Quaternary structure of a SPATE autotransporter protein

The temperature-sensitive hemagglutinin (Tsh) is a representative of the growing subfamily of secreted bacterial virulence factors, known as serine protease autotransporters of the Enterobacteriaceae(SPATEs). Expressed by avian and human pathogenic strains of Escherichia coli Tsh acts as a serine protease and an adhesin to erythrocytes, hemoglobin, and extracellular matrix proteins. Mature Tsh is comprised of a 106-kDa secreted domain (Tsh(beta)) and a 33-kDa outer membrane beta-domain (Tsh(beta)). Based on the size of beta-domains and functional properties of their passenger domains, all SPATEs are considered to be conventional autotransporters. However, it is unsettled if the conventional autotransporters exist as monomers, oligomers, or multimers (e.g., hexamers). To determine the quaternary structure of Tsh in vitro, we purified Tsh(beta) from the outer membranes and showed that it is natively folded because it is heat modifiable and resistant to protease digestion. Blue-native polyacrylamide gel electrophoresis of Tsh(beta) indicated that Tsh(beta) exists as a monomer or a dimer. The cross-linking analysis demonstrated that purified Tsh(beta) exists as a monomer. The size-exclusion chromatography and cross-linking analyses of purified Tsh also showed that the passenger domain of Tsh is a monomer. Overall, our data indicated that Tsh is a monomeric protein in vitro and support the concept that the SPATE autotransporters exist as monomers rather than as multimers. Implications of our findings on the mechanism of autotransporter secretion across the outer membrane are discussed.