In recent years there has been considerable progress towards the development of expression systems for the display of heterologous polypeptides and, to a lesser extent, oligosaccharides on the surface of bacteria or yeast. The availability of protein display vectors has in turn provided the impetus for a range of exciting technologies. Polypeptide libraries can be displayed in bacteria and screened by cell sorting techniques, thus simplifying the isolation of proteins with high affinity for ligands. Expression of antigens on the surface of nonvirulent microorganisms is an attractive approach to the development of high-efficacy recombinant live vaccines. Finally, cells displaying protein receptors or antibodies are of use for analytical applications and bioseparations.
SummaryHaemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Hap s ). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the Cterminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap  ) predicted formation of a -barrel with an N-terminal transmembrane ␣-helix followed by 14 transmembrane -strands. Additional analysis revealed that the final -strand contains an amino acid motif common to other -barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Hap s was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Hap s . These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of -barrel proteins to the bacterial outer membrane.
The Escherichia coli K-12 outer membrane protein OmpT is a prototype of a unique family of bacterial endopeptidases known as the omptins. This family includes OmpT and OmpP of E. coli, SopA of Shigella flexneri, PgtE of Salmonella enterica, and Pla of Yersinia pestis. Despite their sequence similarities, the omptins vary in their reported functions. The OmpT protease is characterized by narrow cleavage specificity defined by the extracellular loops of the beta-barrel protruding above the lipid bilayer. It employs a distinct proteolytic mechanism that involves a histidine and an aspartate residue. Most of the omptin proteins have been implicated in bacterial pathogenesis. As a result, the omptins are potential targets for antimicrobial drug and vaccine development. This review summarizes recent developments in omptins structure and function, emphasizes their role in pathogenesis, proposes evolutionary relation among the existing omptins, and offers possible directions for future research.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.