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Isolation and Analysis of Suppressor Mutations in Tumor-Targeted msbB Salmonella

  • K. Brooks Low
  • Sean R. Murray
  • John Pawelek
  • David Bermudes
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1409)

Abstract

Tumor-targeted Salmonella offers a promising approach to the delivery of therapeutics for the treatment of cancer. The Salmonella strains used, however, must be stably attenuated in order to provide sufficient safety for administration. Approaches to the generation of attenuated Salmonella strains have included deletion of the msbB gene that is responsible for addition of the terminal myristol group to lipid A. In the absence of myristoylation, lipid A is no longer capable of inducing septic shock, resulting in a significant enhancement in safety. However, msbB Salmonella strains also exhibit an unusual set of additional physiological characteristics, including sensitivities to NaCl, EGTA, deoxycholate, polymyxin, and CO2. Suppressor mutations that compensate for these sensitivities include somA, Suwwan, pmrA C , and zwf. We describe here methods for isolation of strains with compensatory mutations that suppress these types of sensitivities and techniques for determining their underlying genetic changes and analysis of their effects in murine tumor models.

Key words

Salmonella Tumor-targeting Antitumor efficacy msbB Myristoylation Lipid A somA Suwwan pmrAC zwf Suppressor analysis Epistasis Pseudo-reversion Tolerance acquisition Compensatory mutations Rescue mutations Chemical conditionality 

Notes

Acknowledgments

This work was supported by start-up funds from the California State University, Northridge College of Mathematics and Science (for DB). KBL, JP, and DB express their admiration for the late Helen Coley Nauts (1907–2001) and appreciation for her meeting with them in April 2000 to discuss the work of her late father William B. Coley. We also thank the anonymous reviewers for their helpful comments.

References

  1. 1.
    Hall SS (1997) A commotion in the blood: Life, death, and the immune system. Henry Holt, New YorkGoogle Scholar
  2. 2.
    Nauts HC, Swift WE, Coley BL (1946) The treatment of malignant tumors by bacterial toxins as developed by the late William B. Coley, MD, reviewed in the light of modern research. Cancer Res 6:205–216PubMedGoogle Scholar
  3. 3.
    Coley WB (1891) Contribution to the knowledge of sarcoma. Ann Surg 14:199–220PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Fehleisen F (1883) Die etiologie des erysipels. Berlin. (as cited by Coley in Ref. 3)Google Scholar
  5. 5.
    Parker RC, Plummer HC, Siebenmann CO, Chapman MG (1947) Effect of histolyticus infection and toxin on transplantable mouse tumors. Proc Soc Exp Biol Med 66:461–467CrossRefPubMedGoogle Scholar
  6. 6.
    Möse JR, Möse G (1964) Oncolysis by clostridia. I. Activity of Clostridium butyricum (M-55) and other nonpathogenic clostridia against the Ehrlich carcinoma. Cancer Res 24:212–216Google Scholar
  7. 7.
    Carey RW, Holland JF, Whang HY, Neter E, Bryant B (1967) Clostridial oncolysis in man. Eur J Cancer 3:37–46CrossRefGoogle Scholar
  8. 8.
    Fox ME, Lemmon M, Mauchline ML, Davis TO, Giaccia AJ, Minton NP, Brown JM (1996) Anaerobic bacteria as a delivery system for cancer gene therapy: In vitro activation of 5-fluorocytosine by genetically engineered clostridia. Gene Ther 3:173–178PubMedGoogle Scholar
  9. 9.
    Dang LH, Bettegowda C, Huso DL, Kinzler KW, Vogelstein B (2001) Combination bacteriolytic therapy for the treatment of experimental tumors. Proc Natl Acad Sci U S A 98:15155–15160PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Dolgin E (2011) From spinach scare to cancer care. Nat Med 17:273–275CrossRefPubMedGoogle Scholar
  11. 11.
    Forbes NS (2010) Engineering the perfect (bacterial) cancer therapy. Nat Rev Cancer 10:785–794PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Bermudes D, Low KB, Pawelek JM (2000) Tumor-targeted Salmonella. Highly selective delivery vectors. Adv Exp Med Biol 465:57–63CrossRefPubMedGoogle Scholar
  13. 13.
    Bermudes D, Low KB, Pawelek J (2000) Tumor-targeted Salmonella: Strain development and expression of the HSV TK effector gene. In: Walther W, Stein U (eds) Gene therapy: methods and protocols, vol 35. Humana, Totowa, NJ, pp 419–436Google Scholar
  14. 14.
    Darveau R (1999) Infection, inflammation and cancer. Nat Biotechnol 17:19CrossRefPubMedGoogle Scholar
  15. 15.
    Low KB, Ittensohn M, Le T, Platt J, Sodi S, Amoss M, Ash O, Carmichael E, Chakraborty A, Fisher J, Lin SL, Luo X, Miller SI, Zheng Limou King I, Pawelek JM, Bermudes D (1999) Lipid A mutant Salmonella with suppressed virulence and TNFα induction retain tumor-targeting in vivo. Nat Biotechnol 17:37–41PubMedGoogle Scholar
  16. 16.
    Pawelek JM, Low KB, Bermudes D (1997) Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 57:4537–4544PubMedGoogle Scholar
  17. 17.
    Pawelek JM, Low KB, Bermudes D (2003) Bacteria as tumour-targeting vectors. Lancet Oncol 4:548–556CrossRefPubMedGoogle Scholar
  18. 18.
    ClinicalTrials.gov. Identifier: NCT01118819, Safety study of Clostridium novyi-NT spores to treat patients with solid tumors that have not responded to standard therapiesGoogle Scholar
  19. 19.
    ClinicalTrials.gov. Identifier: NCT01598792, Safety study of recombinant Listeria monocytogenes (Lm) based vaccine virus vaccine to treat oropharyngeal Cancer (REALISTIC)Google Scholar
  20. 20.
    ClinicalTrials.gov. Identifier: NCT01675765, CRS-207 Cancer vaccine in combination with chemotherapy as front-line treatment for malignant pleural mesotheliomaGoogle Scholar
  21. 21.
    Toso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP, Schwartzentruber DJ, Sherry RM, Topalian SL, Yang JC, Stock F, Freezer LJ, Morton KE, Seipp C, Haworth L, Mavroukakis S, White D, MacDonald S, Mao J, Sznol M, Rosenberg SA (2002) Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 20:142–152PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, Cavagnolo R, Cahill A, Clairmont C, Sznol M (2003) Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Ther 10:737–744CrossRefPubMedGoogle Scholar
  23. 23.
    ClinicalTrials.gov. Identifier: NCT00004988, Treatment of patients with cancer with genetically modified Salmonella typhimurium bacteriaGoogle Scholar
  24. 24.
    Zhao M, Yang M, Li X-M, Jiang P, Li S, Xu M, Hoffman RM (2005) Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci U S A 102:755–760PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Zhao M, Yang M, Ma H, Li X, Tan X, Li S, Yang Z, Hoffman RM (2006) Targeted therapy with a Salmonella typhimurium leucine-arginine auxotroph cures orthotopic human breast tumors in nude mice. Cancer Res 66:7647–7652CrossRefPubMedGoogle Scholar
  26. 26.
    Zhao M, Geller J, Ma H, Yang M, Penman S, Hoffman RM (2007) Monotherapy with a tumor-targeting mutant of Salmonella typhimurium cures orthotopic metastatic mouse models of human prostate cancer. Proc Natl Acad Sci U S A 104:10170–10174PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Hiroshima Y, Zhao M, Zhang Y, Maawy A, Hassanein MK, Uehara F, Miwa S, Yano S, Momiyama M, Suetsugu A, Chishima T, Tanaka K, Bouvet M, Endo I, Hoffman RM (2013) Comparison of efficacy of Salmonella typhimurium A1-R and chemotherapy on stem-like and non-stem human pancreatic cancer cells. Cell Cycle 12:2774–2780PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    ClinicalTrials.gov. Identifier: NCT01099631, IL-2 expressing, attenuated Salmonella typhimurium in unresectable hepatic spreadGoogle Scholar
  29. 29.
    Low KB, Ittensohn M, Luo X, Zheng L-M, King I, Pawelek JM, Bermudes D (2004) Construction of VNP20009, a novel, genetically stable antibiotic sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods Mol Med 90:47–60Google Scholar
  30. 30.
    Murray SR, Bermudes D, de Felipe KS, Low KB (2001) Extragenic suppressors of growth defects in msbB Salmonella. J Bacteriol 183:5554–5561PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Murray SR, Suwwan de Felipe K, Obuchowski PL, Pike J, Bermudes D, Low KB (2004) Hot spot for a large deletion in the 18–19 Cs region confers a multiple phenotype in Salmonella enterica serovar Typhimurium strain ATCC 14028. J Bacteriol 186:8516–8523PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Murray SR, Ernst RK, Bermudes D, Miller SI, Low KB (2007) PmrA(Con) confers pmrHFIJKL-dependent EGTA and polymyxin resistance on msbB Salmonella by decorating Lipid A with phosphoethanolamine. J Bacteriol 189:5161–5169PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Karsten V, Murray SR, Pike J, Troy K, Ittensohn M, Kondradzhyan M, Low KB, Bermudes D (2009) msbB deletion confers acute sensitivity to CO2 in Salmonella enterica serovar Typhimurium that can be suppressed by a loss-of-function mutation in zwf. BMC Microbiol 189:170. doi: 10.1186/1471-2180-9-170.33 CrossRefGoogle Scholar
  34. 34.
    Karow M, Georgopoulos C (1992) Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB. J Bacteriol 174:702–710PubMedCentralPubMedGoogle Scholar
  35. 35.
    Engel H, Smink AJ, van Wijngaarden L, Keck W (1992) Murine-metabolizing enzymes from Escherichia coli: existence of a second lytic transglycosylase. J Bacteriol 174:6394–6403PubMedCentralPubMedGoogle Scholar
  36. 36.
    Kahn SA, Everest P, Servos S, Foxwell N, Zahringer U, Brade H, Rietschel ET, Dougan G, Charles IG, Maskell D (1998) A lethal role for lipid a in Salmonella infections. Mol Microbiol 29:571–579Google Scholar
  37. 37.
    Carty S, Sreekumar K, Raetz C (1999) Effect of cold shock on lipid A biosynthesis in Escherichia coli. J Biol Chem 274:9677–9685Google Scholar
  38. 38.
    Baker SJ, Markowitz S, Fearon ER, Willson JK, Vogelstein B (1990) Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 249:912–915CrossRefPubMedGoogle Scholar
  39. 39.
    Beadle GW, Ephrussi B (1936) Development of eye colors in Drosophila: transplantation experiments with suppressor of vermillion. Proc Natl Acad Sci U S A 22:536–540PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    Crick F, Barnett L, Brenner S, Watts-Tobin JR (1961) General nature of the genetic code for proteins. Nature 192:1227–1232CrossRefPubMedGoogle Scholar
  41. 41.
    Bossi L, Roth JR (1981) Four-base codons ACCA, ACCU and ACCC are recognized by the frameshift suppressor sufJ. Cell 24:489–496CrossRefGoogle Scholar
  42. 42.
    Ruiz N, Falcone B, Kahne D, Silhavy TJ (2005) Chemical conditionality: a genetic strategy to probe organelle assembly. Cell 121:307–317CrossRefPubMedGoogle Scholar
  43. 43.
    Ruiz N, Kahne D, Silhavy TJ (2006) Advances in understanding bacterial outer-membrane biogenesis. Nat Rev Microbiol 4:57–66CrossRefPubMedGoogle Scholar
  44. 44.
    Silhavy TJ, Kahane D, Walker S (2010) The bacterial cell envelope. In: Shapiro L, Losick R (eds) Cell Biology of Bacteria. Cold Spring Harbor Laboratory Press, Plainview, NY, pp 79–94Google Scholar
  45. 45.
    Wu T, Malinverni J, Ruiz N, Kim S, Silhavy TJ, Kahne D (2005) Identification of a multi-component complex required for outer membrane biogenesis. Cell 121:235–245CrossRefPubMedGoogle Scholar
  46. 46.
    Beckwith J (2009) Genetic suppressors and recovery of repressed biochemical memory. J Biol Chem 284:12585–12592PubMedCentralCrossRefPubMedGoogle Scholar
  47. 47.
    Hartman PE, Roth JR (1973) Mechanisms of suppression. Adv Genet 17:1–105CrossRefPubMedGoogle Scholar
  48. 48.
    Michels CA (2002) Suppression analysis. Chapter 8, In: CA Michels (Eds) Genetic techniques for biological research: A case study approach. Wiley and Sons. pp. 91–98. doi: 10.1002/0470846623Google Scholar
  49. 49.
    Prelich G (1999) Suppression mechanisms: themes and variations. Trends Genet 15:261–266CrossRefPubMedGoogle Scholar
  50. 50.
    Okuda S, Tokuda H (2011) Lipoprotein sorting in bacteria. Annu Rev Microbiol 65:239–259CrossRefPubMedGoogle Scholar
  51. 51.
    Raivio TL, Silhavy TJ (2001) Periplasmic stress and ECF sigma factors. Annu Rev Microbiol 55:591–624CrossRefPubMedGoogle Scholar
  52. 52.
    Ruiz N, Silhavy TJ (2005) Sensing external stress: watchdogs of the Escherichia coli cell envelope. Curr Opin Microbiol 8:122–126CrossRefPubMedGoogle Scholar
  53. 53.
    De Las Penas A, Connolly L, Gross CA (1997) SigmaE is an essential sigma factor in Escherichia coli. J Bacteriol 179:6862–6864PubMedCentralPubMedGoogle Scholar
  54. 54.
    Alba BM, Gross CA (2004) Regulation of the Escherichia coli sigma-dependent envelope stress response. Mol Microbiol 52:613–619CrossRefPubMedGoogle Scholar
  55. 55.
    Button JE, Silhavy TJ, Ruiz N (2007) A suppressor of cell death caused by the loss of sigmaE downregulates extracytoplasmic stress responses and outer membrane vesicle production in Escherichia coli. J Bacteriol 189:1523–1530PubMedCentralCrossRefPubMedGoogle Scholar
  56. 56.
    Hayden JD, Ades SE (2008) The extracytoplasmic stress factor, σE, is required to maintain cell envelope integrity in Escherichia coli. PLoS One 3(2), e1573. doi: 10.1371/journal.pone.0001573 PubMedCentralCrossRefPubMedGoogle Scholar
  57. 57.
    Rowley G, Spector M, Kormanec J, Roberts M (2006) Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens. Nat Rev Microbiol 4:383–394CrossRefPubMedGoogle Scholar
  58. 58.
    Paradis-Bleau C, Markovski M, Uehara T, Lupoli TJ, Walker S, Kahne DE, Bernhardt TG (2010) Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases. Cell 143:1110–112026PubMedCentralCrossRefPubMedGoogle Scholar
  59. 59.
    Typas A, Banzhaf M, van Saparoea B, Verheul J, Bilboy J, Nichols RJ, Zietek M, Beilharz K, Kannenberg K, von Rechenberg M, Breukink E, den Blaauwen T, Gross CA, Vollmer W (2010) Regulation of peptidoglycan synthesis by outer-membrane proteins. Cell 143:1097–1109PubMedCentralCrossRefPubMedGoogle Scholar
  60. 60.
    Qi S-Y, Sukupolvi S, O’Connor CD (1991) Outer membrane permeability of Escherichia coli K12: Isolation, cloning and mapping of suppressors of a defined antibiotic-hypersensitive mutant. Mol Gen Genet 229:421–427CrossRefPubMedGoogle Scholar
  61. 61.
    Tsai SP, Hartin RJ, Ryu J-I (1989) Transformation in restriction-deficient Salmonella typhimurium LT2. J Gen Microbiol 135:2561–2567PubMedGoogle Scholar
  62. 62.
    Kleckner N, Bender J, Gottesman S (1991) Uses of transposons with emphasis on the Tn10. Methods Enzymol 204:139–180CrossRefPubMedGoogle Scholar
  63. 63.
    Miller JH (1992) A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Plainview, NYGoogle Scholar
  64. 64.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Plainview, NYGoogle Scholar
  65. 65.
    Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganism. J Mol Biol 3:208–21848CrossRefGoogle Scholar
  66. 66.
    Kolodkin AL, Capage MA, Golub EI, Low KB (1983) F sex factor of Escherichia coli K-12 codes for a single-stranded DNA binding protein. Proc Natl Acad Sci U S A 80:4422–4426PubMedCentralCrossRefPubMedGoogle Scholar
  67. 67.
    Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088PubMedCentralCrossRefPubMedGoogle Scholar
  68. 68.
    O’Callaghan D, Charbit A (1990) High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation. Mol Gen Genet 223:156–158Google Scholar
  69. 69.
    McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F, Hou S, Layman D, Leonard S, Nguyen C, Scott K, Holmes A, Grewal N, Mulvaney E, Ryan E, Sun H, Florea L, Miller W, Stoneking T, Nhan M, Waterston RK (2001) Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852–856CrossRefPubMedGoogle Scholar
  70. 70.
    Wu TT (1966) A model for three-point analysis of random generalized transduction. Genetics 54:405–410PubMedCentralPubMedGoogle Scholar
  71. 71.
    Clairmont C, Lee KC, Pike J, Ittensohn M, Low KB, Pawelek J, Bermudes D, Brecher SM, Margitich D, Turnier J, Li Z, Luo X, King I, Zheng L-M (2000) Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 181:1996–2002CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • K. Brooks Low
    • 1
  • Sean R. Murray
    • 2
    • 3
  • John Pawelek
    • 4
  • David Bermudes
    • 2
    • 3
  1. 1.Department of Therapeutic Radiology, School of MedicineYale UniversityNew HavenUSA
  2. 2.Biology DepartmentCalifornia State University, NorthridgeNorthridgeUSA
  3. 3.Interdisciplinary Research Institute for the Sciences (IRIS)California State University, NorthridgeNorthridgeUSA
  4. 4.Department of Dermatology, School of MedicineYale UniversityNew HavenUSA

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