Predator-prey relationships among isolates of Salmonella enterica serovar typhimurium

Abstract only availableWhen two genetically related (but not identical) strains of Salmonella enterica serovar Typhimurium are grown together in the same culture, there is a tendency for competition to occur as nutrients deplete, possibly causing one strain to outgrow another. The Cancer Research Center has access to thousands of strains of Salmonella that have been sealed and stored under harsh environmental conditions for almost forty years. Our goal is to explore mutations that have arisen among these archived strains, and to test whether these mutations would provide a selective advantage over non-archived S. Typhimurium. We tested for selective advantages by competing different combinations of archived and non-archived strains in liquid media. This was done by selecting two different strains, preparing them in 1:100 ratios, and recording changes in population over a two to four week period. In every competition, the archival strain outgrew the non-archival when initially in minority. From our data, we can conclude that these archived strains must have gained some phenotypic qualities that provide additional fitness under environmental pressure. Further research might reveal information on specific phenotypic characteristics that our Salmonella strains may have utilized to survive under such nutrient-poor conditions.Life Sciences Undergraduate Research Opportunity Progra

Phenotypic Evolution of Therapeutic Salmonella enterica Serovar Typhimurium after Invasion of TRAMP Mouse Prostate Tumor

Salmonella has been of interest in cancer research due to its intrinsic ability to selectively target and colonize within tumors, leading to tumor cell death. Current research indicates promising use of Salmonella in regular administrations to remove tumors in mouse models while minimizing toxic side effects. However, selection of mutants during such long-term tumor colonization is a safety concern, and understanding selection of certain phenotypes within a tumor is an important consideration in predicting the long-term success of bacterium-based cancer treatment strategies. Thus, we have made an initial examination of selected phenotypes in a therapeutic Salmonella enterica serovar Typhimurium population developed from an archival wild-type LT2 strain and intraperitoneally injected into a 6-month-old TRAMP (transgenic adenocarcinoma of mouse prostate) mouse. We compared the original injected strain to isolates recovered from prostate tumors and those recovered from the spleen and liver of non-tumor-bearing TRAMP mice through phenotypic assessments of bacteriophage susceptibility, motility, growth rates, morphology, and metabolic activity. Tumor isolate traits, particularly the loss of wild-type motility and flagella, reflect the selective pressure of the tumor, while the maintenance of bacteriophage resistance indicates no active selection to remove this robust trait. We posit that the Salmonella population adopts certain strategies to minimize energy consumption and maximize survival and proliferation once within the tumor. We find these insights to be nonnegligible considerations in the development of cancer therapies involving bacteria and suggest further examinations into the evolution of therapeutic strains during passage through tumors.Center for Cancer Research (National Cancer Institute (U.S.)

Diversity of progeny from a single colony of Salmonella typhimurium after 19 months in sealed agar stabs

Abstract only availableRecent studies at the Cancer Research Center revealed numerous mutations in Salmonella typhimurium that had been sealed in agar stab vials and stored for over forty years. The bacteria that were conserved in over 20,000 vials were all progeny of the same S. typhimurium strain. However, they were not progeny from a single colony (thus, a single parental cell), but from cultures used in genetic studies in several laboratories. To continue the evolutionary and mutational study of S.typhimurium, a new set of 100 similar agar stabs were inoculated 19 months ago from a single colony (thus, a single parental cell), and sealed. Cells from this set were assayed to see if mutations had occurred. Through motility tests, colony growth on three media, re-streaking of unique colonies, and phage spot testing, genetic variability was observed after 19 months storage. In this amount of time enough mutation did occur to display diverse phenotypes among progeny of the single strain of S. typhimurium. To confront any concerns that the mutations may have been present 19 months ago, a -80 °C stock of the parent colony was used as a control. While the phenotypic changes were significantly less then the vials stored for forty years, it is obvious that 19 months was enough time for genetic variability to occur in S. typhimurium from a single parent. Support from Cancer Research Center. Special thanks to Dustin Newman and Alison Fea for technical instruction.Cancer Research Cente

Effectiveness of therapeutic Salmonella Typhimurium in selectively targeting human cancer cell lines [abstract]

Abstract only availableStudies indicate that non-virulent strains of Salmonella Typhimurium have tumor-targeting activity. Indeed, S. Typhimurium has been observed to selectively target cancer tissue by a ratio of over 1000:1. Most of these studies focused on the cancer cell selectivity of one strain, the genetically modified S. typhimurium VNP20009. One such study found that a single IV injection of VNP20009 produced tumor growth inhibition of 57 - 95% in mice. Another study conducted by Thamm and associates found that administration of VNP20009 results in dectable bacterial colonization of tumor tissue and partial anti-tumor activity in tumor-bearing dogs. Despite its selectivity, VNP20009 was shown to be too toxic when given to patients in phase I clinical tests. Scientists at Columbia's Cancer Research Center developed a therapeutic strain, CRC2636, an archival strain of S. typhimurium that has been shown to destroy PC-3M without extensive lysis of the cancer cells, a factor thought to contribute to the toxicity of VNP20009. Our research strategy involved analyzing the effectiveness of CRC2636 in selectively targeting prostate, breast, and colon cancer cell lines when incubated with their normal counterparts. In order to track CRC2636 in the attachment and invasion studies, we electroporated pRST plasmids that constitutively expressed the fluorescent mCherry protein into our therapeutic strain. Attachment studies were done on a time course from 10 minutes up to 4 hours and invasion studies were done up to 16 hours. Quantitative results were obtained by counting the number of attached bacterial cells to the cancerous and normal cells at the various time points tested.Cancer Research Cente

Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells

BACKGROUND: A collection of over 20,000 Salmonella typhimurium LT2 mutants, sealed for four decades in agar stabs, is a unique resource for study of genetic and evolutionary changes. Previously, we reported extensive diversity among descendants including diversity in RpoS and catalase synthesis, diversity in genome size, protein content, and reversion from auxotrophy to prototrophy. RESULTS: Extensive and variable losses and a few gains of catabolic functions were observed by this standardized method. Thus, 95 catabolic reactions were scored in each of three plates in wells containing specific carbon and nitrogen substrates. CONCLUSION: While the phenotype microarray did not reveal a distinct pattern of mutation among the archival isolates, the data did confirm that various isolates have used multiple strategies to survive in the archival environment. Data from the MacConkey plates verified the changes in carbohydrate metabolism observed in the Biolog™ system

Comparative selectivity of various Salmonella typhimurium strains in targeting prostate cancer cells [abstract]

Abstract only availableRecent studies indicate that non-virulent strains of serovar typhimurium () have tumor-targeting activity. Indeed, S. typhimurium has been observed to selectively target cancer tissue by a ratio of over 1000:1. However, most of these studies focused on the cancer cell selectivity of one strain, the genetically modified VNP20009. One such study found that a single IV injection of VNP20009 produced tumor growth inhibition of 57-95% in mice. Another study conducted by Thamm and associates found that administration of VNP20009 results in detectable bacterial colonization of tumor tissue and partial anti-tumor activity in tumor-bearing dogs. However, VNP20009 was shown to be too toxic when given to cancer patients in phase I clinical tests. Scientists at Columbia's Cancer Research Center discovered an archival strain of (CRC1674) that destroys PC-3M prostate cancer cells without extensive lysis of the cancer cells, a factor thought to contribute to the toxicity of VNP20009. This project studies the comparative selectivity of four strains for prostate cancer cells. In order to study attachment to prostate cancer cells, shorter incubation times were used, up to a period of 4 hours. Invasion assays involved incubation periods up to 24 hours. To confirm distinct selectivity towards prostate cancer cells only, the study also included attachment and invasion assays using noncancerous prostate cells. We have discovered attachment after 5 minutes of co-incubation and are currently investigating long-term effects of co-incubation with prostate cancer and normal cell lines.Life Sciences Undergraduate Research Opportunity Progra

Multiple genetic switches spontaneously modulating bacterial mutability

Background: All life forms need both high genetic stability to survive as species and a degree of mutability to evolve for adaptation, but little is known about how the organisms balance the two seemingly conflicting aspects of life: genetic stability and mutability. The DNA mismatch repair (MMR) system is essential for maintaining genetic stability and defects in MMR lead to high mutability. Evolution is driven by genetic novelty, such as point mutation and lateral gene transfer, both of which require genetic mutability. However, normally a functional MMR system would strongly inhibit such genomic changes. Our previous work indicated that MMR gene allele conversion between functional and non-functional states through copy number changes of small tandem repeats could occur spontaneously via slipped-strand mis-pairing during DNA replication and therefore may play a role of genetic switches to modulate the bacterial mutability at the population level. The open question was: when the conversion from functional to defective MMR is prohibited, will bacteria still be able to evolve by accepting laterally transferred DNA or accumulating mutations? Results: To prohibit allele conversion, we "locked" the MMR genes through nucleotide replacements. We then scored changes in bacterial mutability and found that Salmonella strains with MMR locked at the functional state had significantly decreased mutability. To determine the generalizability of this kind of mutability 'switching' among a wider range of bacteria, we examined the distribution of tandem repeats within MMR genes in over 100 bacterial species and found that multiple genetic switches might exist in these bacteria and may spontaneously modulate bacterial mutability during evolution. Conclusions: MMR allele conversion through repeats-mediated slipped-strand mis-pairing may function as a spontaneous mechanism to switch between high genetic stability and mutability during bacterial evolution.Evolutionary BiologyGenetics & HereditySCI(E)9ARTICLEnull1

Effect of Salmonella typhimurium on human cancer line tumors in mice [abstract]

Abstract only availableResearch of bacteria-driven degeneration of tumor cells, initially pioneered by Dr William Coley in 1893 using heat-killed mixtures of streptococci, has led to the scrutiny of the enterobacteria species Salmonella typhimurium on various cancer lines since the mid-1990s. Salmonella is facultative anaerobe that typically causes enteritis in humans, but attenuated strains have been observed to specifically target cancer tissue when injected into the circulatory system of mammalian hosts. The LT2 (Lilleengan Type 2) Salmonella was isolated by Lilleengan in 1953 and was found to favor cancer cells and promote anti-tumor activity in 2005 (unpublished data). LT2 was the first Salmonella typhimurium to be sequenced, facilitating the creation of genomic microarrays that are currently being used to determine which genes are essential for tumor-killing mechanisms. Experimental Approaches: Therapeutic Salmonella has been developed and stored at the Cancer Research Center (CRC), which consists of approximately two thousand auxotrophic Salmonella typhimurium mutants stored in sealed stab vials for over 50 years at room temperature. Although therapeutic Salmonella has been shown to be effective against prostate cancer cell lines, little is known about the therapeutic breadth of Salmonella cancer therapy. Using tissue cultures, therapeutic Salmonella candidate CRC2631 will be tested on various human cancer cell lines. My studies will focus on colon cancer cell line HT29. Predicted Outcomes: Salmonella attachment and invasion of the HT29 cells is expected, which will be characterized by fluorescent microscopy time course experiment and gentamycin exclusion assays. Data from Salmonella attachment and invasion of prostate cancer lines will be used for comparison. Overall significance: The examination of salmonella-treated tumors will test the therapeutic range of CRC2631 Salmonella therapy, thus revealing what types of cancers can be effectively treated using therapeutic Salmonella. The nature (biological and chemical) of therapeutic Salmonella selection of the cancer cells can be determined through analysis of the mechanism of bacterial attachment or lack thereof, in addition to identification of molecules present or not present in the tested cancer cell lines that lead to the discrimination and destruction of cancer cells but not normal tissue.Cancer Research Cente