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Abstract Background In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. Results The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. Conclusions This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

Amino acid deprivation, particularly of nonessential amino acids that can be synthesized by normal cells but not by cancer cells with specific defects in the biosynthesis pathway, has emerged as a potential strategy in cancer therapeutics. In normal cells, arginine is synthesized from citrulline in two steps via two enzymes: argininosuccinate synthetase (ASS1) and argininosuccinate lyase. Several cancer cells exhibit arginine auxotrophy due to the loss or down-regulation of ASS1. These cells undergo starvation-induced cell death in the presence of arginine-degrading enzymes such as arginine deaminase (ADI). Thus, ADI has emerged as a potential therapeutic in cancer therapy. However, the use of ADI has two major disadvantages: ADI of bacterial origin is strongly antigenic in mammals, and ADI has a short circulation half-life (∼5 h). In this study, we engineered tumor-targeting Salmonella Gallinarum to express and secrete ADI and deployed this strain into mice implanted with ASS1-defective mouse colorectal cancer (CT26) through an intravenous route. A notable antitumor effect was observed, suggesting that the disadvantages were overcome as ADI was expressed constitutively by tumor-targeting bacteria. A combination with chloroquine, which inhibits the induction of autophagy, further enhanced the effect. Anti-cancer effect of Salmonella Gallinarum expressing an arginine deiminase (ADI) on arginine-dependent tumors in situ., Competing Interests: Competing interests The authors declare no competing interests. Institutional Review Board Statement The animal study protocol was approved by the Ethics Committee of Chonnam National University–Institutional Animal Use and Care Committee (CNU IACUC-H-2020-7)., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan., (© 2024. The Author(s).)

We report here a novel anti-cancer therapy based on an avian-host-specific serotype Salmonella enterica serovar Gallinarum ( S. Gallinarum ) deficient in ppGpp synthesis. To monitor the tumor targeting, a bioluminescent ΔppGpp S. Gallinarum was constructed and injected intravenously into mice bearing syngeneic and human xenograft tumors. Strong bioluminescent signals were detected specifically in all grafted tumors at 2 days post-injection (dpi). The bacterial counts in normal and tumor tissue at 1 dpi revealed that ΔppGpp S. Gallinarum reached >10 8 CFU/g in tumor tissue and 10 6 -10 7 CFU/g in endothelial organs; counts were much lower in other organs. At 16 dpi, ΔppGpp S. Gallinarum counts in tumor tissue decreased to ∼10 6 CFU/g, while those in the other organs became undetectable. A strong anti-cancer effect was observed after the injection of ΔppGpp S. Gallinarum into BALB/c mice grafted with CT26 colon cancer cells. This could be attributed to reduced virulence, which allowed the administration of at least a 10-fold greater dose (10 8 CFU) of ΔppGpp S. Gallinarum than other attenuated strains of S. enterica serovar Typhimurium (≤10 7 CFU). An advantage of the avian-specific S. Gallinarum as a cancer therapeutic should be a reduced capacity to cause infections or harm in humans., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors.)

In bacteria and primitive eukaryotes, sulfonamide antibiotics block the folate pathway by inhibiting dihydropteroate synthase (FolP) that combines para-aminobenzoic acid (pABA) and dihydropterin pyrophosphate (DHPP) to form dihydropteroic acid (DHP), a precursor for tetrahydrofolate synthesis. However, the emergence of resistant strains has severely compromised the use of pABA mimetics as sulfonamide drugs. Salmonella enterica serovar Gallinarum (S. Gallinarum) is a significant source of antibiotic-resistant infections in poultry. Here, a sulfonamide-resistant FolP mutant library of S. Gallinarum was generated through random mutagenesis. Among resistant strains, substitution of amino acid Arginine 171 with Proline (R171P) in the FolP protein conferred the highest resistance against sulfonamide. Substitution of Phe28 with Leu or Ile (F28L/I) led to modest sulfonamide resistance. Structural modeling indicates that R171P and Phenylalanine 28 with leucine or isoleucine (F28L/I) substitution mutations are located far from the substrate-binding site and cause insignificant conformational changes in the FolP protein. Rather, in silico studies suggest that the mutations altered the stability of the protein, potentially resulting in sulfonamide resistance. Identification of specific mutations in FolP that confer resistance to sulfonamide would contribute to our understanding of the molecular mechanisms of antibiotic resistance., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Antimicrobial resistant pathogens are a global health threat driven by the indiscriminate use of antimicrobials. Antimicrobial resistance can be acquired by resistance genes encoded by mobile genetic elements. In this study, we identified a strain of Salmonella enterica serovar Gallinarum (SG4021) from an infected chicken in Korea and characterized the presence of resistance genes in its plasmid by whole genome sequencing. The sequence was then compared with that of a plasmid (P2) from strain SG_07Q015, the only other strain of S. Gallinarum isolated in Korea for which a genome sequence is available. The results revealed that both strains harbored nearly identical DNA carrying antibiotic resistance gene cassettes inserted into integron In2 of the transposable element Tn21, namely an aadA1 resistance gene conferring resistance to aminoglycosides and a sul1 resistance gene conferring resistance to sulfonamide. Interestingly, despite the presence of sul1 in SG4021, an antibiotic sensitivity test revealed that it was sensitive to sulfonamides. Further analysis revealed that this disparity was due to the insertion of a ~ 5 kb ISCR16 sequence downstream of the promoter driving sul1 expression in SG4021. Using various mutants, we showed that the insertion of ISCR16 blocked the expression of the sul1 gene from the upstream promoter. Therefore, the functionality of antimicrobial resistance genes determines phenotypic antimicrobial resistance., (© 2023. The Author(s).)

Bacterial cancer therapy is a promising next-generation modality to treat cancer that often uses tumor-colonizing bacteria to deliver cytotoxic anticancer proteins. However, the expression of cytotoxic anticancer proteins in bacteria that accumulate in the nontumoral reticuloendothelial system (RES), mainly the liver and spleen, is considered detrimental. This study examined the fate of the Escherichia coli strain MG1655 and an attenuated strain of Salmonella enterica serovar Gallinarum ( S. Gallinarum) with defective ppGpp synthesis after intravenous injection into tumor-bearing mice (~10 8 colony forming units/animal). Approximately 10% of the injected bacteria were detected initially in the RES, whereas approximately 0.01% were in tumor tissues. The bacteria in the tumor tissue proliferated vigorously to up to 10 9 colony forming units/g tissue, whereas those in the RES died off. RNA analysis revealed that tumor-associated E. coli activated rrnB operon genes encoding the rRNA building block of ribosome needed most during the exponential stage of growth, whereas those in the RES expressed substantially decreased levels of this gene and were cleared soon presumably by innate immune systems. Based on this finding, we engineered ΔppGpp S. Gallinarum to express constitutively a recombinant immunotoxin comprising TGFα and the Pseudomonas exotoxin A (PE38) using a constitutive exponential phase promoter, the ribosomal RNA promoter rrnB P1. The construct exerted anticancer effects on mice grafted with mouse colon (CT26) or breast (4T1) tumor cells without any notable adverse effects, suggesting that constitutive expression of cytotoxic anticancer protein from rrnB P1 occurred only in tumor tissue.

The biosynthesis of host lipids and/or lipid droplets (LDs) has been studied extensively as a putative therapeutic target in diverse viral infections. However, directly targeting the LD lipolytic catabolism in virus-infected cells has not been widely investigated. Here, we show the linkage of the LD-associated lipase activation to the breakdown of LDs for the generation of free fatty acids (FFAs) at the late stage of diverse RNA viral infections, which represents a broad-spectrum antiviral target. Dysfunction of membrane transporter systems due to virus-induced cell injury results in intracellular malnutrition at the late stage of infection, thereby making the virus more dependent on the FFAs generated from LD storage for viral morphogenesis and as a source of energy. The replication of SARS-CoV-2 and influenza A virus (IAV), which is suppressed by the treatment with LD-associated lipases inhibitors, is rescued by supplementation with FFAs. The administration of lipase inhibitors, either individually or in a combination with virus-targeting drugs, protects mice from lethal IAV infection and mitigates severe lung lesions in SARS-CoV-2-infected hamsters. Moreover, the lipase inhibitors significantly reduce proinflammatory cytokine levels in the lungs of SARS-CoV-2- and IAV-challenged animals, a cause of a cytokine storm important for the critical infection or mortality of COVID-19 and IAV patients. In conclusion, the results reveal that lipase-mediated intracellular LD lipolysis is commonly exploited to facilitate RNA virus replication and furthermore suggest that pharmacological inhibitors of LD-associated lipases could be used to curb current COVID-19- and future pandemic outbreaks of potentially troublesome RNA virus infection in humans., (© 2022. The Author(s).)

Type I restriction-modification enzymes are oligomeric proteins composed of methylation (M), DNA sequence-recognition (S), and restriction (R) subunits. The different bipartite DNA sequences of 2-4 consecutive bases are recognized by two discerned target recognition domains (TRDs) located at the two-helix bundle of the two conserved regions (CRs). Two M-subunits and a single S-subunit form an oligomeric protein that functions as a methyltransferase (M 2 S 1 MTase). Here, we present the crystal structure of the intact MTase from Vibrio vulnificus YJ016 in complex with the DNA-mimicking Ocr protein and the S-adenosyl-L-homocysteine (SAH). This MTase includes the M-domain with a helix tail (M-tail helix) and the S 1/2 -domain of a TRD and a CR α-helix. The Ocr binds to the cleft of the TRD surface and SAH is located in the pocket within the M-domain. The solution- and negative-staining electron microscopy-based reconstructed (M 1 S 1/2 ) 2 structure reveals a symmetric (S 1/2 ) 2 assembly using two CR-helices and two M-tail helices as a pivot, which is plausible for recognizing two DNA regions of same sequence. The conformational flexibility of the minimal M 1 S 1/2 MTase dimer indicates a particular state resembling the structure of M 2 S 1 MTases., (Copyright © 2022. Published by Elsevier B.V.)

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 (P tetA ) was 100-fold higher in expression strength than tetR promoter (P tetR ). 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 P tetA and P tetR , and Doxy releases TetR from tetO to de-repress P tetA and P tetR ., Results: In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (P tetA :P tetR = 4~6:1) compared with that of pJL87 (P tetA :P tetR = 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., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

Background: Clostridioides difficile infection (CDI) is an infectious nosocomial disease caused by Clostridioides difficile, an opportunistic pathogen that occurs in the intestine after extensive antibiotic regimens., Results: Nine C. difficile strains (CBA7201-CBA7209) were isolated from nine patients diagnosed with CDI at the national university hospital in Korea, and the whole genomes of these strains were sequenced to identify their genomic characteristics. Comparative genomic analysis was performed using 51 reference strains and the nine isolated herein. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that all 60 C. difficile strains belong to the genus Clostridioides, while core-genome tree indicated that they were divided into five groups, which was consistent with the results of MLST clade analysis. All strains were confirmed to have a clindamycin antibiotic resistance gene, but the other antibiotic resistance genes differ depending on the MLST clade. Interestingly, the six strains belonging to the sequence type 17 among the nine C. difficile strains isolated here exhibited unique genomic characteristics for PaLoc and CdtLoc, the two toxin gene loci identified in this study, and harbored similar antibiotic resistance genes., Conclusion: In this study, we identified the specific genomic characteristics of Korean C. difficile strains, which could serve as basic information for CDI prevention and treatment in Korea., (© 2021. The Author(s).)

Fibroblast growth factor 23 (FGF23), a hormone generally derived from bone, is important in phosphate and vitamin D homeostasis. In acute kidney injury (AKI) patients, high-circulating FGF23 levels are associated with disease progression and mortality. However, the organ and cell type of FGF23 production in AKI and the molecular mechanism of its excessive production are still unidentified. For insight, we investigated folic acid (FA)-induced AKI in mice. Interestingly, simultaneous with FGF23, orphan nuclear receptor ERR-γ expression is increased in the liver of FA-treated mice, and ectopic overexpression of ERR-γ was sufficient to induce hepatic FGF23 production. In patients and in mice, AKI is accompanied by up-regulated systemic IL-6, which was previously identified as an upstream regulator of ERR-γ expression in the liver. Administration of IL-6 neutralizing antibody to FA-treated mice or of recombinant IL-6 to healthy mice confirms IL-6 as an upstream regulator of hepatic ERR-γ-mediated FGF23 production. A significant ( P < 0.001) interconnection between high IL-6 and FGF23 levels as a predictor of AKI in patients that underwent cardiac surgery was also found, suggesting the clinical relevance of the finding. Finally, liver-specific depletion of ERR-γ or treatment with an inverse ERR-γ agonist decreased hepatic FGF23 expression and plasma FGF23 levels in mice with FA-induced AKI. Thus, inverse agonist of ERR-γ may represent a therapeutic strategy to reduce adverse plasma FGF23 levels in AKI., Competing Interests: The authors declare no competing interest.

Previously, we uncovered a novel mechanism in which senescence is controlled by mitochondrial functional recovery upon Ataxia-telangiectasia mutated (ATM) inhibition. However, it remains elusive how ATM controls signaling pathways to achieve restorative effect. In this study, we performed microarray and found that p53 pathway was differentially expressed upon ATM inhibition. We found that ATM inhibition yields senescence amelioration through p53-dependent manner. The restorative effect was also afforded by direct p53 inhibition. Furthermore, mitochondrial metabolic reprogramming via p53 inhibition was a prerequisite for senescence amelioration. Taken together, our data indicated that p53 pathway functions as potential target for ATM-mediated senescence amelioration., (Copyright © 2020 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Neutropenic sepsis is a fatal consequence of chemotherapy, and septic complications are the principal cause of mortality. Chemotherapy-induced neutropenia leads to the formation of microscopic ulcers in the gastrointestinal epithelium that function as a portal of entry for intraluminal bacteria, which translocate across the intestinal mucosal barrier and gain access to systemic sites, causing septicemia. A cyclophosphamide-induced mouse model was developed to mimic the pathophysiologic sequence of events that occurs in patients with neutropenic sepsis. The TLR5 agonist bacterial flagellin derived from Vibrio vulnificus extended the survival of cyclophosphamide-treated mice by reducing the bacterial load in internal organs. The protective effect of flagellin was mediated by the antimicrobial protein lipocalin 2 (Lcn2), which is induced by TLR5-NF-κB activation in hepatocytes. Lcn2 sequestered iron from infecting bacteria, particularly siderophore enterobactin-dependent members of the Enterobacteriaceae family, thereby limiting their proliferation. Lcn2 should be considered for the treatment of neutropenic sepsis and gastrointestinal damage during chemotherapy to prevent or minimize the adverse effects of cancer chemotherapy.

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors.

Senescence is defined as irreversible cell cycle arrest and constitutes a major driving force in diseases related to aging or premature aging. Recent studies indicate that activation of the serine/threonine protein kinase B-raf (BRAF) plays important roles in oncogene-induced senescence. However, it remains elusive whether BRAF inhibition might be effective for abrogating senescence. In this study, we assessed several BRAF inhibitors to identify compounds that ameliorate senescence and revealed SB590885 as an effective agent. Senescence-ameliorating effect upon BRAF inhibition was evident from the observation that SB590885 treatment increased cellular proliferation but diminished senescent phenotypes. Moreover, BRAF inhibition induced the mitochondrial functional recovery along with the metabolic reprogramming, which comprises two salient features that are altered in senescent cells. Furthermore, mitochondrial metabolic reprogramming via BRAF inhibition was a prerequisite for senescence amelioration. Taken together, our data revealed a novel mechanism in which senescence amelioration is mediated by mitochondrial metabolic reprogramming upon BRAF inhibition., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain; however, the roles of GABA in antimicrobial host defenses are largely unknown. Here we demonstrate that GABAergic activation enhances antimicrobial responses against intracellular bacterial infection. Intracellular bacterial infection decreases GABA levels in vitro in macrophages and in vivo in sera. Treatment of macrophages with GABA or GABAergic drugs promotes autophagy activation, enhances phagosomal maturation and antimicrobial responses against mycobacterial infection. In macrophages, the GABAergic defense is mediated via macrophage type A GABA receptor (GABA A R), intracellular calcium release, and the GABA type A receptor-associated protein-like 1 (GABARAPL1; an Atg8 homolog). Finally, GABAergic inhibition increases bacterial loads in mice and zebrafish in vivo, suggesting that the GABAergic defense plays an essential function in metazoan host defenses. Our study identified a previously unappreciated role for GABAergic signaling in linking antibacterial autophagy to enhance host innate defense against intracellular bacterial infection.

Macrophages release iron into the bloodstream via a membrane-bound iron export protein, ferroportin (FPN). The hepatic iron-regulatory hormone hepcidin controls FPN internalization and degradation in response to bacterial infection. Salmonella typhimurium can invade macrophages and proliferate in the Salmonella-containing vacuole (SCV). Hepcidin is reported to increase the mortality of Salmonella-infected animals by increasing the bacterial load in macrophages. Here we assess the iron levels and find that hepcidin increases iron content in the cytosol but decreases it in the SCV through FPN on the SCV membrane. Loss-of-FPN from the SCV via the action of hepcidin impairs the generation of bactericidal reactive oxygen species (ROS) as the iron content decreases. We conclude that FPN is required to provide sufficient iron to the SCV, where iron serves as a cofactor for the generation of antimicrobial ROS rather than as a nutrient for 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 ., Competing Interests: CONFLICTS OF INTEREST The authors do not have any conflicts of interest.

Background: Alexander disease (AxD) is an astrogliopathy that predominantly affects the white matter of the central nervous system (CNS), and is caused by a mutation in the gene encoding the glial fibrillary acidic protein (GFAP), an intermediate filament primarily expressed in astrocytes and ependymal cells. The main pathologic feature of AxD is the presence of Rosenthal fibers (RFs), homogeneous eosinophilic inclusions found in astrocytes. Because of difficulties in procuring patient' CNS tissues and the presence of RFs in other pathologic conditions, there is a need to develop an in vivo assay that can determine whether a mutation in the GFAP results in aggregation and is thus disease-causing., Methods: We found a GFAP mutation (c.382G > A, p.Asp128Asn) in a 68-year-old man with slowly progressive gait disturbance with tendency to fall. The patient was tentatively diagnosed with AxD based on clinical and radiological findings. To develop a vertebrate model to assess the aggregation tendency of GFAP, we expressed several previously reported mutant GFAPs and p.Asp128Asn GFAP in zebrafish embryos., Results: The most common GFAP mutations in AxD, p.Arg79Cys, p.Arg79His, p.Arg239Cys and p.Arg239His, and p.Asp128Asn induced a significantly higher number of GFAP aggregates in zebrafish embryos than wild-type GFAP., Conclusions: The p.Asp128Asn GFAP mutation is likely to be a disease-causing mutation. Although it needs to be tested more extensively in larger case series, the zebrafish assay system presented here would help clinicians determine whether GFAP mutations identified in putative AxD patients are disease-causing.

COACH syndrome is an autosomal recessive developmental disorder, a subtype of Joubert syndrome and related disorders, characterized by cerebellar vermis hypoplasia, oligophrenia, ataxia, coloboma, and hepatic fibrosis. Although mutations in TMEM67 (transmembrane protein 67)/MKS3 (Meckel-Gruber syndrome, type 3) were reported to cause COACH syndrome, this causality has not verified by functional studies. In a 20-year-old Korean man, we found cerebellar ataxia, isolated elevation in serum γ-glutamyl transpeptidase (γ-GTP) activity, oligophrenia, the molar tooth sign (MTS) in the brain MR images and congenital hepatic fibrosis (CHF). Two novel compound heterozygous mutations were found in TMEM67 in the patient: i) missense mutation (c.395 G > C and p.Gly132Ala) in exon 3, and ii) deletion in exon 26 (c.2758delT and p.Tyr920ThrfsX40). Western blotting showed that the p.Tyr920ThrfsX40 mutation accelerates turnover of the TMEM67 protein. Although wild-type human TMEM67 RNA rescued phenotypes of zebrafish embryos injected with anti-sense oligonucleotide morpholinos against tmem67, the two human TMEM67 RNAs individually harboring the two mutations did not. Finally, Wnt signaling, but not Hedgehog signaling, was suppressed in tmem67 morphants. To the best of our knowledge, this is the first report verifying the causality between COACH syndrome and TMEM67, which will further our understanding of molecular pathogenesis of the syndrome.

The anticancer strategy underlying the use of immunotoxins is as follows: the cancer-binding domain delivers the toxin to a cancer cell, after which the toxin enters and kills the cell. TGFα-PE38 is an immunotoxin comprising transforming growth factor alpha (TGFα), a natural ligand of epidermal growth factor receptor (EGFR), and a modified Pseudomonas exotoxin A (PE38) lacking N terminal cell-binding domain, a highly potent cytotoxic protein moiety. Tumor cells with high level of EGFR undergo apoptosis upon treatment with TGFα-PE38. However, clinical trials demonstrated that this immunotoxin delivered by an intracerebral infusion technique has only a limited inhibitory effect on intracranial tumors mainly due to inconsistent drug delivery. To circumvent this problem, we turned to tumor-seeking bacterial system. Here, we engineered Salmonella typhimurium to selectively express and release TGFα-PE38. Engineered bacteria were administered to mice implanted with mouse colon or breast tumor cells expressing high level of EGFR. We observed that controlled expression and release of TGFα-PE38 from intra-tumoral Salmonellae by either an engineered phage lysis system or by a bacterial membrane transport signal led to significant inhibition of solid tumor growth. These results demonstrated that delivery by tumor-seeking bacteria would greatly augment efficacy of immunotoxin in cancer therapeutics.

We report a method of cancer immunotherapy using an attenuated Salmonella typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues. Engineered FlaB-secreting bacteria effectively suppressed tumor growth and metastasis in mouse models and prolonged survival. By using Toll-like receptor 5 (TLR5)-negative colon cancer cell lines, we provided evidence that the FlaB-mediated tumor suppression upon bacterial colonization is associated with TLR5-mediated host reactions in the tumor microenvironment. These therapeutic effects were completely abrogated in TLR4 and MyD88 knockout mice, and partly in TLR5 knockout mice, indicating that TLR4 signaling is a requisite for tumor suppression mediated by FlaB-secreting bacteria, whereas TLR5 signaling augmented tumor-suppressive host reactions. Tumor microenvironment colonization by engineered Salmonella appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via TLR4 signaling. Subsequent secretion of FlaB from colonizing Salmonella resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Together, these findings provide evidence that nonvirulent tumor-targeting bacteria releasing multiple TLR ligands can be used as cancer immunotherapeutics., (Copyright © 2017, American Association for the Advancement of Science.)

Inflammaging is defined as low-grade, chronic, systemic inflammation in aging, in the absence of overt infection. Age-associated deterioration of gastrointestinal function could be ascribed to the inflammaging, although evidence is yet to emerge. Here we show that microvessels in aging mouse intestine were progressively deprived of supportive structures, microvessel-associated pericytes and adherens junction protein vascular endothelial (VE)-cadherin, and became leaky. This alteration was ascribed to up-regulation of angiopoetin-2 in microvascular endothelial cells. Up-regulation of the angiopoietin-2 was by TNF-α, originated from M2-like residential CD206 + macrophages, proportion of which increases as animal ages. It was concluded that antigenic burdens encountered in intestine throughout life create the condition of chronic stage of inflammation, which accumulates M2-like macrophages expressing TNF-α. The TNF-α induces vascular leakage to facilitate recruitment of immune cells into intestine under the chronic inflammatory setting.

Small heterodimer partner (SHP) is a transcriptional corepressor regulating diverse metabolic processes. Here, we show that SHP acts as an intrinsic negative regulator of iron homeostasis. SHP-deficient mice maintained on a high-iron diet showed increased serum hepcidin levels, decreased expression of the iron exporter ferroportin as well as iron accumulation compared to WT mice. Conversely, overexpression of either SHP or AMP-activated protein kinase (AMPK), a metabolic sensor inducing SHP expression, suppressed BMP6-induced hepcidin expression. In addition, an inhibitory effect of AMPK activators metformin and AICAR on BMP6-mediated hepcidin gene expression was significantly attenuated by ablation of SHP expression. Interestingly, SHP physically interacted with SMAD1 and suppressed BMP6-mediated recruitment of the SMAD complex to the hepcidin gene promoter by inhibiting the formation of SMAD1 and SMAD4 complex. Finally, overexpression of SHP and metformin treatment of BMP6 stimulated mice substantially restored hepcidin expression and serum iron to baseline levels. These results reveal a previously unrecognized role for SHP in the transcriptional control of iron homeostasis.

Enteropathogenic E. coli causes attaching and effacing (A/E) intestinal lesions. The genes involved in the formation of A/E lesions are encoded within a chromosomal island comprising of five major operons, LEE1-5. The global regulator H-NS represses the expression of these operons. Ler, a H-NS homologue, counteracts the H-NS-mediated repression. Using a novel genetic approach, we identified the amino acid residues in Ler that are involved in the interaction with H-NS: I20 and L23 in the C-terminal portion of α-helix 3, and I42 in the following unstructured linker region.

Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

Bacteria-based anticancer therapies aim to overcome the limitations of current cancer therapy by actively targeting and efficiently removing cancer. To achieve this goal, new approaches that target and maintain bacterial drugs at sufficient concentrations during the therapeutic window are essential. Here, we examined the tumor tropism of attenuated Salmonella typhimurium displaying the RGD peptide sequence (ACDCRGDCFCG) on the external loop of outer membrane protein A (OmpA). RGD-displaying Salmonella strongly bound to cancer cells overexpressing αvβ3, but weakly bound to αvβ3-negative cancer cells, suggesting the feasibility of displaying a preferential homing peptide on the bacterial surface. In vivo studies revealed that RGD-displaying Salmonellae showed strong targeting efficiency, resulting in the regression in αvβ3-overexpressing cancer xenografts, and prolonged survival of mouse models of human breast cancer (MDA-MB-231) and human melanoma (MDA-MB-435). Thus, surface engineering of Salmonellae to display RGD peptides increases both their targeting efficiency and therapeutic effect.

Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.

Escherichia coli and attenuated Salmonella both naturally accumulate in a tumor mass, yet have distinct therapeutic efficacy: the E. coli K-12 strain (MG1655) cannot induce as significant a tumor suppression as attenuated Salmonella typhimurium, despite similar levels of accumulation in the tumor. To elucidate the mechanism of the robust antitumor effect of S. typhimurium, the cytokine profiles elicited by bacterial colonization in tumors were analyzed. C57BL/6 mice bearing MC38 tumors were injected with Salmonella or MG1655 in the tail vein. Tumors were collected 3 days post-infection and homogenized. Inflammasome-related signals were measured by real-time PCR, ELISA and western blot analysis. Only attenuated Salmonella triggered significant levels of the inflammatory cytokine IL-1β in the tumor, whereas tumor growth was significantly suppressed. In addition, transcript levels of the core molecules of inflammasome signaling, IPAF, NLRP3 and P2X7, were significantly elevated only in Salmonella-treated tumors. Upon direct interaction between Salmonella and BMDM, BMDM expressed inflammasome-related proteins such as NLRP3, IPAF and caspase-1 p10, and secreted a significant amount of IL-1β in supernatants. Coincubation assays with BMDM and Salmonella-treated MC38 cells (damaged cancer cells) revealed secretion of IL-1β only when TLR4 and inflammasome were activated by both LPS and damaged cancer cells. ATP released from damaged cancer cells was also identified as a mechanism of NLRP3 activation. In conclusion, Salmonella activate the inflammasome pathway using damage signals released from cancer cells and through direct interaction with macrophages., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

Although strains of attenuated Salmonella typhimurium and wild-type Escherichia coli show similar tumor-targeting capacities, only S. typhimurium significantly suppresses tumor growth in mice. The aim of the present study was to examine bacteria-mediated immune responses by conducting comparative analyses of the cytokine profiles and immune cell populations within tumor tissues colonized by E. coli or attenuated Salmonellae. CT26 tumor-bearing mice were treated with two different bacterial strains: S. typhimurium defective in ppGpp synthesis (ΔppGpp Salmonellae) or wild-type E. coli MG1655. Cytokine profiles and immune cell populations in tumor tissue colonized by these two bacterial strains were examined at two time points based on the pattern of tumor growth after ΔppGpp Salmonellae treatment: 1) when tumor growth was suppressed ('suppression stage') and 2) when they began to re-grow ('re-growing stage'). The levels of IL-1β and TNF-α were markedly increased in tumors colonized by ΔppGpp Salmonellae. This increase was associated with tumor regression; the levels of both IL-1β and TNF-α returned to normal level when the tumors started to re-grow. To identify the immune cells primarily responsible for Salmonellae-mediated tumor suppression, we examined the major cell types that produce IL-1β and TNF-α. We found that macrophages and dendritic cells were the main producers of TNF-α and IL-1β. Inhibiting IL-1β production in Salmonellae-treated mice restored tumor growth, whereas tumor growth was suppressed for longer by local administration of recombinant IL-1β or TNF-α in conjunction with Salmonella therapy. These findings suggested that IL-1β and TNF-α play important roles in Salmonella-mediated cancer therapy. A better understanding of host immune responses in Salmonella therapy may increase the success of a given drug, particularly when various strategies are combined with bacteriotherapy.

Monobodies are binding scaffold proteins originating from a human fibronectin domain III (Fn3) scaffold that can be easily engineered with specificity and affinity. Human EphA2 (hEphA2) is an early detection marker protein for various tumors including lung, breast, and colon cancer. In this study, we isolated two hEphA2-specific monobodies (E1 and E10) by screening a yeast surface display library. They showed the same amino acid sequence except in the DE loop and had high affinity (~2 nM Kd) against hEphA2. E1 bound only hEphA2 and mEphA2, although it bound hEphA2 with an affinity 2-fold higher than that of mEphA2. However, E10 also bound the mEphA6 and mEphA8 homologs as well as hEphA2 and mEphA2. Thus, E1 but not E10 was highly specific for hEphA2. E1 specifically bound human cells and xenograft tumor tissues expressing hEphA on the cell surface. In vivo optical imaging showed strong targeting of Cy5.5-labeled E1 to mouse tumor tissue induced by PC3 cells, a human prostate cancer cell line that expresses a high level of hEphA2. In conclusion, the highly specific monobody E1 is useful as a hEphA2 probe candidate for in vivo diagnosis and therapy.

Bacteria can be engineered to deliver anticancer proteins to tumors via a controlled expression system that maximizes the concentration of the therapeutic agent in the tumor. L-asparaginase (L-ASNase), which primarily converts asparagine to aspartate, is an anticancer protein used to treat acute lymphoblastic leukemia. In this study, Salmonellae were engineered to express L-ASNase selectively within tumor tissues using the inducible araBAD promoter system of Escherichia coli. Antitumor efficacy of the engineered bacteria was demonstrated in vivo in solid malignancies. This result demonstrates the merit of bacteria as cancer drug delivery vehicles to administer cancer-starving proteins such as L-ASNase to be effective selectively within the microenvironment of cancer tissue.

σ38 in Escherichia coli is required for expression of a subset of stationary phase genes. However, the promoter elements for σ38-dependent genes are virtually indistinguishable from that for σ70-dependent house-keeping genes. hdeABp is a σ38-dependent promoter and LEE5p is a σ70-dependent promoter, but both are repressed by H-NS, a bacterial histone-like protein, which acts at promoter upstream sequence. We swapped the promoter upstream sequences of the two promoters and found that the σ dependency was switched. This was further verified using lacUV5 core promoter. The results suggested that the determinant for σ38-dependent promoter lies in the promoter upstream sequence.

Drug delivery systems (DDSs) incorporating bacterial minicells have been evaluated as a very powerful tool in view of biocompatibility. However, limited studies have been carried out on these systems, mainly using minicells from Salmonella sp. and Escherichia coli. Thus, we generated a new minicell-producing strain from an endotoxin-free Corynebacterium glutamicum by the inactivation of genes related to cell division. The two knockout strains, ΔparA and Δncgl1366, showed distinct abilities to produce minicells. The resulting minicells were purified via sequential antibiotic treatments and centrifugations, which resulted in reproducible yields.

Caveolar endocytosis has an important function in the cellular uptake of some bacterial toxins, viruses, and circulating proteins. However, the molecular machinery involved in caveolae-dependent bacterial endocytosis is poorly defined. In the present study, we identify a new molecular mechanism for the caveolin-1-dependent entry of Salmonella into host cells via the direct regulation of actin reorganization. In contrast to the interaction of caveolae with other pathogens, the caveolae did not form Salmonella-containing vesicles or endosomes in the host cells. Instead, the caveolae rapidly moved to the apical plasma membrane upon actin condensation during early invasion. Interestingly, the injected bacterial protein SopE interacted with Rac1 to regulate actin reorganization, and both proteins colocalized and directly interacted with caveolin-1 in caveolae during early invasion. After the complete internalization of Salmonella, SopE levels decreased both in the caveolae and in the host cytoplasm; Rac1 activity was also decreased. Downregulation of caveolin-1 by siRNA treatment led to reduction of Salmonella invasion compared with control siRNA-treated cells. These results suggest a new model in which caveolin-1 might be involved in Salmonella entry via its interaction with SopE and Rac1, leading to enhanced membrane ruffling for phagocytosis into host cells., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Histone-like nucleoid structuring protein (H-NS) is a small but abundant protein present in enteric bacteria and is involved in compaction of the DNA and regulation of the transcription. Recent reports have suggested that H-NS binds to a specific AT rich DNA sequence than to intrinsically curved DNA in sequence independent manner. We detected two high-specificity H-NS binding sites in LEE5 promoter of EPEC centered at -110 and -138, which were close to the proposed consensus H-NS binding motif. To identify H-NS binding sequence in LEE5 promoter, we took a random mutagenesis approach and found the mutations at around -138 were specifically defective in the regulation by H-NS. It was concluded that H-NS exerts maximum repression via the specific sequence at around -138 and subsequently contacts a subunit of RNAP through oligomerization.

Objectives: The in vivo efficacy of a cefotaxime-ciprofloxacin combination against Vibrio vulnificus and the effects on rtxA1 expression of commonly used antibiotics are unknown., Methods: In vitro time-kill studies were performed to evaluate synergism. Female BALB/c mice were injected subcutaneously with 1×10(7) or 1×10(8) cfu of V. vulnificus. Antibiotic therapy was initiated at 2 h after inoculation in the following four therapy groups: cefotaxime; ciprofloxacin; cefotaxime-plus-ciprofloxacin; and cefotaxime-plus-minocycline. The cytotoxicity of V. vulnificus for HeLa cells was measured using the lactate dehydrogenase assay; rtxA1 transcription was measured in a transcriptional reporter strain using a β-galactosidase assay., Results: In vitro time-kill assays exhibited synergism between cefotaxime and ciprofloxacin. In the animal experiments, the 96-h survival rate for the cefotaxime-plus-ciprofloxacin group (85%; 17/20) was significantly higher than that of the cefotaxime-plus-minocycline (35%; 7/20) and cefotaxime alone (0%; 0/20) groups (P<0.05 for both). Bacterial counts in the liver and spleen were significantly lower in the cefotaxime-plus-ciprofloxacin group 24 and 48 h after treatment, relative to the other groups. At sub-inhibitory concentrations, ciprofloxacin inhibited more effectively rtxA1 transcription and mammalian cell cytotoxicity than either minocycline or cefotaxime (P<0.05 for both)., Conclusions: Ciprofloxacin is more effective at reducing rtxA1 transcription and subsequent cytotoxicity than either minocycline or cefotaxime, and the combination of ciprofloxacin and cefotaxime was more effective in clearing V. vulnificus in vivo than previously used regimens. These data suggest that the combination of ciprofloxacin and cefotaxime is an effective option for the treatment of V. vulnificus sepsis in humans.

Ler, a homolog of H-NS in enteropathogenic Escherichia coli (EPEC), plays a critical role in the expression of virulence genes encoded by the pathogenic island, locus of enterocyte effacement (LEE). Although Ler acts as an antisilencer of multiple LEE operons by alleviating H-NS-mediated silencing, it represses its own expression from two LEE1 P1 promoters, P1A and P1B, that are separated by 10 bp. Various in vitro biochemical methods were used in this study to elucidate the mechanism underlying transcription repression by Ler. Ler acts through two AATT motifs, centered at position -111.5 on the coding strand and at +65.5 on the noncoding strand, by simultaneously repressing P1A and P1B through DNA-looping. DNA-looping was visualized using atomic force microscopy. It is intriguing that an antisilencing protein represses transcription, not by steric exclusion of RNA polymerase, but by DNA-looping. We propose that the DNA-looping prevents further processing of open promoter complex (RPO) at these promoters during transcription initiation.

In response to microbial infection, expression of the defensin-like peptide hepcidin (encoded by Hamp) is induced in hepatocytes to decrease iron release from macrophages. To elucidate the mechanism by which Salmonella enterica var. Typhimurium (S. typhimurium), an intramacrophage bacterium, alters host iron metabolism for its own survival, we examined the role of nuclear receptor family members belonging to the NR3B subfamily in mouse hepatocytes. Here, we report that estrogen-related receptor γ (ERRγ, encoded by Esrrg) modulates the intramacrophage proliferation of S. typhimurium by altering host iron homeostasis, and we demonstrate an antimicrobial effect of an ERRγ inverse agonist. Hepatic ERRγ expression was induced by S. typhimurium-stimulated interleukin-6 signaling, resulting in an induction of hepcidin and eventual hypoferremia in mice. Conversely, ablation of ERRγ mRNA expression in liver attenuated the S. typhimurium-mediated induction of hepcidin and normalized the hypoferremia caused by S. typhimurium infection. An inverse agonist of ERRγ ameliorated S. typhimurium-mediated hypoferremia through reduction of ERRγ-mediated hepcidin mRNA expression and exerted a potent antimicrobial effect on the S. typhimurium infection, thereby improving host survival. Taken together, these findings suggest an alternative approach to control multidrug-resistant intracellular bacteria by modulating host iron homeostasis.

Several optical imaging techniques have been used to monitor bacterial tropisms for cancer. Most such techniques require genetic engineering of the bacteria to express optical reporter genes. This study investigated a novel tumor-targeting strain of bacteria, Rhodobacter sphaeroides 2.4.1 (R. sphaeroides), which naturally emits near-infrared fluorescence, thereby facilitating the visualization of bacterial tropisms for cancer. To determine the penetration depth of bacterial fluorescence, various numbers of cells (from 10(8) to 10(10)  CFU) of R. sphaeroides and two types of Escherichia coli, which stably express green fluorescent protein (GFP) or red fluorescent protein (RFP), were injected s.c. or i.m. into mice. Bacterial tropism for cancer was determined after i.v. injection of R. sphaeroides (10(8)  CFU) into mice implanted s.c. with eight types of tumors. The intensity of the fluorescence signal in deep tissue (muscle) from R. sphaeroides was much stronger than from E. coli-expressing GFP or RFP. The near-infrared fluorescence signal from R. sphaeroides was visualized clearly in all types of human or murine tumors via accumulation of bacteria. Analyses of C-reactive protein and procalcitonin concentrations and body weights indicated that i.v. injection of R. sphaeroides does not induce serious systemic immune reactions. This study suggests that R. sphaeroides could be used as a tumor-targeting microorganism for the selective delivery of drugs to tumor tissues without eliciting a systemic immune reaction and for visualizing tumors., (© 2014 The Societies and Wiley Publishing Asia Pty Ltd.)

Advances in genetic engineering tools have contributed to the development of strategies for utilizing biologically derived vesicles as nanomedicines for achieving cell-specific drug delivery. Here, we describe bioengineered bacterial outer membrane vesicles (OMVs) with low immunogenicity that can target and kill cancer cells in a cell-specific manner by delivering small interfering RNA (siRNA) targeting kinesin spindle protein (KSP). A mutant Escherichia coli strain that exhibits reduced endotoxicity toward human cells was engineered to generate OMVs displaying a human epidermal growth factor receptor 2 (HER2)-specific affibody in the membrane as a targeting ligand. Systemic injection of siRNA-packaged OMVs caused targeted gene silencing and induced highly significant tumor growth regression in an animal model. Importantly, the modified OMVs were well tolerated and showed no evidence of nonspecific side effects. We propose that bioengineered OMVs have great potential as cell-specific drug-delivery vehicles for treating various cancers.

Bacterial cancer therapy relies on the fact that several bacterial species are capable of targeting tumor tissue and that bacteria can be genetically engineered to selectively deliver therapeutic proteins of interest to the targeted tumors. However, the challenge of bacterial cancer therapy is the release of the therapeutic proteins from the bacteria and entry of the proteins into tumor cells. This study employed an attenuated Salmonella typhimurium to selectively deliver the mitochondrial targeting domain of Noxa (MTD) as a potential therapeutic cargo protein, and examined its anti-cancer effect. To release MTD from the bacteria, a novel bacterial lysis system of phage origin was deployed. To facilitate the entry of MTD into the tumor cells, the MTD was fused to DS4.3, a novel cell-penetrating peptide (CPP) derived from a voltage-gated potassium channel (Kv2.1). The gene encoding DS4.3-MTD and the phage lysis genes were placed under the control of PBAD , a promoter activated by L-arabinose. We demonstrated that DS4.3-MTD chimeric molecules expressed by the Salmonellae were anti-tumoral in cultured tumor cells and in mice with CT26 colon carcinoma.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, which causes death of motor neurons in the anterior horn of the spinal cord. Genetic cause of SMA is the deletion or mutation of SMN1 gene, which encodes the SMN protein. Although SMA patients include SMN2 gene, a duplicate of SMN1 gene, predominant production of exon 7 skipped isoform from SMN2 pre-mRNA, fails to rescue SMA patients. Here we show that hnRNP M, a member of hnRNP protein family, when knocked down, promotes exon 7 skipping of both SMN2 and SMN1 pre-mRNA. By contrast, overexpression of hnRNP M promotes exon 7 inclusion of both SMN2 and SMN1 pre-mRNA. Significantly, hnRNP M promotes exon 7 inclusion in SMA patient cells. Thus, we conclude that hnRNP M promotes exon 7 inclusion of both SMN1 and SMN2 pre-mRNA. We also demonstrate that hnRNP M contacts an enhancer on exon 7, which was previously shown to provide binding site for tra2β. We present evidence that hnRNP M and tra2β contact overlapped sequence on exon 7 but with slightly different RNA sequence requirements. In addition, hnRNP M promotes U2AF65 recruitment on the flanking intron of exon 7. We conclude that hnRNP M promotes exon 7 inclusion of SMN1 and SMN2 pre-mRNA through targeting an enhancer on exon 7 through recruiting U2AF65. Our results provide a clue that hnRNP M is a potential therapeutic target for SMA., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Tumor-specific expression of antitumor drugs can be achieved using attenuated Salmonella typhimurium harboring the PBAD promoter, which is induced by L-arabinose. However, L-arabinose does not accumulate because it is metabolized to D-xylulose-5-P by enzymes encoded by the ara operon in Salmonellae. To address this problem, we developed an engineered strain of S. typhimurium in which the ara operon is deleted. Linear DNA transformation was performed using λ red recombinase to exchange the ara operon with linear DNA carrying an antibiotic-resistance gene with homology to regions adjacent to the ara operon. The ara operon-deleted strain and its parental strain were transformed with a plasmid encoding Renilla luciferase variant 8 (RLuc8) or cytolysin A (clyA) under the control of the PBAD promoter. Luciferase assays demonstrated that RLuc8 expression was 49-fold higher in the ara operon-deleted S. typhimurium than in the parental strain after the addition of L-arabinose. In vivo bioluminescence imaging showed that the tumor tissue targeted by the ara operon-deleted Salmonella had a stronger imaging signal (~30-fold) than that targeted by the parental strain. Mice with murine colon cancer (CT26) that had been injected with the ara operon-deleted S. typhimurium expressing clyA showed significant tumor suppression. The present report demonstrates that deletion of the ara operon of S. typhimurium enhances L-arabinose accumulation and thereby drives PBAD-promoted expression of cytotoxic agents and imaging agents. This is a promising approach for tumor therapy and imaging.

We propose a bacteria-based microrobot (bacteriobot) based on a new fusion paradigm for theranostic activities against solid tumors. We develop a bacteriobot using the strong attachment of bacteria to Cy5.5-coated polystyrene microbeads due to the high-affinity interaction between biotin and streptavidin. The chemotactic responses of the bacteria and the bacteriobots to the concentration gradients of lysates or spheroids of solid tumors can be detected as the migration of the bacteria and/or the bacteriobots out of the central region toward the side regions in a chemotactic microfluidic chamber. The bacteriobots showed higher migration velocity toward tumor cell lysates or spheroids than toward normal cells. In addition, when only the bacteriobots were injected to the CT-26 tumor mouse model, Cy5.5 signal was detected from the tumor site of the mouse model. In-vitro and in-vivo tests verified that the bacteriobots had chemotactic motility and tumor targeting ability. The new microrobot paradigm in which bacteria act as microactuators and microsensors to deliver microstructures to tumors can be considered a new theranostic methodology for targeting and treating solid tumors.

Mammalian peroxiredoxin V (PrdxV) is a multifunctional protein that protects cells from DNA damage and inhibits stress-induced apoptosis. However, PrdxV is also known to be involved in modulating lipopolysaccharide (LPS)-induced host cell signaling, but its precise role is not fully understood. In this study, we used stably transfected RAW264.7 cells and transiently transfected 293-mTLR4-MD2-CD14 cells expressing wild-type (WT) or mutant (C48S) PrdxV to characterize the function and mechanism of action of PrdxV in LPS-induced immune responses. We found that PrdxV selectively reduces production of interleukin 6 (IL-6) by inhibiting activation of signal transducer and activator of transcription 5 (Stat5) through interaction with Jak2. Notably, this activity of PrdxV was dependent on its catalytic Cys48 residue, but not its peroxidase activity. The binding of to Jak2 effectively inhibited Jak2 phosphorylation, but PrdxV did not act as efficiently as SOCS1 (suppressor of cytokine signaling 1). Our results suggest that PrdxV is a key mediator contributing to the regulation of LPS/TLR4-induced immune responses., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

A number of recent reports have demonstrated that attenuated Salmonella typhimurium are capable of targeting both primary and metastatic tumors. The use of bacteria as a vehicle for the delivery of anticancer drugs requires a mechanism that precisely regulates and visualizes gene expression to ensure the appropriate timing and location of drug production. To integrate these functions into bacteria, we used a repressor-regulated tetracycline efflux system, in which the expression of a therapeutic gene and an imaging reporter gene were controlled by divergent promoters (tetAP and tetRP) in response to extracellular tetracycline. Attenuated S. typhimurium was transformed with the expression plasmids encoding cytolysin A, a therapeutic gene, and renilla luciferase variant 8, an imaging reporter gene, and administered intravenously to tumor-bearing mice. The engineered Salmonella successfully localized to tumor tissue and gene expression was dependent on the concentration of inducer, indicating the feasibility of peripheral control of bacterial gene expression. The bioluminescence signal permitted the localization of gene expression from the bacteria. The engineered bacteria significantly suppressed both primary and metastatic tumors and prolonged survival in mice. Therefore, engineered bacteria that carry a therapeutic and an imaging reporter gene for targeted anticancer therapy can be designed as a theranostic agent.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the unintentional byproduct of various industrial processes, is classified as human carcinogen and could disrupt reproductive, developmental and endocrine systems. Induction of cyp1a1 is used as an indicator of TCDD exposure. We sought to determine tissues that are vulnerable to TCDD toxicity using a transgenic zebrafish (Danio rerio) model. We inserted a nuclear enhanced green fluorescent protein gene (EGFP) into the start codon of a zebrafish cyp1a gene in a fosmid clone using DNA recombineering. The resulting recombineered fosmid was then used to generate cyp1a reporter zebrafish, embryos of which were exposed to TCDD. Expression pattern of EGFP in the reporter zebrafish mirrored that of endogenous cyp1a mRNA. In addition, exposure of the embryos to TCDD at as low as 10 pM for 72 h, which does not elicit morphological abnormalities of embryos, markedly increased GFP expression. Furthermore, the reporter embryos responded to other AhR ligands as well. Exposure of the embryos to TCDD revealed previously reported (the cardiovascular system, liver, pancreas, kidney, swim bladder and skin) and unreported target tissues (retinal bipolar cells, otic vesicle, lateral line, cloaca and pectoral fin bud) for TCDD. Transgenic cyp1a reporter zebrafish we have developed can further understanding of ecotoxicological relevance and human health risks by TCDD. In addition, they could be used to identify agonists of AhR and antidotes to TCDD toxicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)