title: Dominant negative phenotype of Bacillus thuringiensis Cry1Ab, Cry11Aa and Cry4Ba mutants suggest hetero-oligomer formation among different Cry toxins.
abstract: BACKGROUND
  Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix α-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1∶1, 0.5∶1, mutant∶wt) indicating that oligomer formation is a key step in toxicity of Cry toxins.
  
  
  RESULTS
  The DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix α-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix α-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10∶1 or 100∶1 ratios (mutant∶wt) probably by competition of binding with toxin receptors.
  
  
  CONCLUSIONS
  We show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins.
authors: Departmento de Microbiologia Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
authors: Daniela
authors: D
authors: Carmona
authors: Claudia
authors: C
authors: Rodríguez-Almazán
authors: Carlos
authors: C
authors: Muñoz-Garay
authors: Leivi
authors: L
authors: Portugal
authors: Claudia
authors: C
authors: Pérez
authors: Ruud A
authors: RA
authors: de Maagd
authors: Petra
authors: P
authors: Bakker
authors: Mario
authors: M
authors: Soberón
authors: Alejandra
authors: A
authors: Bravo
references: Biochemistry. 2002 May 14;41(19):6178-84
references: 11994014
references: Biochem J. 2009 Nov 11;424(2):191-200
references: 19732034
references: Lett Appl Microbiol. 2010 Jul;51(1):42-7
references: 20536706
references: Appl Environ Microbiol. 1996 Feb;62(2):583-6
references: 8593057
references: Peptides. 2011 Mar;32(3):595-600
references: 20558220
references: Eur J Biochem. 1986 Dec 1;161(2):273-80
references: 3023091
references: PLoS One. 2009;4(5):e5545
references: 19440244
references: J Biol Chem. 2009 Nov 20;284(47):32750-7
references: 19808680
references: Appl Environ Microbiol. 2008 May;74(9):2565-72
references: 18326669
references: PMC Biophys. 2010 May 13;3(1):10
references: 20465833
references: Biochemistry. 2008 Oct 28;47(43):11263-72
references: 18826260
references: J Invertebr Pathol. 2010 Jul;104(3):231-3
references: 20361977
references: J Biol Chem. 1999 Dec 31;274(53):37736-42
references: 10608833
references: J Membr Biol. 2006;212(1):61-8
references: 17206518
references: J Cell Sci. 1983 Mar;60:181-97
references: 6874728
references: Cell Mol Life Sci. 2009 Apr;66(8):1337-49
references: 19132293
references: Biochem J. 2006 Feb 15;394(Pt 1):77-84
references: 16255715
references: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13803-8
references: 14623961
references: J Biol Chem. 2007 Jul 20;282(29):21222-9
references: 17537728
references: Biochemistry. 2008 May 6;47(18):5101-10
references: 18407662
references: Appl Environ Microbiol. 2009 Dec;75(23):7548-50
references: 19820153
references: J Biol Chem. 2010 Apr 23;285(17):12497-503
references: 20177063
references: Gene. 1991 Dec 1;108(1):115-9
references: 1662180
references: Science. 2001 Apr 27;292(5517):695-7
references: 11326092
references: J Econ Entomol. 2008 Dec;101(6):1950-9
references: 19133479
references: Appl Environ Microbiol. 1993 Mar;59(3):815-21
references: 8481007
references: J Biol Chem. 1989 Nov 15;264(32):19103-7
references: 2509473
references: Appl Environ Microbiol. 1993 Nov;59(11):3922-7
references: 16349095
references: J Bacteriol. 2003 Sep;185(18):5491-9
references: 12949101
references: Circulation. 2003 Jan 28;107(3):485-9
references: 12551875
references: Appl Environ Microbiol. 2009 Jul;75(14):4661-7
references: 19465527
references: BMC Biochem. 2006 May 22;7:16
references: 16716213