Abstract
Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I', (residues 101-363 deleted), and IpaC Delta H (residues 63-170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261-363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed.
Highlights
Shigella flexneri causes a self-limiting gastroenteritis called shigellosis, which is characterized by severe localized inflammation and ulceration of the colonic mucosa [1]
Additional activities associated with Invasion plasmid antigen C (IpaC) include oligomerization in solution [8], reconstitution into complexes with IpaB in vitro [8], in vivo formation of complexes containing IpaB that promote the uptake of latex beads by cultured cells [6], and reconstitution with IpaB and IpaD to form a complex that may allow entry of noninvasive Escherichia coli into cultured cells [13]
All three proteins are efficiently secreted by the S. flexneri SF621 type III secretion system (TTSS) and are able to interact with IpaB, indicating that biological functions associated with the N terminus remain intact [14]
Summary
Invasion plasmid antigens; IpaC, invasion plasmid antigen C; IpaD, invasion plasmid antigen D; ANS, 8-anilino-1-napthalene sulfonic acid; TTSS, type III secretion system; MEM, minimum Eagle’s medium; SipC, Salmonella invasion protein C. Truncated versions of IpaC, IpaC IЈ (residues 101–363 deleted) and IpaC ⌬H (residues 63–170 deleted), were overexpressed in E. coli and purified, permitting structural analysis and a more comprehensive investigation into the role of the individual domains described above. The interaction of these mutants and full-length IpaC with a liposome model was examined. In addition to these structural studies, an investigation into the actin nucleation properties of IpaC and these deletion constructs was conducted. Interpretation of IpaC structure with regard to the functional properties of the wild-type protein is discussed
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