Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.
Shigella rely on a type III secretion system as the primary virulence factor for invasion and colonization of human hosts. Although there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many type III secretion system proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent N, N-dimethyldodecylamine N-oxide (LDAO) and non-ionic detergent n-octyl-oligo-oxyethylene efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC "activation" in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO.
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322.
Abram R
AR
Bernard
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322.
Shari M
SM
Duarte
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047.
Prashant
P
Kumar
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322. Electronic address: nick.dickenson@usu.edu.
Nicholas E
NE
Dickenson
D000942:Antigens, Bacterial / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003902:Detergents / Q000737:chemistry; D004123:Dimethylamines; D004355:Drug Stability; D008027:Light; D008051:Lipid Bilayers; D008081:Liposomes; D018832:Molecular Chaperones; D010743:Phospholipids / Q000737:chemistry; D012542:Scattering, Radiation
D000942:Antigens, Bacterial; D003432:Cross-Linking Reagents; D003902:Detergents; D004123:Dimethylamines; D008051:Lipid Bilayers; D008081:Liposomes; D018832:Molecular Chaperones; D010743:Phospholipids; C092342:IpaC protein, Shigella; C014518:dodecyldimethylamine oxide
circular dichroism; light scattering (dynamic); liposomes; physical characterization; physical stability