Systemic infection by Salmonella typhimurium requires coordinated expression of virulence genes found primarily in Salmonella Pathogenecity Islands (SPIs). We have previously reported that the intracellular signal that induces these virulence genes is a stringent signal molecule, ppGpp [Song et al. J Biol Chem 2003;279:34183]. In this study, we found that relA and spoT double mutant Salmonella (DeltappGpp strain), which is defective in ppGpp synthesis, was virtually avirulent in BALB/c mice. Subsequently, the live vaccine potential of the avirulent DeltappGpp Salmonella strain was determined. A single immunization with live DeltappGpp Salmonella efficiently protected mice from challenge with wild-type Salmonella at a dose 10(6)-fold above the LD50 30 days after immunization. Various assays revealed that immunization of mice with the DeltappGpp strain elicited both systemic and mucosal antibody responses, in addition to cell-mediated immunity.
"Lastly, the Salmonella glmS balanced-lethal system was tested in an animal model with wild-type and GlmS− mutant Salmonellae carrying S.tGlmS+p. Since S. typhimurium is highly virulent in rodents, an attenuated strain of S. typhimurium defective in ΔppGpp synthesis ,  was used (ppGpp synthesized by relA and spoT is required for virulence of S. Typhimurium
). The ppGpp-null mutant (relA::kan, spot::cat) and the ppGpp-null mutant carrying the glmS mutation, which are both resistant to kanamycin and chloramphenicol, were transformed with ampicillin-resistant s.tglmS+ p. Mice carrying grafted CT26 (mouse colon cancer) cells were constructed as described previously. "
[Show abstract][Hide abstract] ABSTRACT: During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS(-) mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS(+)p) complemented the phenotype of the GlmS(-) mutant, and that GlmS(+) p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS (+)pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number.
PLoS ONE 03/2013; 8(3):e60511. DOI:10.1371/journal.pone.0060511 · 3.23 Impact Factor
"The attenuated S. typhimurium strains SHJ2037 and A1R were cultured in Luria-Bertani (LB) broth medium (Difco Laboratories, USA) with vigorous aeration at 37°C, as previously described (Na et al., 2006; Zhao et al., 2006). SHJ2037 (relA::cat, spoT::kan) is a ∆ppGpp strain and A1R is an auxotrophic mutant for leucine and arginine, kindly provided by R. M. Hoffman (AntiCancer, Inc., USA) (Zhao et al., 2006). "
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Microbiology 06/2012; 50(3):502-10. DOI:10.1007/s12275-012-2090-9 · 1.44 Impact Factor
"It has also been shown that ppGpp plays a key role in coupling virulence to metabolic status in several other pathogenic bacteria including Mycobacterium tuberculosis [10,11], Listeria monocytogenes , Legionella pneumophilia [13,14], Vibrio cholera  and Pseudomonas aeruginosa . A complete understanding of the pathways and mechanisms by which ppGpp mediates bacterial virulence may suggest targets for antimicrobial therapies . "
[Show abstract][Hide abstract] ABSTRACT: Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (ST(EX)), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wild-type S. Typhimurium and a ppGpp null strain under growth conditions which model ST(EX). In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium ST(EX) primary transcriptome than previously recognised.
Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment.
The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research.
Emily G. Notch, Britton C. Goodale, Roxanna Barnaby, Bonita Coutermarsh, Brent Berwin, Vivien F. Taylor, Brian P. Jackson, Bruce A. Stanton
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