Overexpression of somatostatin receptor type 2 in neuroendocrine tumors for improved Ga68-DOTATATE imaging and treatment.

BACKGROUND: Neuroendocrine tumors are found throughout the body, including the pancreas. These tumors are phenotypically and genetically heterogeneous and can be difficult to accurately image using current imaging standards. However, positron emission tomography/computed tomography with radiolabeled somatostatin analogs has shown clinical success because many neuroendocrine tumors overexpress somatostatin receptor subtype 2. Unfortunately, patients with poorly differentiated neuroendocrine tumors often have a diminished level of somatostatin receptor subtype 2. We found that histone deacetylase inhibitors can upregulate the functional expression of somatostatin receptor subtype 2. METHODS: We evaluated the effect of histone deacetylase inhibitors on somatostatin receptor subtype 2 expression at the mRNA and protein level in neuroendocrine tumor cell lines. The effect of histone deacetylase inhibitors on surface somatostatin receptor subtype 2 was also investigated by fluorescence-activated cell sorting analysis. Changes in somatostatin receptor subtype 2 expression in neuroendocrine tumor xenografts after treatment were imaged using Ga68-DOTATATE positron emission tomography/computed tomography. RESULTS: The functional increase of somatostatin receptor subtype 2 in neuroendocrine tumors after histone deacetylase inhibitor treatment was confirmed through in vitro experiments and small animal Ga68-DOTATATE positron emission tomography/computed tomography imaging. Histone deacetylase inhibitors increased somatostatin receptor subtype 2 transcription and protein expression in neuroendocrine tumor cell lines. Small animal Ga68-DOTATATE positron emission tomography/computed tomography imaging confirmed the enhancement of radiopeptide uptake after histone deacetylase inhibitor administration. CONCLUSION: This study demonstrates a new method to potentially improve imaging and treatments that target somatostatin receptor subtype 2 in neuroendocrine tumors.

Origin and bioactivities of thiosulfinated FK228.

During a large laboratory-scale purification of FK228 from the fermentation broth of Burkholderia thailandensis MSMB43, a small amount of thiosulfinated FK228 (TS-FK228) was unexpectedly purified only after the broth was mixed with silica gel. Evidence supports the postulations that TS-FK228 was derived from FK228 through spontaneous chemical reaction with silica gel, and TS-FK228 existed as two isomers 1 and 2. TS-FK228 demonstrated similar inhibitory activity and profile against human class I histone deacetylases but exhibited a much higher antiproliferative activity against representative human cancer cell lines when compared to FK228.

Genomics-guided discovery of a new and significantly better source of anticancer natural drug FK228.

FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No. 968 up to 19 mg/L in a pilot industry-scale batch fermentation. Here we report a genomics-guided discovery of Burkholderia thailandensis MSMB43 as a new and significantly better source of FK228. The genome of B. thailandensis MSMB43 was found to contain a functional biosynthetic gene cluster highly homologous to that of FK228 in C. violaceum No. 968, and the bacterium indeed produces authentic FK228. By simple fermentation in shaking flasks in a preferred M8 medium, B. thailandensis MSMB43 produced FK228 up to 67.7 mg/L; by fed-batch fermentation in a 20-L fermentor in M8 medium, B. thailandensis MSMB43 produced FK228 up to 115.9 mg/L, which is 95 fold higher than that of C. violaceum No. 968 under the same laboratory fermentation conditions. RT-PCR analysis indicated that the high FK228 yield of B. thailandensis MSMB43 was due to high expression of biosynthetic genes, represented by Bth_depA, during the fermentation process. Further genetic manipulation resulted in a recombinant strain, B. thailandensis MSMB43/pBMTL3-tdpR, which harbors a broad host-range vector expressing the thailandepsin biosynthetic pathway regulatory gene tdpR. This engineered strain produced up to 168.5 mg/L of FK228 in fed-batch fermentation in a 20-L fermentor in M8 medium. Therefore, the wild-type B. thailandensis MSMB43 or its engineered derivative could potentially be a good starting point for an industrial process to improve FK228 production for its expanding use in therapy.

Chromobacterium spp. mediate their anti-Plasmodium activity through secretion of the histone deacetylase inhibitor romidepsin.

The Chromobacterium sp. Panama bacterium has in vivo and in vitro anti-Plasmodium properties. To assess the nature of the Chromobacterium-produced anti-Plasmodium factors, chemical partition was conducted by bioassay-guided fractionation where different fractions were assayed for activity against asexual stages of P. falciparum. The isolated compounds were further partitioned by reversed-phase FPLC followed by size-exclusion chromatography; high resolution UPLC and ESI/MS data were then collected and revealed that the most active fraction contained a cyclic depsipeptide, which was identified as romidepsin. A pure sample of this FDA-approved HDAC inhibitor allowed us to independently verify this finding, and establish that romidepsin also has potent effect against mosquito stages of the parasite's life cycle. Genomic comparisons between C. sp. Panama and multiple species within the Chromobacterium genus further demonstrated a correlation between presence of the gene cluster responsible for romidepsin production and effective antiplasmodial activity. A romidepsin-null Chromobacterium spp. mutant loses its anti-Plasmodium properties by losing the ability to inhibit P. falciparum HDAC activity, and romidepsin is active against resistant parasites to commonly deployed antimalarials. This independent mode of action substantiates exploring a chromobacteria-based approach for malaria transmission-blocking.

Mechanistic studies of DepR in regulating FK228 biosynthesis in Chromobacterium violaceum no. 968.

DepR, a LysR-type transcriptional regulator encoded by the last gene of the putative min operon (orf21-20-19-depR) located at the downstream region of the anticancer agent FK228 biosynthetic gene cluster in Chromobacterium violaceum No. 968, positively regulates the biosynthesis of FK228. In this work, the mechanism underlining this positive regulation was probed by multiple approaches. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay (DIFA) identified a conserved 35-nt DNA segment in the orf21-orf22 intergenic region where the purified recombinant DepR binds to. Quantitative reverse transcription PCR (RT-qPCR) and green fluorescent protein (GFP) promoter probe assays established that transcription of phasin gene orf22 increases in the depR deletion mutant of C. violaceum (CvΔdepR) compared to the wild-type strain. FK228 production in the orf22-overexpressed strain C. violaceum was reduced compared with the wild-type strain. DepR has two conserved cysteine residues C199 and C208 presumed to form a disulfide bridge upon sensing oxidative stress. C199X point mutations that locked DepR in a reduced conformation decreased the DNA-binding affinity of DepR; T232A or R278A mutation also had a negative impact on DNA binding of DepR. Complementation of CvΔdepR with any of those versions of depR carrying a single codon mutation was not able to restore FK228 production to the level of wild-type strain. All evidences collectively suggested that DepR positively regulates the biosynthesis of FK228 through indirect metabolic networking.

Histone deacetylase inhibitor thailandepsin-A activates Notch signaling and suppresses neuroendocrine cancer cell growth in vivo.

Novel therapies for neuroendocrine (NE) cancers are desperately needed as they frequently present as metastatic disease and cause debilitating symptoms by secreting excessive hormones. Induction of Notch isoforms has a tumor suppressive effect in NE cancer cell lines, and we have observed that histone deacetylase inhibitors (HDACi) potently activate Notch. In this study, we describe the potential for Burkholderia thailandensis-derived class I HDACi thailandepsin A (TDP-A) as a Notch activator and therapeutic agent against NE cancer. IC50 for TDP-A was determined to be 4-6 nM in NE cancer cell lines (BON, MZ-CRC-1, and TT) without cytotoxicity to lung fibroblasts. The binding characteristics of TDP-A to its target HDAC1 was examined using bioluminescence resonance energy transfer (BRET). Western blot and flow cytometry analysis showed that TDP-A induces cell cycle arrest and apoptosis in a dose-dependent manner. TDP-A dose-dependently activated the Notch pathway as measured by increasing functional CBF1-luciferase reporter signal and mRNA and protein expressions of Notch isoforms, which were attenuated by pretreatment with γ-secretase inhibitor DAPT. Furthermore, TDP-A lead to changes in expression level of downstream targets of Notch pathway and reduced expression of NE cancer markers. An in vivo study demonstrated that TDP-A suppressed NE cancer progression. These results show that TDP-A, as a Notch activator, is a promising agent against NE cancers.

Developing new targeting strategy for androgen receptor variants in castration resistant prostate cancer.

The presence of androgen receptor variant 7 (AR-V7) variants becomes a significant hallmark of castration-resistant prostate cancer (CRPC) relapsed from hormonal therapy and is associated with poor survival of CRPC patients because of lacking a ligand-binding domain. Currently, it still lacks an effective agent to target AR-V7 or AR-Vs in general. Here, we showed that a novel class of agents (thailanstatins, TSTs and spliceostatin A analogs) can significantly suppress the expression of AR-V7 mRNA and protein but in a less extent on the full-length AR expression. Mechanistically, TST-D is able to inhibit AR-V7 gene splicing by interfering the interaction between U2AF65 and SAP155 and preventing them from binding to polypyrimidine tract located between the branch point and the 3' splice site. In vivo, TST-D exhibits a potent tumor inhibitory effect on human CRPC xenografts leading to cell apoptosis. The machinery associated with AR gene splicing in CRPC is a potential target for drugs. Based on their potency in the suppression of AR-V7 responsible for the growth/survival of CRPC, TSTs representing a new class of anti-AR-V agents warrant further development into clinical application.

HDAC Inhibitor-Mediated Epigenetic Regulation of Glaucoma-Associated TGFß2 in the Trabecular Meshwork.

PURPOSE: Elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG) results from glaucomatous damage to the trabecular meshwork (TM). The glaucoma-associated factor TGFß2 is increased in aqueous humor and TM of POAG patients. We hypothesize that histone acetylation has a role in dysregulated TGFß2 expression. METHODS: Protein acetylation was compared between nonglaucomatous TM (NTM) and glaucomatous TM (GTM) cells using Western immunoblotting (WB). Nonglaucomatous TM cells were treated with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor for 4 days. Total and nuclear proteins, RNA, and nuclear protein-DNA complexes were harvested for WB, quantitative PCR (qPCR), and chromatin immunoprecipitation (ChIP) assays, respectively. Paired bovine eyes were perfused with TDP-A versus DMSO, or TDP-A versus TDP-A plus the TGFß pathway inhibitor LY364947 for 5 to 9 days. Intraocular pressure, TM, and perfusate proteins were compared. RESULTS: We found increased acetylated histone 3 and total protein acetylation in the GTM cells and TDP-A treated NTM cells. Chromatin immunoprecipitation assays showed that TDP-A induced histone hyperacetylation associated with the TGFß2 promoter. This change of acetylation significantly increased TGFß2 mRNA and protein expression in NTM cells. In perfusion-cultured bovine eyes, TDP-A increased TGFß2 in the perfusate as well as elevated IOP. Histologic and immunofluorescent analyses showed increased extracellular matrix and cytoskeletal proteins in the TM of TDP-A treated bovine eyes. Cotreatment with the TGFß pathway inhibitor LY364947 blocked TDP-A-induced ocular hypertension. CONCLUSIONS: Our results suggest that histone acetylation has an important role in increased expression of the glaucoma-associated factor TGFß2. Histone hyperacetylation may be the initiator of glaucomatous damage to the TM.

Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation.

Thailanstatin A (TST-A) is a potent antiproliferative natural product discovered by our group from Burkholderia thailandensis MSMB43 through a genome-guided approach. The limited supply of TST-A, due to its low titer in bacterial fermentation, modest stability and very low recovery rate during purification, has hindered the investigations of TST-A as an anticancer drug candidate. Here we report the significant yield improvement of TST-A and its direct precursor, thailanstatin D (TST-D), through metabolic engineering of the thailanstatin biosynthetic pathway in MSMB43. Deletion of tstP, which encodes a dioxygenase involved in converting TST-A to downstream products including FR901464 (FR), resulted in 58% increase of the TST-A titer to 144.7 ± 2.3 mg/L and 132% increase of the TST-D titer to 14.6 ± 0.5 mg/L in the fermentation broth, respectively. Deletion of tstR, which encodes a cytochrome P450 involved in converting TST-D to TST-A, resulted in more than 7-fold increase of the TST-D titer to 53.2 ± 12.1 mg/L in the fermentation broth. An execution of 90 L pilot-scale fed-batch fermentation of the tstP deletion mutant in a 120-L fermentor led to the preparation of 714 mg of TST-A with greater than 98.5% purity. The half-life of TST-D in a phosphate buffer was found to be at least 202 h, significantly longer than that of TST-A or FR, suggesting superior stability. However, the IC50 values of TST-D against representative human cancer cell lines were determined to be greater than those of TST-A, indicating weaker antiproliferative activity. This work enabled us to prepare sufficient quantities of TST-A and TST-D for our ongoing translational research.

Target engagement and drug residence time can be observed in living cells with BRET.

The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment.

Engineered Production of Tryprostatins in E. coli through Reconstitution of a Partial ftm Biosynthetic Gene Cluster from Aspergillus sp.

Tryprostatin A and B are indole alkaloid-based fungal products that inhibit mammalian cell cycle at the G2/M phase. They are biosynthetic intermediates of fumitremorgins produced by a complex pathway involving a nonribosomal peptide synthetase (FtmA), a prenyltransferase (FtmB), a cytochrome P450 hydroxylase (FtmC), an O-methyltransferase (FtmD), and several additional enzymes. A partial fumitremorgin biosynthetic gene cluster (ftmABCD) from Aspergillus sp. was reconstituted in Escherichia coli BL21(DE3) cells, with or without the co-expression of an Sfp-type phosphopantetheinyltransferase gene (Cv_sfp) from Chromobacterium violaceum No. 968. Several recombinant E. coli strains produced tryprostatin B up to 106 mg/l or tryprostatin A up to 76 mg/l in the fermentation broth under aerobic condition, providing an effective way to prepare those pharmaceutically important natural products biologically.

Disulfide cross-linked micelles of novel HDAC inhibitor thailandepsin A for the treatment of breast cancer.

Histone deacetylase (HDAC) inhibitors are an emerging class of targeted therapy against cancers. Thailandepsin A (TDP-A) is a recently discovered class I HDAC inhibitor with broad anti-proliferative activities. In the present study, we aimed to investigate the potential of TDP-A in the treatment of breast cancer. We demonstrated that TDP-A inhibited cell proliferation and induced apoptosis in breast cancer cells at low nanomolar concentrations. TDP-A activated the intrinsic apoptotic pathway through increase of pro-apoptotic protein Bax, decrease of anti-apoptotic Bcl-2, and cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). TDP-A also induced cell cycle arrest at the G2/M phase, and promoted the production of reactive oxygen species (ROS). We have successfully encapsulated TDP-A into our recently developed disulfide cross-linked micelles (DCMs), improving its water solubility and targeted delivery. TDP-A loaded DCMs (TDP-A/DCMs) possess the characteristics of high loading capacity (>20%, w/w), optimal and monodisperse particle size (16 ± 4 nm), outstanding stability with redox stimuli-responsive disintegration, sustained drug release, and preferential uptake in breast tumors. In the MDA-MB-231 breast cancer xenograft model, TDP-A/DCMs were more efficacious than the FDA-approved FK228 at well-tolerated doses. Furthermore, TDP-A/DCMs exhibited synergistic anticancer effects when combined with the proteasome inhibitor bortezomib (BTZ) loaded DCMs (BTZ/DCMs). Our results indicate that TDP-A nanoformulation alone or in combination with BTZ nanoformulation are efficacious against breast cancer.

Determination of thailandepsin B in rat plasma with a LC-MS/MS method.

BACKGROUND: Thailandepsin B (TDP-B) is a potent histone deacetylase inhibitor under development. A reliable bioanalytical method for the quantification of TDP-B in plasma samples is required. RESULTS: The stabilizer mixture containing hydrochloric acid, formic acid and dichlorvos was applied to stabilize TDP-B in matrix samples. The method validation was conducted over the curve range of 1.00 to 1000 ng/ml. The intrabatch and interbatch precision and accuracy of the quality control samples showed ≤10.9% relative standard deviation and -6.7% to 15.0% relative error. Moreover, a possible metabolite M of TDP-B was tentatively characterized. CONCLUSION: A reliable LC-MS/MS method was developed and validated for the quantification of TDP-B and was successfully applied to a rat pharmacokinetic study.

Identification and characterization of the spiruchostatin biosynthetic gene cluster enable yield improvement by overexpressing a transcriptional activator.

Spiruchostatins A and B are members of the FK228-family of natural products with potent histone deacetylase inhibitory activities and antineoplastic activities. However, their production in the wild-type strain of Pseudomonas sp. Q71576 is low. To improve the yield, the spiruchostatin biosynthetic gene cluster (spi) was first identified by rapid genome sequencing and characterized by genetic mutations. This spi gene cluster encodes a hybrid biosynthetic pathway similar to that encoded by the FK228 biosynthetic gene cluster (dep) in Chromobacterium violaceum No. 968. Each gene cluster contains a pathway regulatory gene (spiR vs. depR), but these two genes encode transcriptional activators of different classes. Overexpression of native spiR or heterologous depR in the wild-type strain of Pseudomonas sp. Q71576 resulted in 268 or 1,285 % increase of the combined titer of spiruchostatins A and B, respectively. RT-PCR analysis indicates that overexpression of heterologous depR upregulates the expression of native spiR.

The novel histone deacetylase inhibitor thailandepsin A inhibits anaplastic thyroid cancer growth.

BACKGROUND: Anaplastic thyroid cancer (ATC) remains refractory to available surgical and medical interventions. Histone deacetylase (HDAC) inhibitors are an emerging targeted therapy with antiproliferative activity in a variety of thyroid cancer cell lines. Thailandepsin A (TDP-A) is a novel class I HDAC inhibitor whose efficacy remains largely unknown in ATC. Therefore, we aimed to characterize the effect of TDP-A on ATC. METHODS: Human-derived ATC cells were treated with TDP-A. IC50 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay, and cell proliferation was measured by viable cell count. Molecular mechanisms of cell growth inhibition were investigated by Western blot analysis of canonical apoptosis markers, intrinsic and extrinsic apoptosis regulators, and cell cycle regulatory proteins. Cell cycle staging was determined with propidium iodide flow cytometry. RESULTS: TDP-A dose- and time-dependently reduced cell proliferation. Increased cleavage of the apoptosis markers Caspase-9, Caspase-3, and poly adenosine diphosphate ribose polymerase were observed with TDP-A treatment. Levels of the intrinsic apoptosis pathway proteins BAD, Bcl-XL, and BAX remained unchanged. Importantly, the extrinsic apoptosis activator cleaved Caspase-8 increased dose-dependently, and the antiapoptotic proteins Survivin and Bcl-2 decreased. Among the cell cycle regulatory proteins, levels of CDK inhibitors p21/WAF1 and p27/KIP increased. Flow cytometry showed that ATC cells were arrested in G2/M phase with diminished S phase after TDP-A treatment. CONCLUSIONS: TDP-A induces a notable dose- and time-dependent antiproliferative effect on ATC, which is mainly attributed to extrinsic apoptosis with concomitant cell cycle arrest. TDP-A therefore warrants further preclinical and clinical investigations.

The structural basis of an NADP⁺-independent dithiol oxidase in FK228 biosynthesis.

The disulfide bond is unusual in natural products and critical for thermal stability, cell permeability and bioactivity. DepH from Chromobacterium violaceum No. 968 is an FAD-dependent enzyme responsible for catalyzing the disulfide bond formation of FK228, an anticancer prodrug approved for the treatment of cutaneous T-cell lymphoma. Here we report the crystal structures of DepH and DepH complexed with a substrate analogue S,S'-dimethyl FK228 at 1.82 Å and 2.00 Å, respectively. Structural and biochemical analyses revealed that DepH, in contrast to the well characterized low molecular weight thioredoxin reductases (LMW TrxRs), is an NADP(+)-independent dithiol oxidase. DepH not only lacks a conserved GGGDXAXE motif necessary for NADP(+) binding in the canonical LMW TrxRs, but also contains a 11-residue sequence which physically impedes the binding of NADP(+). These observations explain the difference between NADP(+)-independent small molecule dithiol oxidases and NADP(+)-dependent thioredoxin reductases and provide insights for understanding the catalytic mechanism of dithiol oxidases involved in natural product biosynthesis.

Genome-guided discovery of diverse natural products from Burkholderia sp.

Burkholderia species have emerged as a new source of diverse natural products. This mini-review covers all of the natural products discovered in recent years from Burkholderia sp. by genome-guided approaches--these refer to the use of bacterial genome sequence as an entry point for in silico structural prediction, wet lab experimental design, and execution. While reliable structural prediction based on cryptic biosynthetic gene cluster sequence was not always possible due to noncanonical domains and/or module organization of a deduced biosynthetic pathway, a molecular genetic method was often employed to detect or alter the expression level of the gene cluster to achieve an observable phenotype, which facilitated downstream natural product purification and identification. Those examples of natural product discovery from Burkholderia sp. provide practical guidance for future exploration of Gram-negative bacteria as a new source of natural products.

Structural determinants stabilizing the axial channel of ClpP for substrate translocation.

Acyldepsipeptides (ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N-terminal region that forms the axial gates. We found that adoption of a ß-hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram-positive and Gram-negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes.

Activation of ClpP protease by ADEP antibiotics: insights from hydrogen exchange mass spectrometry.

The bacterial protease ClpP consists of 14 subunits that assemble into two stacked heptameric rings. The central degradation chamber can be accessed via axial pores. In free ClpP, these pores are obstructed by the N-terminal regions of the seven subunits at either end of the barrel. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind in hydrophobic clefts surrounding the pore region, causing the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. Recently published X-ray structures yielded conflicting models regarding the conformation adopted by the N-terminal regions in the open state. Here, we use hydrogen/deuterium exchange (HDX) mass spectrometry to obtain complementary insights into the ClpP behavior with and without ADEP1. Ligand binding causes rigidification of the equatorial belt, accompanied by destabilization in the vicinity of the binding clefts. The N-terminal regions undergo rapid deuteration with only minor changes after ADEP1 binding, revealing a lack of stable H-bonding. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a "hydrophobic plug" causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way, ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore. This proposed mechanism helps reconcile the conflicting models that had been put forward earlier.

Inactivation of cyclic Di-GMP binding protein TDE0214 affects the motility, biofilm formation, and virulence of Treponema denticola.

As a ubiquitous second messenger, cyclic dimeric GMP (c-di-GMP) has been studied in numerous bacteria. The oral spirochete Treponema denticola, a periodontal pathogen associated with human periodontitis, has a complex c-di-GMP signaling network. However, its function remains unexplored. In this report, a PilZ-like c-di-GMP binding protein (TDE0214) was studied to investigate the role of c-di-GMP in the spirochete. TDE0214 harbors a PilZ domain with two signature motifs: RXXXR and DXSXXG. Biochemical studies showed that TDE0214 binds c-di-GMP in a specific manner, with a dissociation constant (Kd) value of 1.73 µM, which is in the low range compared to those of other reported c-di-GMP binding proteins. To reveal the role of c-di-GMP in T. denticola, a TDE0214 deletion mutant (TdΔ214) was constructed and analyzed in detail. First, swim plate and single-cell tracking analyses showed that TdΔ214 had abnormal swimming behaviors: the mutant was less motile and reversed more frequently than the wild type. Second, we found that biofilm formation of TdΔ214 was substantially repressed (∼6.0-fold reduction). Finally, in vivo studies using a mouse skin abscess model revealed that the invasiveness and ability to induce skin abscesses and host humoral immune responses were significantly attenuated in TdΔ214, indicative of the impact that TDE0214 has on the virulence of T. denticola. Collectively, the results reported here indicate that TDE0214 plays important roles in motility, biofilm formation, and virulence of the spirochete. This report also paves a way to further unveil the roles of the c-di-GMP signaling network in the biology and pathogenicity of T. denticola.