Genetically-modified Strain Research Articles

Analysis of the properties of Komagataella phaffii CF-st401, a genetically modified producer of the sweet protein brazzein by using in silico methods

Currently, in order to reduce the consumption of mono- and disaccharides in diets, sweeteners are widely used. At the same time, none of the sweeteners approved for food industry usage matches the organoleptic properties of natural sugars. This circumstance was the basis for the development of technology for producing a new type of food ingredient with a sweet taste - brazzein using the producer strain Komagataella phaffii CF-st401. The purpose of the study was the application of in silico methods to assess the safety of K. phaffii CF-st401 genetically modified (GM) microorganism, which is the producer of the "Sweet protein Brazzein". Material and methods. The research object was the map of K. phaffii CF-st401 plasmid obtained by Biryuch LLC as a result of sequencing the plasmid of GM strain K. phaffii CF-st401, including: a synthetic nucleotide sequence similar to the sequence encoding the brazzein protein in the plant (Pentadiplandra brazzeana) and optimized for expression in the DNA of the recipient strain; the nucleotide sequence of plasmid M4794 from Saccharomyces cerevisiae and a linear fragment of the flanking DNA regions from K. phaffii used for integration into the genome of the recipient strain K. phaffii YIB Δleu2 VKPM Y-476, as well as amino acid sequence data of recombinant brazzein. By using bioinformatics methods (in silico), we investigated the DNA structure of the vector sequence of K. phaffii CF-st401, including the presence of operons responsible for toxin production, antibiotic resistance, and allergenicity. Results. As a result of the studies of K. phaffii CF-st401 vector plasmid, introduced into K. phaffii YIB Δleu2 VKPM Y-4761, it was shown that its regions responsible for the structure of the "Sweet protein Brazzein" coincide by more than 70% with elements of the brazzein P56552 reference protein from the plant P. brazzeana. The absence of selective markers and allergenicity in the vector plasmid was confirmed. Conclusion. The analysis of the structure of the DNA vector sequence of the K. phaffii CF-st401 GM strain confirmed the feasibility of using bioinformatics methods to predict the properties of technological microorganisms when assessing their safety for consumers.

Safety and efficacy of a feed additive consisting of endo-1,4-β-d-mannanase produced by Paenibacillus lentus DSM 33618 (Hemicell® HT/HT-L) for chickens and turkeys for fattening, chickens reared for laying, turkeys reared for breeding, minor poultry species to point of lay, pigs for fattening, weaned piglets and minor porcine species (Elanco GmbH).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of endo-1,4-β-d-mannanase (Hemicell® HT/HT-L) produced by a genetically-modified strain of Paenibacillus lentus (DSM 33618) as a zootechnical feed additive for chickens and turkeys for fattening, chickens reared for laying, turkeys reared for breeding, minor poultry species to point of lay, pigs for fattening, piglets (weaned) and minor porcine species. The production strain was obtained from a Paenibacillus lentus recipient strain that has been evaluated previously by EFSA and considered to be safe. The genetic modification does not raise safety concerns and there were no antibiotic resistance genes from the genetic modification in the production strain. Viable cells and the DNA of the production strain were not found in the intermediate product used to formulate the additive. Hemicell® HT/HT-L produced by Paenibacillus lentus DSM 33618 is considered safe for the above-mentioned target species at the intended conditions of use. The use of Hemicell® HT/HT-L as a feed additive raises no concerns for the consumer or for the environment. Hemicell® HT/HT-L is not irritant to the skin and eyes but is regarded as a dermal sensitiser and a potential respiratory sensitiser. The additive has a potential to be efficacious at 32,000U/kg in chickens for fattening, chickens reared for laying, minor poultry species for fattening or reared for laying/breeding, pigs for fattening and minor porcine species, and at 48,000U/kg in turkeys for fattening, turkeys reared for breeding and weaned piglets.

First monitoring for unauthorized genetically modified bacteria in food enzymes from the food market

Recently, several methods were developed to detect genetically modified (GM) bacteria, allowing to screen for the potential presence of bacteria and to prove bacterial GM contaminations in fermentation products (such as FE). To obtain a first overview of the possible contamination of FE with GM bacteria, a general workflow was proposed, combining recently developed detection methods. Those methods include a 16S screening and viability assessment to target bacterial contaminations, as well as a first line and second line qPCR screening targeting bacterial GMM in FE preparations. Subsequently, the proposed workflow was applied on a total of 51 representative FE preparations that were collected from the EU market. On this basis, several FE contaminated with a bacterial GM strain, including the presence of full-length AMR genes, could be identified. In addition, the presence of two specific GMM (both viable and associated recombinant DNA) could be confirmed in multiple FE as well as the presence of viable GMM. On this basis, numerous bacterial contaminations, including GMM carrying full-length antimicrobial resistance (AMR) genes, were detected and confirmed. The generated results during this study have highlighted the importance to control such FE preparations for the potential contamination with their bacterial producer organism, including GMM. • FE preparations are often produced by GMM. • Analysis of commercial EU FE preparations to obtain a first picture of GMM presence. • The presence of numerous unauthorized GMM, carrying AMR genes, was demonstrated.

Biodiversity risk assessment of genetically modified Chaetoceros gracilis for outdoor cultivation.

A genetically modified (GM) strain of the diatom Chaetoceros gracilis expressing the phosphite dehydrogenase gene (ptxD), which is a useful gene both for the biological containment and the avoidance of microbial contamination, was characterized to estimate the risk against the biodiversity by laboratory experiments. GM strain could grow in the medium containing phosphite as a sole source of phosphorus, while its general characteristics such as growth, salt tolerance, heat and dehydration resistance in the normal phosphate-containing medium were equivalent to those of wild type (WT) strain. The increase in potential toxicity of GM strain against plant, crustacean, fish and mammal was also disproved. The dispersal ability of WT strain cultured in an outdoor raceway pond was investigated for 28 days by detecting the psb31 gene in vessels, settled at variable distances (between 5 and 60 m) from the pond. The diatom was detected only in one vessel placed 5 m apart. To estimate the influence on the environment, WT and GM strains were inoculated into freshwater, seawater and soil. The influence on the microbiome in those samples was assessed by 16S rRNA gene amplicon sequencing, in addition to the analysis of the survivability of those strains in the freshwater and the seawater. The results indicated that the effect to the microbiome and the survivability were comparable between WT and GM strains. All results showed that the introduction of the ptxD gene into the diatom had a low risk on biodiversity.

Characterization of Genetically Modified Microorganisms Using Short- and Long-Read Whole-Genome Sequencing Reveals Contaminations of Related Origin in Multiple Commercial Food Enzyme Products.

Despite their presence being unauthorized on the European market, contaminations with genetically modified (GM) microorganisms have repeatedly been reported in diverse commercial microbial fermentation produce types. Several of these contaminations are related to a GM Bacillus velezensis used to synthesize a food enzyme protease, for which genomic characterization remains currently incomplete, and it is unknown whether these contaminations have a common origin. In this study, GM B. velezensis isolates from multiple food enzyme products were characterized by short- and long-read whole-genome sequencing (WGS), demonstrating that they harbor a free recombinant pUB110-derived plasmid carrying antimicrobial resistance genes. Additionally, single-nucleotide polymorphism (SNP) and whole-genome based comparative analyses showed that the isolates likely originate from the same parental GM strain. This study highlights the added value of a hybrid WGS approach for accurate genomic characterization of GMM (e.g., genomic location of the transgenic construct), and of SNP-based phylogenomic analysis for source-tracking of GMM.

Biosorption of cyanate by two strains of Chlamydomonas reinhardtii: evaluation of the removal efficiency and antioxidants activity

Two strains of the chlorophyte Chlamydomonas reinhardtii, a wild type (WT) and a transgenic strain (C.CYN) contained an exogenous cyanase gene (CYN), were used to investigate the growth and cyanate biosorption capability through the analysis of the adsorption equilibrium isotherm. The potential antioxidants activity of the algal strains was also investigated under cyanate concentration. The antioxidants activity of both C.CYN and WT were enhanced by the application of cyanate. Two adsorption isotherm models and the sorption kinetics were used to check the efficiency of the cyanate removal process. The results showed the biosorbent efficiency of Chlamydomonas in the removal of KCNO from aqueous solution. The C.CYN strain has great efficiency to remove cyanate as compared to the WT. The maximum percentage of cyanate removal was 83.75% for the C.CYN and 50% for the WT as treated with 0.8 mg.ml−1 KCNO. The data were adapted to the nonlinear Langmuir model on the basis of the coefficient of determination. The calculated qmax was 0.54 and 0.42 µg.mg−1 for C.CYN and WT which correlated to the experimental one (0.67 and 0.4 µg.mg−1, respectively). Our data highlight the application of the transgenic algal strain toward the removal of highly toxic materials as cyanate. Novelty statement The main objective of this work is to find out an efficient genetically-modified Chlamydomonas strain to remove the highly toxic cyanate compound from contaminated area. Moreover, to evaluate the biosorption ability of this transgenic strain with its wild one via two adsorption isotherm (the Langmuir and Freundlich) models. Also, to estimate the antioxidants activity of these strains under the cyanate toxicity through four different assays.

Risk assessment of glucoamylase and xylanase complex from Aspergillus awamori Xyl T-15

The introduction of methods for food production using microbial synthesis, including those obtained with the help of genetically modified (GM) microorganisms, at the present stage, allows to increase production volumes and reduce the cost of food. At the same time, such products in accordance with TR CU 021/2011 "On food safety" are classified as a "novel food"» and can be placed on the market only after its risk estimation for health. The emergence of new data and research methods in the last years has made it necessary to improve the risk assessment system for this category of food. The aim of the research is to develope risk assessment approaches of food obtained by microbial synthesis on the example of the GM strain Aspergillus awamori Xyl T-15 and the enzyme preparation (EP) (a complex of glucoamylase and xylanase) produced by it. Material and methods. Outbred ICR mice (CD-1) and Wistar rats (males and females) were used in the experimental studies. Investigations of GM strain A. awamori Xyl T-15 virulence and its ability to disseminate internal organs have been carried out. Acute and subacute (during 80 days) toxicity of EP (a complex of glucoamylase and xylanase) have been studied. Results. The presented experimental data allow us to make a conclusion about the avirulence of the A. awamori Xyl T-15 strain, the lack of ability to disseminate internal organs (invasiveness). At the same time, the strain is characterized by the ability to produce mycotoxins (ochratoxin, fumonisin B2, T-2 and HT-2 toxins). The EP, a complex of glucoamylase and xylanase from A. awamori Xyl T-15, has a low oral acute toxicity for rats (LD50>5000 mg/kg). I ntragastric EP administration at doses of 10, 100 and 1000 mg/kg of body weight during 80 days had not revealed adversely affect on the rate of weight gain in animals, indicators of anxiety and cognitive function, and some studied biochemical indicators. At a dose of 100 mg/kg b.w. or more, there were changes in the relative mass of organs (lungs, kidneys, adrenal glands), small shifts in the parameters of erythropoiesis and leukocyte formula, at a dose of 1000 mg/kg b.w. - an increase in oxidative DNA destruction. T he most pronounced and dose-dependent was the effect of the EP on hepatocyte apoptosis. According to this indicator, the not observed adverse effect level (NOAEL) for EP is not more than 100 mg/kg b.w. in terms of protein. The main target organ for the toxic effect of EP is the liver. Conclusion. The data obtained demonstrate the necessity to conduct an additional analysis of the risks of possible negative effects of EP, namely, to study its impact on the gut microbiocenosis and the immune status of experimental animals, to analyze the presence of determinants of pathogenicity and antibiotic resistance, DNA of selective marker genes of A. awamori Xyl T-15 strain by PCR analysis and DNA sequencing methods.

Effects of Chinese honeybee foraging on oilseed rape gene flow and honey ingredients

SUMMARYHoneybee foraging can transfer exogenous genes from genetically modified (GM) oilseed rape (Brassica napusL.) to closely related plants, which not only induces potential ecological risks but also contaminates non-GM seeds or honey products with GM ingredients. These events may lead to international trade disputes. Chinese honeybees (Apis cerana ceranaFabricius) and a herbicide (glufosinate)-resistant GM strain ofB. napus(Z7B10) were studied to examine the effects of honeybee short-range foraging on oilseed rape gene flow and honey ingredients. Results showed variable frequencies of gene flow between GM and non-GM oilseed rape cultivars, with the highest frequency under nylon net isolation with artificially stocked honeybees, the lowest frequency under nylon net isolation alone, and an intermediate frequency under natural pollination, suggesting the important role of honeybee foraging in gene flow frequency. Additionally, GM pollen grains were found in honey collected from honeybees foraging on both GM and non-GM oilseed rape cultivars. The phosphinothricin acetyltransferase protein was also detected in both unbroken pollen-containing and pollen-free honey by protein testing strips, suggesting that honeybee foraging on GM oilseed rape could lead to contamination with GM ingredients. Overall, the results provide a direct scientific basis for the ecological risk assessment and safety management of GM oilseed rape.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive.

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed.

Co-Utilization of Glucose and Xylose for Enhanced Lignocellulosic Ethanol Production with Reverse Membrane Bioreactors.

Integrated permeate channel (IPC) flat sheet membranes were examined for use as a reverse membrane bioreactor (rMBR) for lignocellulosic ethanol production. The fermenting organism, Saccharomyces cerevisiae (T0936), a genetically-modified strain with the ability to ferment xylose, was used inside the rMBR. The rMBR was evaluated for simultaneous glucose and xylose utilization as well as in situ detoxification of furfural and hydroxylmethyl furfural (HMF). The synthetic medium was investigated, after which the pretreated wheat straw was used as a xylose-rich lignocellulosic substrate. The IPC membrane panels were successfully used as the rMBR during the batch fermentations, which lasted for up to eight days without fouling. With the rMBR, complete glucose and xylose utilization, resulting in 86% of the theoretical ethanol yield, was observed with the synthetic medium. Its application with the pretreated wheat straw resulted in complete glucose consumption and 87% xylose utilization; a final ethanol concentration of 30.3 g/L was obtained, which corresponds to 83% of the theoretical yield. Moreover, complete in situ detoxification of furfural and HMF was obtained within 36 h and 60 h, respectively, with the rMBR. The use of the rMBR is a promising technology for large-scale lignocellulosic ethanol production, since it facilitates the co-utilization of glucose and xylose; moreover, the technology would also allow the reuse of the yeast for several batches.

Comparison of the selective targeting efficacy of Salmonella typhimurium A1-R and VNP20009 on the Lewis lung carcinoma in nude mice.

Salmonella typhimurium A1-R is auxotrophic for arg and leu, which attenuates growth in normal tissue but allows high tumor targeting and virulence. A1-R is effective against metastatic human prostate, breast, and pancreatic cancer as well as osteosarcoma, fibrosarcoma, and glioma in clinically-relevant mouse models. VNP20009 is also a genetically-modified strain of Salmonella typhimurium that has been tested in Phase I clinical trials, but is more attenuated than S. typhimurium A1-R and in addition of multiple amino-acid auxotrophs, is purine auxotropic with the purI mutation. In the present study, mouse Lewis lung carcinoma-bearing nude mouse models were treated with S. typhimurium A1-R or VNP20009. S. typhimurium A1-R and VNP20009 were both eliminated from the liver and spleen approximately 3-5 days after administration via the tail vein. However, A1-R showed higher tumor targeting and inhibited the Lewis lung carcinoma to a greater extent than VNP20009, with less body weight loss. The mice tolerated S. typhimurium A1-R to at a least 2-fold higher dose than VNP20009 when the bacteria were administered iv. The results of the present study suggest that S. typhimurium A1-R has greater clinical potential than VNP20009.

Abstract 710: The efficacy of tumor-targeting Salmonella typhimurium A1-R on bone metastasis

Abstract We have previously developed a genetically-modified bacterial strain of Salmonella typhimurium (A1-R), which expresses GFP. In this study, we measured the efficacy of A1-R on breast cancer bone metastasis. We established an early-stage bone-metastasis model in nude mice by cardiac injection and a late-stage model by injection into the intramedullary cavity of the tibia using MDA-MB-435 human breast cancer cells (Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo. PLoS One 5, e9712, 2010). Both mouse models of bone metastasis were treated with A1-R. Fluorescence imaging of the mice was performed to visualize the metastatic bone lesions. In a first set of experiments, we showed that A1-R invaded and replicated intracellularly in MDA-MB-435-GFP cells in vitro. A1-R dose-dependently inhibited proliferation of MDA-MB-435-GFP cells. In the early-stage bone-metastasis model, A1-R significantly improved metastasis-free survival. In the advanced-stage bone-metastasis model, A1-R significantly inhibited the growth of the metastatic lesions. These data indicated that A1-R is useful to prevent and inhibit the growth of metastatic breast cancer bone tumors. Citation Format: Shinji Miwa, Ming Zhao, Shuya Yano, Yong Zhang, Fuminari Uehara, Yasunori Matsumoto, Yukihiko Hiroshima, Mako Yamamoto, Hiroaki Kimura, Katsuhiro Hayashi, Hiroyuki Tsuchiya, Robert M. Hoffman. The efficacy of tumor-targeting Salmonella typhimurium A1-R on bone metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 710. doi:10.1158/1538-7445.AM2014-710

Abstract 704: Comparison of Salmonella typhimurium A1-R and VNP20009 on the Lewis Lung carcinoma

Abstract Salmonella typhimurium A1-R (A1-R) is auxotrophic for arg and leu, which attenuates growth in normal tissue but allows high tumor virulence. A1-R is effective against metastatic human prostate, breast, and pancreatic cancer as well as osteosarcoma, fibrosarcoma, and glioma in clinically-relevant mouse models. VNP20009 is also a genetically-modified strain of Salmonella typhimurium that has been tested in a Phase I clinical trial, but has more attenuation mutations than A1-R. Lewis-lung mouse lung cancer-bearing mice were treated with A1-R and VNP20009 in nude mice. A1-R and VNP20009 were both eliminated from the liver and spleen approximately 3-5 days after administration via tail vein, however A1-R showed higher tumor targeting and increased mouse survival than VNP20009 with less body weight loss. The mice tolerated A1-R at least 2-fold higher than NVP20009 when bacteria were administered iv. The results of the present study suggest that A1-R has greater clinical potential than VNP20009. Citation Format: Yong Zhang, Nan Zhang, Robert M. Hoffman, Ming Zhao. Comparison of Salmonella typhimurium A1-R and VNP20009 on the Lewis Lung carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 704. doi:10.1158/1538-7445.AM2014-704

Genetically modified mosquito: The Malaysian public engagement experience

On December 21, 2010, 6000 genetically modified (GM) mosquitoes were released in an uninhabited forest in Malaysia. The purpose of the deliberate release was a limited “marked release and recapture” (MRR) experiment, a standard ecological method in entomology, to evaluate under field conditions, the flight distance and longevity of the sterile male Aedes aegypti strain OX513A(My1), a GM strain. As with any other GM technologies, the release was received with mixed responses. As the scientific community debate over the public engagement strategies for similar GM releases, dengue incidence continues to rise with a heavy toll on morbidity, mortality and healthcare budgets. Meanwhile the wild female Aedes aegypti continues to breed offspring, surviving and evading conventional interventions for vector control.

Abstract 2685: Imaging the various mechanisms of cancer-cell killing by Salmonella typhimurium A1-R

Abstract We have developed a genetically-modified bacterial strain of Salmonella typhimurium, auxotrophic for leucine and arginine, which also expresses green fluorescent protein (GFP), termed S. typhimurium A1-R. S. typhimurium A1-R was found to be effective against metastatic human prostate, breast, and pancreatic cancer cell lines as well as osteosarcoma, fibrosarcoma and glioma cell lines in clinically-relevant nude mouse models. To understand the tumor-killing mechanism of S. typhimurium A1-R-GFP, we have studied their interaction with fluorescence-labeled cancer cells in vitro and in vivo. S. typhimurium-GFP invasion, proliferation, and virulence in the cancer cells were visualized by confocal fluorescence microscopy with the Olympus FV1000. We found that A1-R-induced cancer cell death had different mechanisms in different cancer cell lines. Some S. typhimurium A1-R-treated cancer cell lines became apoptotic and necrotic, and other cell lines burst after extensive intracellular bacterial growth. The time for S. typhimurium A1-R-GFP to kill the majority of the cancer cells varied from line to line, ranging from 2 hours to 72 hours. The understanding of the various mechanisms of cell killing by S. typhimurium A1-R will be important for its use as a general therapeutic for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2685. doi:1538-7445.AM2012-2685

Abstract 2677: Determination of the optimal route of administration of Salmonella typhimurium A1-R to target breast cancer in nude mice

Abstract We have previously developed the genetically-modified strain Salmonella typhimurium A1-R that selectively targets tumors. S. typhimurium A1-R is auxotrophic for leu and arg, which precludes it from growing in normal tissues but allows high tumor virulence. We report here the efficacy and safety of three different routes of S. typhimurium A1-R delivery oral (p.o.), intravenous (i.v.), and intra-tumor (i.t.) in nude mice with orthotopic human breast cancer. Nude mice with MDA-MB-435 human breast cancer expressing red fluorescence protein (RFP) were administered S. typhimurium A1-R by either of 3 routes: (p.o.: 2 × 108 CFU/200 αl; i.v.: 2.5 × 107 CFU/100 αl; i.t.: 2.5 × 107 CFU/50 µl) twice a week. Tumor growth was monitored by florescence imaging and caliper measurement in two dimensions. Treatment with S. typhimurium A1-R via all three routes resulted in effective targeting and tumor growth inhibition. Efficacy was observed earliest in the animals treated by i.v. injection. GFP-expressing S. typhimurium A1-R were imaged to target, colonize, and replicate in the human breast tumor, liver, and spleen as observed by fluorescence imaging. Bacterial colonization of tumor and normal organs at different time points were determined after S. typhimurium delivery by either of the 3 routes. S. typhimurium A1-R targeted tumors at much higher levels than normal organs with all 3 routes of administration. The bacteria replicated and survived longer in the tumor than normal organs with all 3 routes of administration. Two weeks after bacteria administration by all 3 routes, the bacteria disappeared from normal organs, but grew continuously in the tumor. The fewest bacteria were detected in normal organs after oral administration, which suggests oral administration has the highest safety. The i.v. route had the greatest anti-tumor efficacy. There were no obvious toxic effects on the host with any of the routes of administration. Non-tumor-bearing mice tolerated S. typhimurium A1-R, p.o., twice weekly treatment and could survive as long as untreated mice. The results of this study suggest that oral administration was the most safe to the host and the i.v. route was most effective for tumor targeting with S. typhimurium A1-R. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2677. doi:1538-7445.AM2012-2677

Efficacy of a genetically modified Salmonella typhymurium against metastatic osteosarcoma in an orthotopic mouse model

10543 Background: We have previously developed a genetically-modified strain of S. typhimurium, selected for tumor targeting and therapy in vivo. Normal tissue is cleared of these bacteria even in immunodeficient athymic mice with no apparent side effects. Methods: In this study, the tumor-targeting strain of S. typhimurium, termed A1-R, was administered i.v. to nude mice which have primary bone tumor and lung metastasis. Primary bone tumor was obtained by orthotopic intratibial injection of 5 × 105143B-RFP (red fluorescent protein) human osteosarcoma cells. One group of mice was treated with A1-R and another group was used a as control. A1-R (5 × 107 colony-forming units) was injected in the tail vein three times on weekly basis. On day 28, lung samples were excised and observed with the Olympus OV100 Small Animal Imaging System. Results: The size of the primary tumor and RFP intensity of lung metastasis were measured. Primary bone tumor size (fluorescence area [mm2]) was 232 ± 70 in the untreated group and 95 ± 23 in the treated group (P<0.05). RFP intensity of the lung metastasis was 3 ± 1.5 × 106in the untreated group and 0.42 ± 0.33 × 106 in the treated group (P<0.05). No toxicity was observed in the treated mice. Conclusions: Therefore, bacterial treatment was effective for both primary bone tumor and lung metastasis. Treatment with the tumor-targeting but non-toxic mutant of S. typhimuriumis a promising approach to therapy of metastatic osteosarcoma. No significant financial relationships to disclose.

Systemic targeting of primary bone tumor and lung metastasis of high-grade osteosarcoma in nude mice with a tumor-selective strain of Salmonella typhymurium

We report here a new targeting strategy for primary bone tumor and lung metastasis with a modified auxotrophic strain of Salmonella typhimurium. We have previously developed the genetically-modified strain of S. typhimurium, selected for tumor targeting and therapy in vivo. Normal tissue is cleared of these bacteria even in immunodeficient athymic mice with no apparent side effects. In this study, the tumor-targeting strain of S. typhimurium, termed A1-R, was administered i.v. to nude mice which have primary bone tumor and lung metastasis. Primary bone tumor was obtained by orthotopic intratibial injection of 5 x 105 143B-RFP (red fluorescent protein) human osteosarcoma cells. One group of mice was treated with A1-R expressing GFP (green fluorescent protein) and another group was used a as control. A1-R (5 x 107 colony-forming units) was injected in the tail vein three times on weekly basis. On day 28, lung samples were excised and observed with the Olympus OV100 Small Animal Imaging System. The size of the primary tumor and RFP intensity of lung metastasis were measured. Primary bone tumor size (fluorescence area [mm2]) was 232 ± 70 in the untreated group and 95 ± 23 in the treated group (P

Cancer metastasis directly eradicated by targeted therapy with a modified Salmonella typhimurium

Cancer metastasis is the life-threatening aspect of cancer and is usually resistant to standard treatment. We report here a targeted therapy strategy for cancer metastasis using a genetically-modified strain of Salmonella typhimurium. The genetically-modified strain of S. typhimurium is auxotrophic for the amino acids arginine and leucine. These mutations preclude growth in normal tissue but do not reduce bacterial virulence in cancer cells. The tumor-targeting strain of S. typhimurium, termed A1-R, and expressing green fluorescent protein (GFP), was administered to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph node metastases and systemically via the tail vein to target the lung metastasis. The cancer cells expressed red fluorescent protein (RFP) in the cytoplasm and GFP in the nucleus linked to histone H2B, enabling color-coded real-time imaging of the bacteria targeting the metastatic tumors. After 7-21 days of treatment, the metastases were eradicated without the need of chemotherapy or any other treatment. No adverse effects were observed. This new strategy demonstrates the clinical potential of targeting and curing cancer metastasis with engineered bacteria without the need of toxic chemotherapy.

Production of biosurfactant by a genetically-modified strain ofBacillus subtilis expressing green fluorescent protein

Biosurfactant production was investigated using two strains ofBacillus subtilis, being one a reference strain (B. subtilis 1012) and the other a genetically-modified strain (B. subtilis W1012) made able to produce the green fluorescent protein (GFP). A new method based on oil displacement technique was set up to measure the biosurfactant level in the medium. Although the tested microorganisms showed similar results in terms of cell growth parameters, the recombinant strain, besides expressing GFP, exhibited an average yield of extracellular surfactant on biomass (Y B/X, av =239 mgB gx −1) more than twice that of the reference strain. The ability of the genetically-modified strain to simultaneously overproduce biosurfactant and GFP even at low cell concentration makes it an interesting candidate for possible use as a biological index-finger to monitor cell viability in bioremediation and oil recovery operations.