Discovery of 2',6-Bis(4-hydroxybenzyl)-2-acetylcyclohexanone, a Novel FtsZ Inhibitor.

Multi-drug resistance is increasing in the pathogenic bacterium S. pneumoniae, which is mainly responsible for meningitis and community-acquired pneumonia (CAP), highlighting the need for new anti-pneumococcal agents. We have identified a potential anti-pneumococcal agent, enol 3, which acts by hindering the cell division process by perturbing Z-ring dynamics inside the cell. Enol 3 was also shown to inhibit FtsZ polymerization and induce its aggregation in vitro but does not affect the activity of tubulin and alkaline phosphatase. Docking studies show that 3 binds near the T7 loop, which is the catalytic site of FtsZ. Similar effects on Z-ring and FtsZ assembly were observed in B. subtilis, indicating that 3 could be a broad-spectrum anti-bacterial agent useful in targeting Gram-positive bacteria. In conclusion, compound 3 shows strong anti-pneumococcal activity, prompting further pre-clinical studies to explore its potential.

Redox Sensing Modulates the Activity of the ComE Response Regulator of Streptococcus mutans.

Streptococcus mutans, a dental pathogen, encodes the ComDE two-component system comprised of a histidine kinase (ComD) and a response regulator (ComE). This system is necessary for production of bacteriocins and development of genetic competence. ComE interacts with its cognate promoters to activate the transcription of bacteriocin and competence-related genes. Previous transcriptomic studies indicated that expressions of bacteriocin genes were upregulated in the presence of oxygen. To understand the relationship between the aerobic condition and bacteriocin expression, we analyzed the S. mutans ComE sequence and its close homologs. Surprisingly, we noticed the presence of cysteine (Cys) residues located at positions 200 and 229, which are highly conserved among the ComE homologs. Here, we investigated the role of Cys residues of S. mutans ComE in the activation of bacteriocin transcription using the PnlmA promoter that expresses bacteriocin NlmA. We constructed both single mutants and double mutants by replacing the Cys residues with serine and performed complementation assays. We observed that the presence of Cys residues is essential for PnlmA activation. With purified ComE mutant proteins, we found that ComE double mutants displayed a nearly 2-fold lower association rate than wild-type ComE. Furthermore, 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence studies indicated that the double mutants displayed wider conformation changes than wild-type ComE. Finally, we demonstrated that close streptococcal ComE homologs successfully activate the PnlmA expression in vivo. This is the first report suggesting that S. mutans ComE and its homologs can sense the oxidation status of the cell, a phenomenon similar to the AgrA system of Staphylococcus aureus but with different outcomes. IMPORTANCE Streptococci are an important species that prefer to grow under anaerobic or microaerophilic environments. Studies have shown that streptococci growth in an aerobic environment generates oxidative stress responses by activating various defense systems, including production of antimicrobial peptides called bacteriocins. This study highlights the importance of a two-component response regulator (ComE) that senses the aerobic environment and induces bacteriocin production in Streptococcus mutans, a dental pathogen. We believe increased bacteriocin secretion under aerobic conditions is necessary for survival and colonization of S. mutans in the oral cavity by inhibiting other competing organisms. Redox sensing by response regulator might be a widespread phenomenon since two other ComE homologs from pathogenic streptococci that inhabit diverse environmental niches also perform a similar function.

Mutation of Arg191 in FtsZ Impairs Cytokinetic Abscission of Bacillus subtilis Cells.

FtsZ monomers assemble to form a dynamic Z-ring at the midcell position in bacteria that coordinates bacterial cell division. Antibacterial agents plumbagin and SB-RA-2001 were found to bind to FtsZ and to inhibit Z-ring formation in bacteria. Docking analysis indicated similar binding regions for these two inhibitors on FtsZ, and residue R191 was involved in the binding interaction with both compounds. In this work, the importance of R191 in FtsZ assembly and in bacterial cell division was analyzed. R191A-FtsZ exhibited significantly poorer polymerization ability. Further, the mutant FtsZ could poison the assembly of wild-type FtsZ (WT-FtsZ). The expression of R191A-FtsZ in Bacillus subtilis strain PL2084 perturbed Z-ring formation and produced filamentous cells, indicating that the mutation hindered the division of these cells. The results suggested that the R191A mutation is a dominant negative mutation of FtsZ. Molecular dynamics simulations of R191A-FtsZ and WT-FtsZ revealed a kink in helices H5 and H7 in the active site of R191A-FtsZ compared to that of WT-FtsZ, which is required for FtsZ assembly. The findings suggested that R191 is an important residue for FtsZ assembly, which can be targeted for the design of FtsZ inhibitors.

Antimicrobial peptide CRAMP (16-33) stalls bacterial cytokinesis by inhibiting FtsZ assembly.

A cathelin-related antimicrobial peptide (CRAMP) of 37 amino acid residues is thought to regulate innate immunity and provide a host defense mechanism in mammals. Here, a part of the CRAMP peptide, CRAMP (16-33) (GEKLKKIGQKIKNFFQKL), was found to bind to FtsZ and to inhibit the assembly and GTPase activity of FtsZ in vitro. A computational analysis indicated that CRAMP (16-33) binds in the cavity of the T7 loop of FtsZ. Both hydrophobic and ionic interactions were involved in the binding interactions. Further, CRAMP (16-33) inhibited the formation of the FtsZ ring in bacteria, indicating that it inhibited bacterial cell division by inhibiting FtsZ assembly.

SB-RA-2001 inhibits bacterial proliferation by targeting FtsZ assembly.

FtsZ has been recognized as a promising antimicrobial drug target because of its vital role in bacterial cell division. In this work, we found that a taxane SB-RA-2001 inhibited the proliferation of Bacillus subtilis 168 and Mycobacterium smegmatis cells with minimal inhibitory concentrations of 38 and 60 µM, respectively. Cell lengths of these microorganisms increased remarkably in the presence of SB-RA-2001, indicating that it inhibits bacterial cytokinesis. SB-RA-2001 perturbed the formation of the FtsZ ring in B. subtilis 168 cells and also affected the localization of the late cell division protein, DivIVA, at the midcell position. Flow cytometric analysis of the SB-RA-2001-treated cells indicated that the compound did not affect the duplication of DNA in B. subtilis 168 cells. Further, SB-RA-2001 treatment did not affect the localization of the chromosomal partitioning protein, Spo0J, along the two ends of the nucleoids and also had no discernible effect on the nucleoid segregation in B. subtilis 168 cells. The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells. In vitro, SB-RA-2001 bound to FtsZ with modest affinity, promoted the assembly and bundling of FtsZ protofilaments, and reduced the GTPase activity of FtsZ. GTP did not inhibit the binding of SB-RA-2001 to FtsZ, suggesting that it does not bind to the GTP binding site on FtsZ. A computational analysis indicated that SB-RA-2001 binds to FtsZ in the cleft region between the C-terminal domain and helix H7, and the binding site of SB-RA-2001 on FtsZ resembled that of PC190723, a well-characterized inhibitor of FtsZ. The findings collectively suggested that SB-RA-2001 inhibits bacterial proliferation by targeting the assembly dynamics of FtsZ, and this can be exploited further to develop potent FtsZ-targeted antimicrobials.

Distribution of two-component signal transduction systems BlpRH and ComDE across streptococcal species.

Two-component signal transduction (TCS) systems are important regulatory pathways in streptococci. A typical TCS encodes a membrane-anchored sensor kinase (SK) and a cytoplasmic response regulator (RR). Approximately, 20 different types of TCSs are encoded by various streptococci. Among them, two TCSs, in particular BlpRH and ComDE, are required for bacteriocins production and competence development. The SK component of these two TCSs is highly similar and belongs to the protein kinase-10 (HPK-10) subfamily. While these two TCSs are present in streptococci, no systematic studies have been done to differentiate between these two TCSs, and the existence of these pathways in several species of the genus Streptococcus is also unknown. The lack of information about these pathways misguided researchers for decades into believing that the Streptococcus mutans BlpRH system is a ComDE system. Here, we have attempted to distinguish between the BlpRH and ComDE systems based on the location of the chromosome, genomic arrangement, and conserved residues. Using the SyntTax and NCBI databases, we investigated the presence of both TCS systems in the genome of several streptococcal species. We noticed that the NCBI database did not have proper annotations for these pathways in several species, and many of them were wrongly annotated, such as CitS or DpiB instead of BlpH. Nevertheless, our critical analyses led us to classify streptococci into two groups: class A (only the BlpRH system) and class B (both the BlpRH and ComDE systems). Most of the streptococcal groups, including bovis, pyogenic, mutans, salivarius, and suis, encode only the BlpRH system. In contrast, only in the mitis and anginosus groups were both the TCS systems present. The focus of this review is to identify and differentiate between the BlpRH and ComDE systems, and discuss these two pathways in various streptococci.

Involvement of ClpE ATPase in Physiology of Streptococcus mutans.

Streptococcus mutans, a dental pathogen, harbors at least three Clp ATPases (ClpC, ClpE, and ClpX) that form complexes with ClpP protease and participate in regulated proteolysis. Among these, the function of ClpE ATPase is poorly understood. We have utilized an isogenic clpE-deficient strain derived from S. mutans UA159 and evaluated the role of ClpE in cellular physiology. We found that loss of ClpE leads to increased susceptibility against thiol stress but not to oxidative and thermal stress. Furthermore, we found that the mutant displays altered tolerance against some antibiotics and altered biofilm formation. We performed a label-free proteomic analysis by comparing the mutant with the wild-type UA159 strain under nonstressed conditions and found that ClpE modulates a relatively limited proteome in the cell compared to the proteomes modulated by ClpX and ClpP. Nevertheless, we found that ClpE deficiency leads to an overabundance of some cell wall synthesis enzymes, ribosomal proteins, and an unknown protease encoded by SMU.2153. Our proteomic data strongly support some of the stress-related phenotypes that we observed. Our study emphasizes the significance of ClpE in the physiology of S. mutans. IMPORTANCE When bacteria encounter environmental stresses, the expression of various proteins collectively known as heat shock proteins is induced. These heat shock proteins are necessary for cell survival specifically under conditions that induce protein denaturation. A subset of heat shock proteins known as the Clp proteolytic complex is required for the degradation of the misfolded proteins in the cell. The Clp proteolytic complex contains an ATPase and a protease. A specific Clp ATPase, ClpE, is uniquely present in Gram-positive bacteria, including streptococci. Here, we have studied the functional role of the ClpE protein in Streptococcus mutans, a dental pathogen. Our results suggest that ClpE is required for survival under certain antibiotic exposure and stress conditions but not others. Our results demonstrate that loss of ClpE leads to a significantly altered cellular proteome, and the analysis of those changes suggests that ClpE's functions in S. mutans are different from its functions in other Gram-positive bacteria.

Evidence of conformational switch in Streptococcus pneumoniae FtsZ during polymerization.

FtsZ, the master coordinator of bacterial cell division, assembles into filaments in the presence of nucleotide. FtsZ from Streptococcus pneumoniae bears two tryptophan residues (W294 and W378) in its amino acid sequence. The tryptophan fluorescence of FtsZ increases during the assembly of FtsZ. We hypothesized that this increase in the fluorescence intensity was due to the change in the environment of one or both tryptophan residues. To examine this, we constructed two mutants (W294F and W378F) of FtsZ by individually replacing tryptophan with phenylalanine. The mutants displayed similar secondary structures, GTPase activity, and polymerization ability as the wild type FtsZ. During the polymerization, only one tryptophan (W294) showed an increase in its fluorescence intensity. Using time-correlated single-photon counting, the fluorescence lifetime of W294 was found to be significantly higher than W378, indicating that W294 was more buried in the structure than W378. The lifetime of W294 further increased during polymer formation, while that of W378 remained unchanged. Fluorescence quenching experiment suggested that the solvent exposure of W294 reduced during the polymerization of FtsZ. W294 is located near the T-7 loop of the protein, a region important for the monomer-monomer interaction during the formation of a protofilament. The results indicated that the region around W294 of S. pneumoniae FtsZ undergoes a conformational switch during polymerization as seen for FtsZ from other bacteria.

Regulation of Streptococcus pneumoniae FtsZ assembly by divalent cations: paradoxical effects of Ca2+ on the nucleation and bundling of FtsZ polymers.

The assembly and disassembly of the FtsZ ring drives the division of bacteria cells, including Streptococcus pneumoniae, which causes pneumonia and meningitis. In contrast to FtsZ from other bacterial species, Streptococcus pneumoniae (Spn) FtsZ contains two tryptophan residues. Here, we demonstrate that the assembly and disassembly of Streptococcus pneumoniae FtsZ (SpnFtsZ) monomers can be monitored by the intrinsic tryptophan fluorescence of FtsZ. We found that the assembly of SpnFtsZ is closely associated with its GTPase activity. Guanosine 5'-[ß,γ-imido]triphosphate, a nonhydrolyzable analog of GTP, stabilized the FtsZ filaments without inducing their bundling. Using intrinsic tryptophan fluorescence, light scattering, and electron microscopy, we could differentiate the effects of divalent calcium and magnesium on the assembly of FtsZ. Though Mg2+ increased the stability of the FtsZ filaments, it could not prevent the disassembly of the filaments under conditions where GTP was limiting. Thus, our results indicate that Mg2+ primarily enhances the longitudinal assembly of FtsZ. Low concentrations of Ca2+ strongly promoted the bundling of FtsZ filaments and inhibited the disassembly of the filaments, suggesting that low concentrations of Ca2+ enhance the lateral interactions between the FtsZ filaments. Interestingly, Ca2+ delayed the nucleation process of FtsZ assembly, indicating that Ca2+ exerts paradoxical effects on the assembly of FtsZ. However, higher concentrations of Ca2+ did not enhance the bundling of FtsZ filaments. In addition, Ca2+ altered the secondary structure of FtsZ and increased the fluorescence of the FtsZ-1-anilinonaphthalene-8-sulfonic acid complex, indicating that Ca2+ induces conformational changes in FtsZ. The study provides an interesting insight into the assembly of SpnFtsZ and its regulation by divalent cations.

ZapC promotes assembly and stability of FtsZ filaments by binding at a different site on FtsZ than ZipA.

ZapC, a component of the divisome in Escherichia coli, is known to co-localize with FtsZ at the mid-cell position. A deletion or an overexpression of ZapC has been found to induce elongation of bacterial cells implying a role of ZapC in the cell division. ZapC has also been shown to enhance the assembly of purified FtsZ. In this study, ZapC was found to prevent the dilution-induced disassembly of preformed FtsZ polymers and to decorate FtsZ protofilaments along the length. ZapC interacted with FtsZ with a dissociation constant of 30±7nM. Salt had no discernable effect on the binding of ZapC to FtsZ; however, bis-ANS inhibited the binding of ZapC to FtsZ suggesting that the interaction was predominantly hydrophobic in nature. Several of the positive regulators of FtsZ assembly including ZipA are shown to bind FtsZ at the C-terminal tail of FtsZ. Using a 12-residue C-terminal tail peptide (LDIPAFLRKQAD) of FtsZ and a C-terminal tail truncated FtsZ construct, we provided data suggesting that ZapC does not bind at the C-terminal tail of FtsZ. The results indicated that ZapC and ZipA, two functionally similar proteins of the divisome complex, regulate FtsZ assembly through different sites of action on FtsZ.