Structural and functional analysis of the extracellular transport signal of chitinase A of Vibrio cholerae.

摘要

ChiA, an 88 kDa endochitinase encoded by the chiA gene of the Gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded type II secretion system, a mechanism which also transports cholera toxin (CT). Prospective extracellular proteins of V. cholerae contain a functionally similar extracellular transport signal which directs proteins fated for extracellular transport to the eps-encoded transport machinery. To locate the amino acid sequences which encode the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N-terminus beyond amino acid position 75 or from the C-terminus beyond amino acid 555. A mutant ChiA composed of only amino acids 75–555 was secreted by wt V. cholerae, but not by an epsD mutant, establishing that this region independently harbored sufficient structural information to promote secretion by the type II system of V. cholerae. To further delimit amino acids that comprise the extracellular transport signal of ChiA, internal deletions of amino acids within the identified region (amino acids 75–555) were engineered. These studies identified region A (amino acids 75–215) and region B (amino acids 424–555), two non-adjacent regions of ChiA, that were necessary for secretion. These studies also confirmed that none of the nine cysteines of mature ChiA are required for secretion by the type II system of V. cholerae. These data support the model that the extracellular transport signal of ChiA is comprised of two distinct signals, both of which are required for the proper recognition and secretion of ChiA by the type II secretion system. To define the precise structure of the extracellular transport signal and to identify potential amino acids comprising the extracellular transport signal, the three-dimensional structure of ChiA must be determined. As an initial approach to resolve the structure of ChiA, enzymatically-active recombinant ChiA was purified from V. cholerae cultures. The purified protein is currently being analyzed for optimal conditions to produce crystals suitable for X-ray diffraction analysis.