 3-methoxybenzamide pharmacophore, compound 9 may represent a promising lead candidate against Gram-positive bacteria that are worthy of further investigation [ABSTRACT FROM AUTHOR]

Cell division in most bacteria is mediated by the tubulin-like FtsZ protein, which polymerizes in a GTP-dependent manner to form the cytokineticZring. Adiverse repertoire of FtsZ-binding proteins affects FtsZ localization and polymerization to ensure correct Z ring formation. Many of these proteins bind the C-terminal domain (CTD) of FtsZ, which serves as a hub for FtsZ regulation. FtsZ ring-associated proteins, ZapA-D (Zaps), are important FtsZ regulatory proteins that stabilize FtsZ assembly and enhance Z ring formation by increasing lateral assembly of FtsZ protofilaments, which then form the Z ring. There are no structures of a Zap protein bound to FtsZ; therefore, how these proteins affect FtsZ polymerization has been unclear. Recent data showed ZapD binds specifically to the FtsZ CTD. Thus, to obtain insight into the ZapD-CTD interaction and how it may mediate FtsZ protofilament assembly, we determined the Escherichia coli ZapD-FtsZ CTD structure to 2.67 Å resolution. The structure shows that the CTD docks within a hydrophobic cleft in the ZapD helical domain and adopts an unusual structure composed of two turns of helix separated by a proline kink. FtsZ CTD residue Phe-377 inserts into the ZapD pocket, anchoring the CTD in place and permitting hydrophobic contacts between FtsZ residues Ile-374, Pro-375, and Leu-378 with ZapD residues Leu-74, Trp-77, Leu-91, and Leu-174. The structural findings were supported by mutagenesis coupled with biochemical and in vivo studies. The combined data suggest that ZapD acts as a molecular cross-linking reagent between FtsZ protofilaments to enhance FtsZ assembly. [ABSTRACT FROM AUTHOR]

The bacterial tubulin FtsZ is the central component of the cell divisionmachinery, coordinating an ensemble of proteins involved in septal cell wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We found that in Escherichia coli cells, FtsZ exhibits dynamic treadmilling predominantly determined by its guanosine triphosphatase activity.The treadmilling dynamics direct the processivemovement of the septal cellwall synthesismachinery but do not limit the rate of septal synthesis. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cellwallwere substantially altered.Thus, FtsZ treadmilling provides amechanism for achieving uniform septal cell wall synthesis to enable correct polar morphology. [ABSTRACT FROM AUTHOR]

The mechanism by which bacteria divide is not well understood. Cell division is mediated by filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan-synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsAZ filaments treadmilled circumferentially around the division ring and drove the motions of the peptidoglycan-synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion of new cell wall by building increasingly smaller concentric rings of peptidoglycan to divide the cell. [ABSTRACT FROM AUTHOR]

The development of novel antimycobacterial agents is an urgent challenge to eradicate the increasing emergence and rapid spread of multidrug-resistant strains. Filamentous temperature-sensitive protein Z (FtsZ) is a crucial cell division protein. Alteration of FtsZ assembly leads to cell division inhibition and cell death. To find novel antimycobacterial agents, a series of N 1 -(benzo[d]oxazol-2-yl)-N 4 -arylidine compounds 5a-o were synthesized. The activity of the compounds was evaluated against drug-sensitive, multidrug-resistant, and extensive-drug-resistant Mycobacterium tuberculosis. Compounds 5b, 5c, 5l, 5m, and 5o showed promising antimycobacterial activity with minimum inhibitory concentrations (MIC) in the range of 0.48-1.85 µg/mL and with low cytotoxicity against human nontumorigenic lung fibroblast WI-38 cells. The activity of the compounds 5b, 5c, 5l, 5m, and 5o was evaluated against bronchitis causing-bacteria. They exhibited good activity against Streptococcus pneumoniae, Klebsiella pneumoniae, Mycoplasma pneumonia, and Bordetella pertussis. Molecular dynamics simulations of Mtb FtsZ protein-ligand complexes identified the interdomain site as the binding site and key interactions. ADME prediction indicated that the synthesized compounds have drug-likeness. The density function theory studies of 5c, 5l, and 5n were performed to investigate E/Z isomerization. Compounds 5c and 5l are present as E-isomers and 5n as an E/Z mixture. Our experimental outcomes provide an auspicious lead for the design of more selective and potent antimycobacterial drugs., (© 2023 Deutsche Pharmazeutische Gesellschaft.)

We propose that the essential function of the most highly conserved protein in bacterial cytokinesis, FtsZ, is not to generate a mechanical force to drive cell division. Rather, we suggest that FtsZ acts as a signal-processing hub to coordinate cell wall synthesis at the division septum with a diverse array of cellular processes, ensuring that the cell divides smoothly at the correct time and place, and with the correct septum morphology. Here, we explore how the polymerization properties of FtsZ, which have been widely attributed to force generation, can also be advantageous in this signal processing role. We suggest mechanisms by which FtsZ senses and integrates both mechanical and biochemical signals, and conclude by proposing experiments to investigate how FtsZ contributes to the remarkable spatial and temporal precision of bacterial cytokinesis. [ABSTRACT FROM AUTHOR]

The dual effect of FtsZ inhibition and oxidative stress by a group of 1,2-dihydroquinolines that culminate in bactericidal effect on mycobacterium strains is demonstrated. They inhibited the non-pathogenic Mycobacterium smegmatis mc 2 155 with MIC as low as 0.9 μg/mL and induced filamentation. Detailed studies revealed their ability to inhibit polymerization and GTPase activity of MtbFtsZ (Mycobacterial filamentous temperature sensitive Z) with an IC 50 value of ∼40 μM. In addition to such target specific effects, these compounds exerted a global cellular effect by causing redox-imbalance that was evident from overproduction of ROS in treated cells. Such multi-targeting effect with one chemical scaffold has considerable significance in this era of emerging drug resistance and could offer promise in the development of new therapeutic agents against tuberculosis. [ABSTRACT FROM AUTHOR]

The Min proteins from E.coli position the bacterial cell-division machinery through pole-to-pole oscillations. In vitro, Min protein self-organization can be reconstituted in the presence of a lipid membrane as a catalytic surface. However, Min dynamics have so far not been reconstituted in fully membrane-enclosed volumes. Microdroplets interfaced by lipid monolayers were employed as a simple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent a facile platform to investigate Min protein regulated dynamics of the cell-division protein FtsZ-mts. In particular, we show that droplet containers reveal distinct Min oscillation modes, and reveal a dependence of FtsZ-mts structures on compartment size. Finally, co-reconstitution of Min proteins and FtsZ-mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self-organization in confined volumes. [ABSTRACT FROM AUTHOR]

Cell division protein FtsZ is the organizer of the cytokinetic ring in almost all bacteria and a target for the discovery of new antibacterial agents that are needed to counter widespread antibiotic resistance. Bacterial cytological profiling, using quantitative microscopy, is a powerful approach for identifying the mechanism of action of antibacterial molecules affecting different cellular pathways. We have determined the cytological profile on Bacillus subtilis cells of a selection of small molecule inhibitors targeting FtsZ on different binding sites. FtsZ inhibitors lead to long undivided cells, impair the normal assembly of FtsZ into the midcell Z-rings, induce aberrant ring distributions, punctate FtsZ foci, membrane spots and also modify nucleoid length. Quantitative analysis of cell and nucleoid length combined, or the Z-ring distribution, allows categorizing FtsZ inhibitors and to distinguish them from antibiotics with other mechanisms of action, which should be useful for identifying new antibacterial FtsZ inhibitors. Biochemical assays of FtsZ polymerization and GTPase activity combined explain the cellular effects of the FtsZ polymer stabilizing agent PC190723 and its fragments. MciZ is a 40-aminoacid endogenous inhibitor of cell division normally expressed during sporulation in B. subtilis. Using FtsZ cytological profiling we have determined that exogenous synthetic MciZ is an effective inhibitor of B. subtilis cell division, Z-ring formation and localization. This finding supports our cell-based approach to screen for FtsZ inhibitors and opens new possibilities for peptide inhibitors of bacterial cell division. [ABSTRACT FROM AUTHOR]

In most bacteria, the tubulin-like GTPase FtsZ forms an annulus at midcell (the Z-ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z-ring assembly and early FtsZ-directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C-terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane-anchored FtsZ in the regulation of cell wall hydrolysis. [ABSTRACT FROM AUTHOR]

Cell division in bacteria is initiated by FtsZ, which forms a Z ring at the middle of the cell, between the nucleoids. The Z ring is stabilized by Z ring-associated proteins (Zaps), which crosslink the FtsZ filaments and provide strength. The deletion of Zaps leads to the elongation phenotype with an abnormal Z ring. The components of cell division in Helicobacter pylori are similar to other gram negative bacteria except for the absence of few components including Zaps. Here, we used HHsearch to identify homologs of the missing cell division proteins and got potential hits for ZapA and ZapB, as well as for few other cell division proteins. We further validated the function of the putative ZapA homolog by genetic complementation, immuno-colocalization and biochemical analysis. [ABSTRACT FROM AUTHOR]

Traditionally eukaryotes exclusive cytoskeleton has been found in bacteria and other prokaryotes. FtsZ, MreB and CreS are bacterial counterpart of eukaryotic tubulin, actin filaments and intermediate filaments, respectively. FtsZ can assemble to a Z-ring at the cell division site, regulate bacterial cell division; MreB can form helical structure, and involve in maintaining cell shape, regulating chromosome segregation; CreS, found inCaulobacter crescentus(C. crescentus), can form curve or helical filaments in intracellular membrane. CreS is crucial for cell morphology maintenance. There are also some prokaryotic unique cytoskeleton components playing crucial roles in cell division, chromosome segregation and cell morphology. The cytoskeleton components ofMycobacterium tuberculosis(M. tuberculosis), together with their dynamics during exposure to antibiotics are summarized in this article to provide insights into the unique organization of this formidable pathogen and druggable targets for new antibiotics. [ABSTRACT FROM AUTHOR]

Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments. [ABSTRACT FROM AUTHOR]

The modified nucleotides (p)ppGpp play an important role in bacterial physiology. While the accumulation of the nucleotides is vital for adaptation to various kinds of stress, changes in the basal level modulates growth rate and vice versa. Studying the phenotypes unique to the strain lacking (p)ppGpp (ppGpp0) under overtly unstressed growth conditions may be useful to understand functions regulated by basal levels of (p)ppGpp and its physiological significance. In this study, we show that the ppGpp0 strain, unlike the wild type, requires the Lon protease for cell division and viability in LB. Our results indicate the decrease in FtsZ concentration in the ppGpp0 strain makes cell division vulnerable to SulA inhibition. We did not find evidence for SOS induction contributing to the cell division defect in the ppGpp0 ∆lon strain. Based on the results, we propose that basal levels of (p)ppGpp are required to sustain normal cell division in Escherichia coli during growth in rich medium and that the basal SulA level set by Lon protease is important for insulating cell division against a decrease in FtsZ concentration and conditions that can increase the susceptibility of FtsZ to SulA. [ABSTRACT FROM AUTHOR]