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Importance of conserved residues of the serine protease autotransporter beta-domain in passenger domain processing and beta-barrel assembly.

ABSTRACT: Serine protease autotransporters of the family Enterobacteriaceae (SPATE) comprise a family of virulence proteins secreted by enteric Gram-negative bacteria via the autotransporter secretion pathway. A SPATE polypeptide contains a C-terminal translocator domain that inserts into the bacterial outer membrane as a beta-barrel structure and mediates secretion of the passenger domain to the extracellular environment. In the present study, we examined the role of conserved residues located in the SPATE beta-barrel-forming region in passenger domain secretion. Thirty-nine fully conserved residues in Tsh were mutated by single-residue substitution, and defects in their secretion phenotypes were assessed by cell fractionation and immunochemistry. A total of 22 single mutants exhibited abnormal phenotypes in different cellular compartments. Most mutants affecting secretion are charged residues with side chains pointing into the beta-barrel interior. Seven mutants showed notable abnormalities in processing (constructs with the E1231A, E1249A, and R1374A mutations) and beta-barrel assembly or insertion into the outer membrane (constructs with the G1158Y, F1360A, Y1375A, and F1377A mutations). The phenotypes of the beta-barrel assembly/insertion mutants and the presence of a processed Tsh passenger domain in the periplasm support the possibility that the translocator domain must undergo extensive folding prior to insertion into the outer membrane. Results from double-mutation experiments further demonstrate that F1360 and F1377 affect beta-barrel insertion/assembly at different times. In light of these new data, a more refined model for the mechanism of SPATE secretion is presented.

SUBMITTER: Yen YT

PROVIDER: S-EPMC2916275 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Importance of conserved residues of the serine protease autotransporter beta-domain in passenger domain processing and beta-barrel assembly.

Yen Yihfen T YT Tsang Casey C Cameron Todd A TA Ankrah Dennis O DO Rodou Athina A Stathopoulos Christos C

Infection and immunity 20100601 8

Serine protease autotransporters of the family Enterobacteriaceae (SPATE) comprise a family of virulence proteins secreted by enteric Gram-negative bacteria via the autotransporter secretion pathway. A SPATE polypeptide contains a C-terminal translocator domain that inserts into the bacterial outer membrane as a beta-barrel structure and mediates secretion of the passenger domain to the extracellular environment. In the present study, we examined the role of conserved residues located in the SPA ...[more]

PMID: 20515934

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Project description:The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded beta-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen.

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Dataset Information

Importance of conserved residues of the serine protease autotransporter beta-domain in passenger domain processing and beta-barrel assembly.

Publications

Importance of conserved residues of the serine protease autotransporter beta-domain in passenger domain processing and beta-barrel assembly.

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