Salmonella enterica Typhimurium engineered for nontoxic systemic colonization of autochthonous tumors.

Much of the bacterial anticancer therapy being developed relies on the ability of bacteria to specifically colonise tumours. Initial attempts to translate promising Salmonella enterica Typhimurium (S. Typhimurium) preclinical results to the clinical setting failed, primarily due to lack of tumour colonisation and the significant toxicities from systemically administered Gram-negative bacteria. To address the difference in results between preclinical experiments performed in mice with transplant tumours and clinical trials in human volunteers with autochthonous tumours, a genetically engineered mouse model of breast cancer (BALB-neuT) was utilised to develop a strain of virulence-attenuated S. Typhimurium capable of robust colonisation of autochthonous tumours. Several genes that code for bacterial surface molecules, responsible for signalling a toxic immune response against the bacteria, were mutated. The resulting S. Typhimurium strain, BCT2, allowed non-toxic intravenous administration of 3 × 106 colony forming units of bacteria in tumour-burdened mice when combined with a vascular disruption agent to induce intratumoral necrotic space and facilitate bacterial colonisation.

Virulence-attenuated Salmonella engineered to secrete immunomodulators reduce tumour growth and increase survival in an autochthonous mouse model of breast cancer.

The ultimate goal of bacterial based cancer therapy is to achieve non-toxic penetration and colonisation of the tumour microenvironment. To overcome this efficacy-limiting toxicity of anticancer immunotherapy, we have tested a therapy comprised of systemic delivery of a vascular disrupting agent to induce intratumoral necrotic space, cannabidiol to temporarily inhibit angiogenesis and acute inflammation, and a strain of Salmonella Typhimurium that was engineered for non-toxic colonisation and expression of immunomodulators within the tumour microenvironment. This combination treatment strategy was administered to transgenic mice burdened with autochthonous mammary gland tumours and demonstrated a statistically significant 64% slower tumour growth and a 25% increase in mean survival time compared to control animals without treatment. These experiments were accomplished with minimal toxicity as measured by less than 7% weight loss and a return to normal weight gain within three days following intravenous administration of the bacteria. Thus, non-toxic, robust colonisation of the microenvironment was achieved to produce a significant antitumor effect.

A Phase I, Dose Escalation, Single Dose Trial of Oral Attenuated Salmonella typhimurium Containing Human IL-2 in Patients With Metastatic Gastrointestinal Cancers.

Salmonella has been shown to preferentially colonize solid tumors. It is known that toxicity limits the systemic administration of immunomodulatory cytokines that have a significant anticancer effect. Therefore, we tested a unique cancer treatment strategy comprised of oral delivery of Saltikva, an attenuated strain of Salmonella typhimurium that contain the human gene for interleukin-2. In preclinical experimentation, a significant antitumor effect without toxicity was observed. A dose escalation, single dose, Phase I trial was conducted. Dose escalation (10 to 10) while monitoring for dose limiting toxicity and response was performed. Flow cytometry was conducted to determine the immunologic effect. In total 22 patients were administered Saltikva. Eight patients did not complete the trial. No toxicity or adverse events were observed. There was no survival advantage. Flow cytometry demonstrated an increase in circulating natural killer (NK) cells and NK-T cells when comparing the prestudy period. The results of this phase I dose escalation study show that oral attenuated S. typhimurium containing the human interleukin-2 gene caused no significant toxicities up to doses of 10 colony forming unit. There was no evidence of partial or complete response. All patients had progressive disease and eventually succumbed to their illness. Although no survival advantage was seen in this single dose study, the statistically significant increase in circulating NK and NK-T cell demonstrates an immunologic effect from this treatment regimen and suggest that a multiple dose study should be undertaken.

Low dose chemotherapy combined with attenuated Salmonella decreases tumor burden and is less toxic than high dose chemotherapy in an autochthonous murine model of breast cancer.

BACKGROUND: Immunotherapies for cancer treatment have demonstrated substantial promise even though toxicities and development of tumor resistance limit their effectiveness. A combinatorial approach using immunotherapy with other treatment modalities may decrease side effects while maintaining maximal therapeutic effect. We aimed to determine if bacterial immunotherapy in combination with a chemotherapeutic would be efficacious and less toxic than conventional chemotherapy in an established, preclinical, autochthonous tumor model. METHODS: BALB-neuT mice develop autochthonous mammary neoplasms that resemble closely the aggressive Her2-driven cancer found in human patients. Virulence-attenuated S. Typhimurium was used for bacterial immunotherapy. Doxorubicin was the chemotherapeutic agent used at the maximum tolerated dose (5 mg/kg) and low dose (1.25 mg/kg). S. Typhimurium was administered intravenously on day 0 and doxorubicin on days 0, 7, and 14. Experiments concluded on day 35. Mammary pad tumors were measured weekly to ascertain efficacy, and mice were weighed weekly to evaluate toxicity. RESULTS: Mice administered maximum tolerated dose doxorubicin (5 mg/kg) demonstrated a 1.4-fold increase in tumor size by day 35 and showed a nearly 25% weight loss by day 14 revealing severe toxicity. When mice were administered a single dose of S. Typhimurium combined with a low dose of doxorubicin (1.25 mg/kg), tumors increased <3-fold by day 35, and mice showed only 5% weight loss, indicating no clinically relevant toxicity. CONCLUSION: Bacterial immunotherapy combined with low dose chemotherapy decreased the tumor burden when compared with low dose chemotherapy alone and was less toxic than maximum tolerated dose chemotherapy in an established, autochthonous murine model of breast cancer.

Vasculature Disruption Enhances Bacterial Targeting of Autochthonous Tumors.

Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has been developed as a vector to deliver therapeutic agents to tumors. The potential of S. Typhimurium in cancer therapy is largely due to its reported propensity to accumulate at greater than 1,000-fold higher concentrations in tumors relative to healthy tissues. In this study, we compared bacterial colonization of tumors in a subcutaneous transplantation model with a more clinically relevant autochthonous tumor model. Following intravenous administration of attenuated S. Typhimurium strain SL3261, we observed approximately 10,000-fold less bacteria in autochthonous tumors that sporadically develop in transgenic BALB-neuT mice compared to tumors developed from subcutaneous transplantation of 4T1 murine breast cancer cells in BALB/c mice. Treatment of BALB-neuT mice with a vasculature-disrupting agent (VDA) prior to bacterial treatment caused necrosis of tumor tissue and significantly increased the bacterial targeting of autochthonous tumors by approximately 1,000-fold. These observations emphasize the importance of appropriate model selection in developing bacteria-based cancer therapies and demonstrate the potential of combining VDA pre-treatment with bacteria to facilitate targeting of clinically relevant tumors.

Exploring metagenomics in the laboratory of an introductory biology course.

Four laboratory modules were designed for introductory biology students to explore the field of metagenomics. Students collected microbes from environmental samples, extracted the DNA, and amplified 16S rRNA gene sequences using polymerase chain reaction (PCR). Students designed functional metagenomics screens to determine and compare antibiotic resistance profiles among the samples. Bioinformatics tools were used to generate and interpret phylogenetic trees and identify homologous genes. A pretest and posttest were used to assess learning gains, and the results indicated that these modules increased student performance by an average of 22%. Here we describe ways to engage students in metagenomics-related research and provide readers with ideas for how they can start developing metagenomics exercises for their own classrooms.

Attenuated Salmonella enterica Typhimurium reduces tumor burden in an autochthonous breast cancer model.

BACKGROUND/AIM: Cancer treatment with attenuated Salmonella enterica Typhimurium (S. Typhimurium) has gained momentum in recent years. However, the effectiveness of this treatment has not been explored in autochthonous models. We report the efficacy of S. Typhimurium in mice with autochthonous mammary tumors. MATERIALS AND METHODS: S. Typhimurium attenuated by deletion of cyclic adenosine monophosphate signaling, SalpNG.1, was injected into female BALB-neuT tumor-bearing mice. Mice were monitored for efficacy and sacrificed for mechanistic studies. RESULTS: In treated mice, seven-week post-treatment tumor burden was reduced by 85% and median survival was increased by 88%. Efficacy was correlated with increased tumor-infiltrating CD8 and natural killer cells. In addition, SalpNG.1 treatment caused a systemic increase of monocytic myeloid-derived suppressor cells that accumulated to high numbers within tumor tissue. Bacteria were not detected in tumor tissue, suggesting that the observed efficacy was due to a systemic rather than a tumor-specific effect of the bacteria. CONCLUSION: S. Typhimurium treatment reduces tumor burden and increases survival in an autochthonous breast cancer model.

Expression of periplasmic chaperones in Salmonella Typhimurium reduces its viability in vivo.

The efficacy of live attenuated bacterial vectors is dependent upon the fine-tuning of a strain's immunogenicity and its virulence. Strains are often engineered to deliver heterologous antigens, but soluble expression of recombinant proteins can be troublesome. Therefore, secretion systems or chaperone proteins are routinely used to assist in attaining high levels of functional, soluble protein production. However, the effects of chaperone expression on the virulence of attenuated bacterial vectors have not been previously reported. In anticipation of utilizing periplasmic chaperone proteins to facilitate soluble production of immunomodulatory proteins in an attenuated strain of Salmonella Typhimurium, the production of the chaperones was tested for their effect on both culture growth and bacterial persistence in mouse tissues. Although no effect on growth of the bacteria was observed in vitro, the increased expression of the periplasmic chaperones resulted in over-attenuation of the Salmonella in vivo.

Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli.

Programmed death ligand 1 (PD-L1), is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. Single chain variable fragments (scFvs) have several desirable characteristics and are an attractive alternative to monoclonal antibodies for experimental or therapeutic purposes. Three chickens were immunized against murine PD-L1, and mRNA isolated from their spleens was used to generate an immunized immunoglobulin variable region library. Using splice-overlap extension PCR, variable region cDNAs were combined to generate full-length scFvs. M13 phage display of the resulting scFv library identified a functional scFv against PD-L1 (αPD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6×-His tag using immobile metal affinity chromatography. The dissociation constant of αPD-L1 scFv was determined to be 7.11×10(-10)M, and the scFv demonstrated inhibitory biological activity comparable to an antagonistic monoclonal antibody, providing an alternative agent for blocking PD-1/PD-L1 signaling.

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery.

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T(m)), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T(m) from 8.4+/-1.7 degrees C (in undried control samples) to 16.5+/-1.3 degrees C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T(m), which began approximately 1 day after initial rehydration and plateaued at 12.5+/-1.8 degrees C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T(m) and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.

Spatial expression of a mercury-inducible green fluorescent protein within a nanoporous latex-based biosensor coating.

Optimizing the reactivity of cell coatings developed as biosensors or biocatalysts requires measurements of gene expression in the immobilized cells. To quantify and localize gene expression within a latex-based mercury biosensor, a plasmid, pmerGFP, was constructed, which contains the green fluorescent protein (GFP) gene under transcriptional control of the mercury resistance operon regulatory sequences. When cells containing this plasmid were exposed to mercuric chloride, GFP synthesis was induced and could be quantified by fluorescence. E. coli strain JM109 (pmerGFP) was mixed with SF091 latex (Rohm & Haas), Tween 20, and glycerol, and coated as an approximate 20-microm thick nanoporous adhesive coating on a polyester substrate. The cell coat was overlaid with a nanoporous topcoat of latex, Tween 20, and glycerol. Different fluorescent microspheres were used to mark the topcoat and cell coat layers of the coating. Upon exposure to mercury(II), cells within the coating were induced to synthesize GFP, and laser scanning confocal microscopy was used to quantify expression spatially within the cell coat. GFP expression in the coatings increased with increasing mercury concentration (2-20 microM), temperature (21-37 degrees C), and time of incubation (0-39 h). There was a gradient of GFP expression through the cell coat with expression higher near the topcoat-cell coat interface relative to the bottom of the cell coat. The topcoat thickness did not significantly affect GFP expression indicating that diffusion of mercury(II) and oxygen through the topcoat was not limiting.

Painting and printing living bacteria: engineering nanoporous biocatalytic coatings to preserve microbial viability and intensify reactivity.

Latex biocatalytic coatings containing approximately 50% by volume of microorganisms stabilize, concentrate and preserve cell viability on surfaces at ambient temperature. Coatings can be formed on a variety of surfaces, delaminated to generate stand-alone membranes or formulated as reactive inks for piezoelectric deposition of viable microbes. As the latex emulsion dries, cell preservation by partial desiccation occurs simultaneously with the formation of pores and adhesion to the substrate. The result is living cells permanently entrapped, surrounded by nanopores generated by partially coalesced polymer particles. Nanoporosity is essential for preserving microbial viability and coating reactivity. Cryo-SEM methods have been developed to visualize hydrated coating microstructure, confocal microscopy and dispersible coating methods have been developed to quantify the activity of the entrapped cells, and FTIR methods are being developed to determine the structure of vitrified biomolecules within and surrounding the cells in dry coatings. Coating microstructure, stability and reactivity are investigated using small patch or strip coatings where bacteria are concentrated 102- to 103-fold in 5-75 microm thick layers with pores formed by carbohydrate porogens. The carbohydrate porogens also function as osmoprotectants and are postulated to preserve microbial viability by formation of glasses inside the microbes during coat drying; however, the molecular mechanism of cell preservation by latex coatings is not known. Emerging applications include coatings for multistep oxidations, photoreactive coatings, stabilization of hyperthermophiles, environmental biosensors, microbial fuel cells, as reaction zones in microfluidic devices, or as very high intensity (>100 g.L-1 coating volume.h-1) industrial or environmental biocatalysts. We anticipate expanded use of nanoporous adhesive coatings for prokaryotic and eukaryotic cell preservation at ambient temperature and the design of highly reactive "living" paints and inks.

Stationary phase, amino acid limitation and recovery from stationary phase modulate the stability and translation of chloramphenicol acetyltransferase mRNA and total mRNA in Escherichia coli.

The functional stability of the chloramphenicol acetyltransferase (cat) mRNA, as well as the functional stability of the total mRNA pool, change during the course of Escherichia coli culture growth. mRNA half-lives are long during lag phase, decrease during the exponential phase and increase again during the stationary phase of the bacterial growth cycle. The half-lives of cat mRNA and total mRNA also increase three- to fourfold during amino acid starvation when compared to exponential culture growth. Even though the stability of the cat message changes about fourfold during culture growth, the amount of cat mRNA per cell mass does not vary significantly between the culture growth phases, indicating that there are compensating changes in cat gene transcription. Translation of cat mRNA also changes during culture growth. In exponential phase, the rate of cat translation is about 14-fold higher than when the culture is in stationary phase. This is in contrast to the fourfold increase in stability of cat mRNA in the stationary-phase culture compared to the exponentially growing culture and indicates that active translation is not correlated with increased mRNA stability. When a stationary-phase culture was diluted into fresh medium, there was a five- to sevenfold increase in CAT synthesis and a threefold increase in total protein synthesis in the presence or absence of rifampicin. These results suggest that while mRNA becomes generally more stable and less translated in the stationary-phase culture, the mRNA is available for immediate translation when nutrients are provided to the culture even when transcription is inhibited.