Disulfide Bond Oxidoreductase DsbA2 of Legionella pneumophila Exhibits Protein Disulfide Isomerase Activity

Abstract

The extracytoplasmic assembly of the Dot/Icm type IVb secretion system (T4SS) of Legionella pneumophila is dependent on correct disulfide bond (DSB) formation catalyzed by a novel and essential disulfide bond oxidoreductase DsbA2 and not by DsbA1, a second nonessential DSB oxidoreductase. DsbA2, which is widely distributed in the microbial world, is phylogenetically distinct from the canonical DsbA oxidase and the DsbC protein disulfide isomerase (PDI)/reductase of Escherichia coli. Here we show that the extended N-terminal amino acid sequence of DsbA2 (relative to DsbA proteins) contains a highly conserved 27-amino-acid dimerization domain enabling the protein to form a homodimer. Complementation tests with E. coli mutants established that L. pneumophila dsbA1, but not the dsbA2 strain, restored motility to a dsbA mutant. In a protein-folding PDI detector assay, the dsbA2 strain, but not the dsbA1 strain, complemented a dsbC mutant of E. coli. Deletion of the dimerization domain sequences from DsbA2 produced the monomer (DsbA2N), which no longer exhibited PDI activity but complemented the E. coli dsbA mutant. PDI activity was demonstrated in vitro for DsbA2 but not DsbA1 in a nitrocefin-based mutant TEM β-lactamase folding assay. In an insulin reduction assay, DsbA2N activity was intermediate between those of DsbA2 and DsbA1. In L. pneumophila, DsbA2 was maintained as a mixture of thiol and disulfide forms, while in E. coli, DsbA2 was present as the reduced thiol. Our studies suggest that DsbA2 is a naturally occurring bifunctional disulfide bond oxidoreductase that may be uniquely suited to the majority of intracellular bacterial pathogens expressing T4SSs as well as in many slow-growing soil and aquatic bacteria.

Identification of disulfide bond isomerase substrates reveals bacterial virulence factors

“…The lack of DsbC and DsbD orthologs in F. tularensis originally led us to speculate that, in addition to oxidoreductase activity, Ft DsbA also may possess isomerase activity. Our prediction and demonstration of the bifunctional nature of Ft DsbA is not unfounded, as L. pneumophila DsbA2 recently was reported to exhibit both oxidoreductase and isomerase activities (Kpadeh et al ., ). Here, we demonstrated Ft DsbA oxidase and isomerase activities in vitro and in F. tularensis , and showed a positive correlation between isomerase activity and F. tularensis virulence.…”

“…These observations indicate that Ft DsbA isomerase and oxidoreductase activities are largely dependent on ROS inactivation mechanisms, including SOD, catalase‐peroxidase, and acid phosphatase (Chong and Celli, ). Although speculative, our Ft DsbA results and the work of others (Kpadeh et al ., ) suggest that any bacterium with merged isomerase and oxidoreductase activities, and the ability to inactivate phagocyte ROS, could be a successful intracellular pathogen.…”

FipB, an Essential Virulence Factor of Francisella tularensis subsp. tularensis, Has Dual Roles in Disulfide Bond Formation

“…FipB is not the only bifunctional DsbArelated protein. The DsbA2 protein of Legionella pneumophila also has both oxidase and protein disulfide isomerase activities (17). DsbA2 has an amino-terminal domain that functions in dimer formation, but the domain is not related to the FKBP_N domain of Mip proteins.…”

“…An in vitro assay of isomerase activity was used to test for isomerase activity of recombinant FipB and the influence of the addition of FipA (17). Only FipB plus FipA demonstrated isomerase activity over background levels (Fig.…”

Disulfide bond formation in prokaryotes: History, diversity and design

“…In other words it is limited by DsbA:substrates stoichiometry. Nevertheless, it raises the possibility that some bacteria may catalyze oxidation and isomerization by a single protein [136]. …”