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Supplementary Figure 1 from Genetically Engineered Salmonella typhimurium as an Imageable Therapeutic Probe for Cancer

Supplementary Figure 3 from Genetically Engineered Salmonella typhimurium as an Imageable Therapeutic Probe for Cancer

Tumor-targeting bacteria have been studied in terms of their ability to visualize the infection pathway (through imaging probes) or to carry therapeutic molecules to tumors. To integrate these monitoring and therapeutic functions, we engineered attenuated Salmonella typhimurium defective in guanosine 5′-diphosphate-3′-diphosphate synthesis to carry cytotoxic proteins (cytolysin A) and express reporter genes. We successfully visualized the therapeutic process with these engineered bacteria in mice and found that they often mediated complete tumor (CT-26) eradication on cytotoxic gene induction. Furthermore, treatment with the engineered bacteria markedly suppressed metastatic tumor growth. Cancer Res; 70(1); 18–23

Supplementary Figure Legends 1-3 from Genetically Engineered Salmonella typhimurium as an Imageable Therapeutic Probe for Cancer

Supplementary Figure 2 from Genetically Engineered Salmonella typhimurium as an Imageable Therapeutic Probe for Cancer

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

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.

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 (~108 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 109 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.

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.

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-&kappa, B 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.

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 (GABAAR), 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., Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in neuronal systems, but the potential role for such neurotransmitters on the immune system are emerging. Here the authors show GABA signaling is linked to autophagy, enhancing the host response against intracellular bacteria.

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.

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.

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.

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., The effects of iron on vacuole-resident Salmonella in macrophages are unclear. Here the authors show that the bacteria are not subject to nutritional inhibition by iron deprivation, but that iron depletion in the vacuole, via the hepcidin-ferroportin axis, inhibits the bactericidal effect of oxidative burst.

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.

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 (Delta ppGpp 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 Delta ppGpp 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-1 beta and TNF-alpha were markedly increased in tumors colonized by Delta ppGpp Salmonellae. This increase was associated with tumor regression; the levels of both IL-1 beta and TNF-alpha 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-1 beta and TNF-alpha. We found that macrophages and dendritic cells were the main producers of TNF-alpha and IL-1 beta. Inhibiting IL-1 beta production in Salmonellae-treated mice restored tumor growth, whereas tumor growth was suppressed for longer by local administration of recombinant IL-1 beta or TNF-alpha in conjunction with Salmonella therapy. These findings suggested that IL-1 beta and TNF-alpha 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Department of Microbiology, Chonnam National University Medical School, Kwangju 501-746, KoreaReceived May 3, 2013, Accepted May 20, 2013Guanosine-5'-diphosphate 3'-diphosphate (ppGpp) serves as alarmone in bacterial stringent responses. In thisstudy, an affinity column was constructed by immobilizing ppGpp to NHS-Sepharose for isolating ppGpp-binding proteins. A novel ppGpp-binding protein, YjgA, was isolated and characterized by MALDI-TOF MS(matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) coupled with two-dimensionalgel electrophoresis. YjgA and truncated forms of YjgA were cloned and over-expressed in BL21 (DE3). Thebinding affinity of YjgA to ppGpp was determined by equilibrium dialysis. The interaction of YjgA withppGpp was very specific, considering that the dissociation constant of YjgA with ppGpp was measured as 5.2± 2.0 µM, while the affinities to GTP and GDP were about 60 and 30 times weaker than ppGpp. Expression ofyjgA gene in Escherichia coli K-12 MG1655 was examined by reverse transcription polymerase chain reaction(RT-PCR). RT-PCR results revealed that yjgA was expressed from early to late stationary phase. The yjgAdeletion mutant exhibited decreased cell number at stationary phase compared to parent strain and the over-expression of YjgA increased the cell number. These results suggested that YjgA might stimulate cell divisionunder stationary phase. In most prokaryotic genome, about half of the protein candidates are hypothetical, thatare expected to be expressed but there is no experimental report on their functions. The approach utilized in thisstudy may serve as an effective mean to probe the functions of hypothetical proteins. Key Words : YjgA, ppGpp, Stringent responses, Stationary phase, Hypothetical proteinIntroductionProkaryotes show stringent response to various nutritionalchanges, including amino acids (Haseltine and Block, 1973),carbon (Flardh et al., 1994), nitrogen (Silberbach et al.,2005) and phosphate (Bougdour and Gottesman, 2007)limitation. Cashel and Gallant first discovered guanosine-5'-diphosphate 3'-diphosphate (ppGpp) accumulation in bacteriaduring nutrient starvation (Cashel and Gallant, 1969), andRelA and SpoT serve as ppGpp synthetases in E. coli(Cashel et al., 1996). ppGpp binds to the β and β' subunits ofcore RNA polymerase, inhibits stable RNA biosynthesis(rRNA and tRNA) during growth inhibition (Ryals et al.,1982; Toulokhonov et al., 2001), and arrests cell cycles(Ferullo and Lovett, 2008). A complete absence of ppGppcreates its own phenotypic features including multiple aminoacid requirements, poor survival of aged culture, aberrantcell division, morphology, and immotility (Potrykus andCashel, 2008). Importantly, ppGpp influences the pattern ofoverall gene expression that requires for survival in stressconditions (Song et al., 2004). In E. coli, ppGpp acts as aglobal regulatory affects on gene expression through itsinfluence on the levels of global regulators such as IHF,RpoS and RpoH (Lange et al., 2005). The absence of ppGppimpairs or severely delays the accumulation of RpoS, astress response sigma factor (Gentry et al., 1993). In addi-tion to transcription, ppGpp regulates translation by inter-acting with EF-G and IF2 (Mitkevich et al., 2010). ppGpp isinvolved in the acid stress response via lysine decarboxylaseLdc1 (Kanjee et al., 2011), phosphate metabolism viapolynucleotide phosphorylase (Gatewood and Jones, 2010),and replication via DnaG primase (Maciag et al., 2010).With such pleotropic eects, ppGpp seems to be a majorregulator of bacterial growth rate (Potrykus et al., 2011). Inspite of the diverse functions connected to ppGpp, the mech-anism of ppGpp regulation remains controversial (Vrentas etal., 2008). The first genome structure was announced in 1995 andmore than 1,000 genome sequences are currently availablein public domain (Karin et al., 2010). Although the wholegenome provides rich information on metabolic pathwaysand their regulations, about a half of proteins in mostgenomes are annotated as hypothetical proteins (Bhatia etal., 1997). Hypothetical proteins, also referred as ‘putative’,‘uncharacterized’ or ‘unknown’ proteins, are those whoseexistence can be predicted but no experimental reports ontheir functions are available. Even for Escherichia coli K-12,the most studied organism, there are still about 1,500 genesthat have not been experimentally categorized (Koonin andGalperin, 2003). The functions of hypothetic proteins are

The use of quinolone for treatment of rickettsial diseases remains controversial. Recent clinical studies suggest that quinolone is not as effective as others in patients with rickettsial diseases including scrub typhus, although the mechanism is not well understood. In this study, we evaluated the mutation in gyrA associated with quinolone resistance. We prospectively enrolled scrub typhus patients, collected blood samples and clinical data from October, 2010 to November, 2011. Among the 21 patients enrolled, one initially received ciprofloxacin for 3 days but was switched to doxycycline due to clinical deterioration. We obtained the gyrA gene of Orientia tsutsugamushi from 21 samples (20 Boryong strain, 1 Kato strain) and sequenced the quinolone resistance-determining region. All of 21 samples had the Ser83Leu mutation in the gyrA gene, which is known to be associated with quinolone resistance. This suggests that quinolones may be avoided for the treatment of serious scrub typhus.

Vibrio vulnificus causes fatal septicemia in susceptible subjects after the ingestion of raw seafood. In the present study, the roles of cyclic adenosine monophosphate (cAMP) receptor protein (CRP) in V. vulnificus pathogenesis were investigated. A mutation in the V. vulnificus crp gene resulted in a significant down-regulation of various virulence phenotypes, except for RtxA1-mediated cytoskeletal rearrangement. Bacterial growth was impeded by the crp mutation. In addition, colony morphology was converted from opaque to translucent type by this mutation, which implies a decrease in capsule production. The crp mutant also showed significant decrease in motility and adhesion to host cells. V. vulnificus CRP positively regulated production of hemolysin and protease at transcriptional level. All these changes in the crp mutant were fully complemented in trans by a plasmid harboring the wild-type gene. In contrast, CRP negatively regulated the expression of RtxA1. The crp mutant caused the cytoskeletal rearrangement in HeLa cells, which is a hallmark activity of RtxA1 toxin. Taken together, CRP seems to play a dual regulatory role in various virulence traits of V. vulnificus.

Vibrio vulnificus, a halophilic estuarine bacterium causing fatal septicemia and necrotic wound infection, is highly cytotoxic to eukaryotic cells. We have reported that RtxA1 toxin kills host cells only after they come into contact with bacteria and plays an essential role in the pathogenesis of V. vulnificus. This study was performed to elucidate the mechanism by which the RtxA1 toxin mediates the death of HeLa cells. By using confocal microscopy and immunoblot analysis, we show that the 501-kDa RtxA1 toxin is processed into 2 fragments after its secretion into host cells. The largerN-terminal fragment (RtxA1-N; approximately 370 kDa) remained at the host cell membrane, whereas the smaller C-terminal fragment (RtxA1-C; approximately 130 kDa) was internalized into the host cell cytoplasm. RtxA1-N is believed to polymerize and form pores at the host cell membrane and to induce an increase in necrotic volume related to calcium. The RtxA1 toxin caused an increase in the intracellular Ca(2+) concentration and the subsequent activation of JNK. The cell death mechanism occurred via calcium-dependent mitochondrial pathways, which caused calcium sequestration in the mitochondria, accompanied by irreversible mitochondrial membrane dysfunction and adenosine triphosphate depletion, and was later accompanied by the disruption of the integrity of the plasma membrane.

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.

We report a method of cancer immunotherapy using an attenuated

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 alpha v beta 3, but weakly bound to alpha v beta 3-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 alpha v beta 3-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.

In the modern concept of gene regulation, 'DNA looping' is the most common underlying mechanism in the interaction between RNA polymerase (RNAP) and transcription factors acting at a distance. This study demonstrates an additional mechanism by which DNA-bound proteins communicate with each other, by analysing the bacterial histone-like nucleoid-structuring protein (H-NS), a general transcriptional silencer. The LEE5 promoter (LEE5p) of enteropathogenic Escherichia coli was used as a model system to investigate the mechanism of H-NS-mediated transcription repression. We found that H-NS represses LEE5p by binding to a cluster of A tracks upstream of -114, followed by spreading to a site at the promoter through the oligomerization of H-NS molecules. At the promoter, the H-NS makes a specific contact with the carboxy terminal domain of the α subunit of RNAP, which prevents the processing of RNAP-promoter complexes into initiation-competent open promoter complexes, thereby regulating LEE5p from distance.

The use of bacteria has contributed to recent advances in targeted cancer therapy especially for its tumor-specific accumulation and proliferation. In this study, we investigated the molecular events following bacterial therapy using an attenuated Salmonella Typhimurium defective in ppGpp synthesis (ΔppGpp), by analyzing those proteins differentially expressed in tumor tissues from treated and untreated mice. CT26 murine colon cancer cells were implanted in BALB/c mice and allowed to form tumors. The tumor-bearing mice were treated with the attenuated Salmonella Typhimurium. Tumor tissues were analyzed by 2D-PAGE. Fourteen differentially expressed proteins were identified by mass spectrometry. The analysis revealed that cytoskeletal components, including vimentin, drebrin-like protein, and tropomyosin-alpha 3, were decreased while serum proteins related to heme or iron metabolism, including transferrin, hemopexin, and haptoglobin were increased. Subsequent studies revealed that the decrease in cytoskeletal components occurred at the transcriptional level and that the increase in heme and iron metabolism proteins occurred in liver. Most interestingly, the same pattern of increased expression of transferrin, hemopexin, and haptoglobin was observed following radiotherapy at the dosage of 14 Gy.

Transcription start points in bacteria are influenced by the nature of the RNA polymerase·promoter interaction. For Escherichia coli RNA polymerase holoenzyme containing σ70, it is presumed that specific sequence in one or more of the -10, extended -10 and -35 elements of the promoter guides the RNAP to select the cognate start point. Here, we investigated the promoter driving expression of the LEE1 operon in enteropathogenic E. coli and found two promoters separated by 10 bp, LEE1 P1A (+1) and LEE1 P1B (+10) using various in vitro biochemical tools. A unique feature of P1B was the presence of multiple transcription starts from five neighbouring As at the initial transcribed region. The multiple products did not arise from stuttering synthesis. Analytical software based on information theory was employed to determine promoter elements. The concentration of the NTP pool altered the preferred transcription start points, albeit the underlying mechanism is elusive. Under in vivo conditions, dominant P1B, but not P1A, was subject to regulation by IHF.