Tumor Pre-Targeting System Using Streptavidin-Expressing Bacteria.

PURPOSE: A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe. PROCEDURES: We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-arabinose 4 days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed. RESULTS: Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin. CONCLUSIONS: We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

Reprogramming a Doxycycline-Inducible Gene Switch System for Bacteria-Mediated Cancer Therapy.

PURPOSE: Attenuated Salmonella typhimurium is a potential biotherapeutic antitumor agent because it can colonize tumors and inhibit their growth. The present study aimed to develop a doxycycline (Doxy)-inducible gene switch system in attenuated S. typhimurium and assess its therapeutic efficacy in various tumor-bearing mice models. PROCEDURES: A Doxy-inducible gene switch system comprising two plasmids was engineered to trigger the expression of cargo genes (Rluc8 and clyA). Attenuated S. typhimurium carrying Rluc8 were injected intravenously into BALB/c mice bearing CT26 tumors, and bioluminescence images were captured at specified intervals post-administration of doxycycline. The tumor-suppressive effects of bacteria carrying clyA were evaluated in BALB/c mice bearing CT26 tumors and in C57BL/6 mice bearing MC38 tumors. RESULTS: Expression of the fimE gene, induced only in the presence of Doxy, triggered a unidirectional switch of the POXB20 promoter to induce expression of the cargo genes. The switch event was maintained over a long period of bacterial culture. After intravenous injection of transformed Salmonella into mice bearing CT26 tumors, the bacteria transformed with the Doxy-inducible gene switch system for Rluc8 targeted only tumor tissues and expressed the payloads 2 days after Doxy treatment. Notably, bacteria carrying the Doxy-inducible gene switch system for clyA effectively suppressed tumor growth and prolonged survival, even after just one Doxy induction. CONCLUSIONS: These results suggest that attenuated S. typhimurium carrying this novel gene switch system elicited significant therapeutic effects through a single induction triggering and were a potential biotherapeutic agent for tumor therapy.

Synergistic cancer immunotherapy utilizing programmed Salmonella typhimurium secreting heterologous flagellin B conjugated to interleukin-15 proteins.

The use of appropriately designed immunotherapeutic bacteria is an appealing approach to tumor therapy because the bacteria specifically target tumor tissue and deliver therapeutic payloads. The present study describes the engineering of an attenuated strain of Salmonella typhimurium deficient in ppGpp biosynthesis (SAM) that could secrete Vibrio vulnificus flagellin B (FlaB) conjugated to human (hIL15/FlaB) and mouse (mIL15/FlaB) interleukin-15 proteins in the presence of L-arabinose (L-ara). These strains, named SAMphIF and SAMpmIF, respectively, secreted fusion proteins that retained bioactivity of both FlaB and IL15. SAMphIF and SAMpmIF inhibited the growth of MC38 and CT26 subcutaneous (sc) tumors in mice and increased mouse survival rate more efficiently than SAM expressing FlaB alone (SAMpFlaB) or IL15 alone (SAMpmIL15 and SAMphIL15), although SAMpmIF had slightly greater antitumor activity than SAMphIF. The mice treated with these bacteria showed enhanced macrophage phenotype shift, from M2-like to M1-like, as well as greater proliferation and activation of CD4+ T, CD8+ T, NK, and NKT cells in tumor tissues. After tumor eradication by these bacteria, ≥50% of the mice show no evidence of tumor recurrence upon rechallenge with the same tumor cells, indicating that they had acquired long-term immune memory. Treatment of mice of 4T1 and B16F10 highly malignant sc tumors with a combination of these bacteria and an immune checkpoint inhibitor, anti-PD-L1 antibody, significantly suppressed tumor metastasis and increased mouse survival rate. Taken together, these findings suggest that SAM secreting IL15/FlaB is a novel therapeutic candidate for bacterial-mediated cancer immunotherapy and that its antitumor activity is enhanced by combination with anti-PD-L1 antibody.

The anticancer effect of PASylated calreticulin-targeting L-ASNase in solid tumor bearing mice with immunogenic cell death-inducing chemotherapy.

L-Asparaginase (L-ASNase), a bacterial enzyme that degrades asparagine, has been commonly used in combination with several chemical drugs to treat malignant hematopoietic cancers such as acute lymphoblastic leukemia (ALL). In contrast, the enzyme was known to inhibit the growth of solid tumor cells in vitro, but not to be effective in vivo. We previously reported that two novel monobodies (CRT3 and CRT4) bound specifically with calreticulin (CRT) exposed on tumor cells and tissues during immunogenic cell death (ICD). Here, we engineered L-ASNases conjugated with monobodies at the N-termini and PAS200 tags at the C-termini (CRT3LP and CRT4LP). These proteins were expected to possess four monobody and PAS200 tag moieties, which did not disrupt the L-ASNase conformation. These proteins were expressed 3.8-fold more highly in E. coli than those without PASylation. The purified proteins were highly soluble, with much greater apparent molecular weights than expected ones. Their affinity (Kd) against CRT was about 2 nM, 4-fold higher than that of monobodies. Their enzyme activity (∼6.5 IU/nmol) was similar to that of L-ASNase (∼7.2 IU/nmol), and their thermal stability was significantly increased at 55 °C. Their half-life times were > 9 h in mouse sera, about 5-fold longer than that of L-ASNase (∼1.8 h). Moreover, CRT3LP and CRT4LP bound specifically with CRT exposed on tumor cells in vitro, and additively suppressed the tumor growth in CT-26 and MC-38 tumor-bearing mice treated with ICD-inducing drugs (doxorubicin and mitoxantrone) but not with a non-ICD-inducing drug (gemcitabine). All data indicated that PASylated CRT-targeted L-ASNases enhanced the anticancer efficacy of ICD-inducing chemotherapy. Taken together, L-ASNase would be a potential anticancer drug for treating solid tumors.

Genetic Programming by Nitric Oxide-Sensing Gene Switch System in Tumor-Targeting Bacteria.

Recent progress in synthetic biology has enabled bacteria to respond to specific disease signals to perform diagnostic and/or therapeutic tasks. Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) colonization of tumors results in increases in nitric oxide (NO) levels, suggesting that NO may act as a candidate inducer of tumor-specific gene expression. The present study describes a NO-sensing gene switch system for triggering tumor-specific gene expression in an attenuated strain of S. Typhimurium. The genetic circuit was designed to sense NO via NorR, thus initiating the expression of FimE DNA recombinase. This was found to lead sequentially to the unidirectional inversion of a promoter region (fimS), which induced the expression of target genes. Target gene expression in bacteria transformed with the NO-sensing switch system was triggered in the presence of a chemical source of NO, diethylenetriamine/nitric oxide (DETA/NO) in vitro. In vivo results revealed that the gene expression is tumor-targeted, and specific to NO generated by inducible nitric oxide synthase (iNOS) after S. Typhimurium colonization. These results showed that NO was a promising inducer to finely tune the expression of target genes carried by tumor-targeting bacteria.

In vivo imaging of invasive aspergillosis with 18F-fluorodeoxysorbitol positron emission tomography.

Invasive aspergillosis is a critical complication in immunocompromised patients with hematologic malignancies or with viral pneumonia caused by influenza virus or SARS­CoV­2. Although early and accurate diagnosis of invasive aspergillosis can maximize clinical outcomes, current diagnostic methods are time-consuming and poorly sensitive. Here, we assess the ability of 2-deoxy-2-18F-fluorosorbitol (18F-FDS) positron emission tomography (PET) to specifically and noninvasively detect Aspergillus infections. We show that 18F-FDS PET can be used to visualize Aspergillus fumigatus infection of the lungs, brain, and muscles in mouse models. In particular, 18F-FDS can distinguish pulmonary aspergillosis from Staphylococcus aureus infection, both of which induce pulmonary infiltrates in immunocompromised patients. Thus, our results indicate that the combination of 18F-FDS PET and appropriate clinical information may be useful in the differential diagnosis and localization of invasive aspergillosis.

Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria.

PURPOSE: In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter. PROCEDURES: In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR. RESULTS: In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division. CONCLUSIONS: Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Reporter gene-based optoacoustic imaging of E. coli targeted colon cancer in vivo.

Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.

Engineered Attenuated Salmonella typhimurium Expressing Neoantigen Has Anticancer Effects.

Neoantigen vaccines are an immunotherapy strategy for treating cancer. The vaccine degrades quickly, so the strategy must include protection and precise targeting for immune cell stimulation. In this study, we engineered attenuated Salmonella typhimurium, which is highly infiltrative to tumors, to act as a carrier for Neoantigen peptide vaccine. Our system used a constitutive promoter vector, so that a single injection of Salmonella expressing Neoantigen could be used without requiring additional induction injections. In vivo experiments on bacteria-treated mice showed that Neoantigen expressed by the engineered carrier infiltrated tumors and resulted in suppressed tumor growth, higher survival rates and longer survival times, a relative increase of CD4 and CD8 T cells, and cytokine release. These results indicate that engineered Salmonella can be used as a carrier for Neoantigen immunotherapy.

Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA.

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic-hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy.

Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein-engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

Imaging Calreticulin for Early Detection of Immunogenic Cell Death During Anticancer Treatment.

Surface-exposed calreticulin (ecto-CRT) is a well-known "eat-me" signal exhibited by dying cells that contributes to their recognition and destruction by the immune system. We assessed the use of a CRT-specific binding peptide for imaging ecto-CRT during immunogenic cell death and its utility for early prediction of treatment response. Methods: A synthetic CRT-specific peptide, KLGFFKR (CRTpep), was labeled with fluorescein isothiocyanate or 18F, and the characteristics of ecto-CRT were evaluated in a colon cancer cell line in vitro and in vivo. Results: In vitro flow cytometry, immunofluorescence staining, and in vivo small-animal PET imaging results showed that CRTpep detected preapoptotic cells treated with immunogenic drugs or radiation but not those treated with the nonimmunogenic drug or a nontherapeutic dose of immunogenic drug. Conclusion: The present results indicate that the CRT-specific peptide would enable the prediction of therapeutic response, thereby facilitating early decisions on continuation or discontinuation of immunogenic treatment.

Production of 64Cu-labeled monobody for imaging of human EphA2-expressing tumors.

We previously reported on the monobody E1, which specifically targets the tumor marker hEphA2. In this study, we labeled NOTA-conjugated E1 with 64Cu (64Cu-NOTA-E1) and evaluated biologic characteristics. The uptake of 64Cu-NOTA-E1 in PC3 cells (a human prostate cancer cell line) with high expression of hEphA2 increased in a time-dependent manner. In PC3 xenograft mice, 64Cu-NOTA-E1 injected via the tail vein allowed visualization of tumors on positron emission tomography after 1 h and the highest uptake measured at 24 h post-injection. By contrast, the radioactivity of other tissues either did not increase or decreased over 24 h. This indicates that 64Cu-NOTA-E1 has high tumor uptake and retention, with rapid clearance, and low background values in other tissues. Therefore, 64Cu-NOTA-E1 should be suitable as a novel PET imaging agent for hEphA2-expressing tumors.

Bacteria-cancer interactions: bacteria-based cancer therapy.

Recent advances in cancer therapeutics, such as targeted therapy and immunotherapy, have raised the hope for cures for many cancer types. However, there are still ongoing challenges to the pursuit of novel therapeutic approaches, including high toxicity to normal tissue and cells, difficulties in treating deep tumor tissue, and the possibility of drug resistance in tumor cells. The use of live tumor-targeting bacteria provides a unique therapeutic option that meets these challenges. Compared with most other therapeutics, tumor-targeting bacteria have versatile capabilities for suppressing cancer. Bacteria preferentially accumulate and proliferate within tumors, where they can initiate antitumor immune responses. Bacteria can be further programmed via simple genetic manipulation or sophisticated synthetic bioengineering to produce and deliver anticancer agents based on clinical needs. Therapeutic approaches using live tumor-targeting bacteria can be applied either as a monotherapy or in combination with other anticancer therapies to achieve better clinical outcomes. In this review, we introduce and summarize the potential benefits and challenges of this anticancer approach. We further discuss how live bacteria interact with tumor microenvironments to induce tumor regression. We also provide examples of different methods for engineering bacteria to improve efficacy and safety. Finally, we introduce past and ongoing clinical trials involving tumor-targeting bacteria.

Real-time monitoring of NADPH levels in living mammalian cells using fluorescence-enhancing protein bound to NADPHs.

Nicotinamide adenine nucleotide phosphate (NADPH) has been known to be involved in the multiple pathways of cell metabolism. However, conventional quantification assays for NADPH have required breaking down the cell membranes of around one million cells per assay, and monitoring NADPH flux in living cells has been limited by a few available tools. Here, we visualized NADPH levels in human cervical cancer cells HeLa using metagenome-derived blue fluorescent protein (mBFP), which specifically binds to NADPH and enhances the intrinsic fluorescence of NADPH up to 10-fold when imaged by two-photon microscopy to reduce photodamage. Adding an oxidizing agent such as diamide to HeLa cells that expressed mBFP led to an immediate decrease of intracellular NADPH depending on glucose availability in culture media. Furthermore, inhibiting glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway with dehydroandrosterone (DHEA) and knockdown of G6PD transcripts gradually decreased NADPH when diamide was added to living cells. These results demonstrate that introducing a bacterial mBFP gene into mammalian cells is a straightforward approach to monitoring intracellular NADPH flux in real time at the single-cell level. Moreover, this strategy can be expanded to tracking the spatio-temporal changes in NADPH even in single-cell organelles such as mitochondria and chloroplasts, which will allow us to more precisely assess the efficacy of biochemically or biophysically metabolic perturbations in animal and plant cells.

Structure-Guided Generation of a Redox-Independent Blue Fluorescent Protein from mBFP.

Fluorescent proteins, such as the green fluorescent protein, are used for detection of cellular components and events. However, green fluorescent protein and its derivatives have limited usage under anaerobic conditions and require a long maturation time. On the other hand, the NADPH-dependent blue fluorescent protein (BFP) without oxidative modification of residues is instantly functional in both aerobic and anaerobic systems. BFP proteins belong to a short-chain dehydrogenase/reductase (SDR) protein family, and their fluorescent property changes with reaction time in the presence of a substrate. With the aim of developing a better fluorescent reporter independent of redox state, we elucidated the crystal structure of a tetrameric mBFP from soil metagenomes with and without NADPH. Apart from the previously known regions, structure-guided mutational studies have identified several residues that contribute to the fluorescence of mBFP, including two aromatic residues (F97 and Y157) near the nicotinamide moiety of the bound NADPH. A single histidine mutation at Y157 (Y157H) has conferred more stabilized, time-independent fluorescence even in the presence of substrates. Furthermore, we discovered another SDR protein that can also emit blue fluorescence. These results open a new possibility for the development of BFP as a stable cellular reporter for widespread use, independent of subcellular environments.

Rapid and sensitive detection of NADPH via mBFP-mediated enhancement of its fluorescence.

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) functions as a reducing agent involved in many biosynthetic and antioxidant reactions in cells. Therefore, a lots of detection or assaying method of this cofactor are developed and used broadly in various research and application fields. These detection or assay tools, however, have often some problems, such as the low sensitivity, susceptibility to environmental interference and time-consuming pretreatment steps, remaining hurdle to successful quantification of NADPH or its derivatives accurately and immediately. Herein, we present a rapid (assay time < 30 s) and sensitive (detection limit < 2 pmol) detection method of NADPH using metagenome-derived blue fluorescent protein (mBFP), a protein capable of significantly enhancing NADPH fluorescence upon binding to this cofactor. Our method takes advantage of the high specificity of mBFP to NADPH and the immediate fluorescence enhancement upon the addition of mBFP to a solution of interest containing NADPH. We can apply this detection scheme to directly quantitative assessment of NADP(H)-dependent enzyme activities in-vitro, and further accessed to quantitative assay of other nicotine amide cofactors, such as NAD+ and NADH, by coupling assay using NAD(H) kinase. Thus, our method enabled us to quantitatively assess the activity of nicotinamide cofactor-associated enzymes in both bacterial and human cell lysates.

Cell mass-dependent expression of an anticancer protein drug by tumor-targeted Salmonella.

Bacterial cancer therapy relies on the properties of certain bacterial species capable of targeting and proliferating within solid malignancies. If these bacteria could be loaded with antitumor proteins, the efficacy of this approach could be greatly increased. However, because most antitumor proteins are also toxic to normal tissue, they must be expressed by bacteria that specifically target and exclusively localize to tumor tissue. As a strategy for treating solid malignancies, we recently evaluated L-asparaginase (L-ASNase) delivered by tumor-targeted Salmonella. In this system, L-ASNase was expressed under the control of the araBAD promoter (PBAD) of the E. coli arabinose operon, which is induced by injection of L-arabinose. Here, we further improved the performance of recombinant Salmonella in cancer therapy by exploiting the quorum-sensing (QS) system, which uses cell mass-dependent auto-induction logic. This approach obviates the necessity of monitoring intratumoral bacterial status and inducing cargo protein expression by administration of an exogenous compound. Recombinant Salmonella in tumors expressed and secreted active L-ASNase in a cell mass-dependent manner, yielding significant anticancer effects. These results suggest that expression of a therapeutic protein under the control of the QS system represents a promising engineering platform for the production of recombinant proteins in vivo.

Conditional constitutive expression system of a drug protein in vivo by positive feedback loop using an inducer-independent artificial transcription factor.

Bacterial-mediated drug delivery is a potential and promising strategy for the specific treatment of cancer with therapeutic molecules, especially with genetically encoded proteins. These proteins must be tightly regulated due to cytotoxicity and thus are usually expressed under the control of the PBAD and TetA/TetR promoters in vivo. Since protein expression from these systems is triggered by exogenous inducer, periodic intravenous injection of inducer is necessary. However, these treatments can result in non-homogenous and/or inefficient expression of therapeutic proteins in vivo due to impeded diffusion and dilution of the inducer further from the injection site. To overcome these hurdles, we designed a conditional constitutive expression system equipped with the artificial transcription factor, AraCC, which has two operator-binding domains and simultaneously binds to the I1 and I2 operators of the PBAD promoter for gene expression in an arabinose-independent manner. Using this construct and the wild type protein AraC under the control of the PBAD promoter, we constructed a self-positive feedback system to constitutively express the therapeutic protein when the induction of AraC was triggered once using arabinose. This expression system could be useful in various cancer treatment strategies using bacteria to deliver genetically encoded drugs in vivo.