Preparation and characterization of translocator/chaperone complexes and their component proteins from Shigella flexneri.
Shigella flexneri causes a severe form of bacillary dysentery also known as shigellosis. Onset of shigellosis requires bacterial invasion of colonic epithelial cells which is initiated by the delivery of translocator and effector proteins to the host cell membrane and cytoplasm, respectively, by the Shigella type III secretion system (TTSS). The Shigella translocator proteins, IpaB and IpaC, form a pore complex in the host cell membrane to facilitate effector delivery; however, prior to their secretion IpaB and IpaC are partitioned in the bacterial cytoplasm by association with the cytoplasmic chaperone IpgC. To determine their structural and biophysical properties, recombinant IpaB/IpgC and IpaC/IpgC complexes were prepared for their first detailed in vitro analysis. Both IpaB/IpgC and IpaC/IpgC complexes are highly stable and soluble heterodimers whose formation prevents IpaB-IpaC interaction as well as Ipa-dependent disruption of phospholipid membranes. Circular dichroism spectroscopy shows that IpgC binding has a detectable influence on IpaC secondary/tertiary structure and stability. In contrast, IpaB structure is not as dramatically affected by chaperone binding. To more precisely ascertain the influence of chaperone binding on IpaC structure and stability, single tryptophan mutants were generated for detailed fluorescence spectroscopy analysis. These mutants provide a low-resolution picture of how IpaC exists in the Shigella cytoplasm with chaperone binding possibly involving distinct regions within the N- and C-terminal halves of IpaC. This preliminary assessment of the IpaC-IpgC interaction is supported by initial deletion mutagenesis studies. The data provide the first structural analysis of IpgC association with IpaB and IpaC.
Birket
Susan E
SE
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Harrington
Amanda T
AT
Espina
Marianela
M
Smith
Nathan D
ND
Terry
Christina M
CM
Darboe
Numukunda
N
Markham
Aaron P
AP
Middaugh
C Russell
CR
Picking
Wendy L
WL
Picking
William D
WD
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D018832:Molecular Chaperones / Q000378:metabolism*; D012763:Shigella flexneri / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D018832:Molecular Chaperones