1563. Mutational Analysis of Disulfide Bond Oxidoreductase DsbA2 Function in Legionella pneumophila
Session: Poster Abstract Session: Microbial and Host Genetic Factors in Disease
Saturday, October 5, 2013
Room: The Moscone Center: Poster Hall C
  • zegbehposterIDSA2013.pdf (7.8 MB)
  • Background: Legionella pneumophila (Lpn) is a Gram negative, intracellular parasite of free living protozoa that when aerosolized causes Legionnaires’ disease, an occasionally fatal pneumonia. The Dot/Icm type IVb secretion system (T4SS) is the main virulence mechanism and is dependent on correct disulfide bond (DSB) formation catalyzed by a novel and essential DSB oxidoreductase DsbA2 and not by DsbA1, a nonessential DSB oxidoreductase. DsbA2 is a bifunctional enzyme containing a highly conserved N-terminal dimerization domain enabling the protein to form a homodimer. Deletion of the dimerization domain produced the monomer (DsbA2N), which no longer exhibits protein disulfide isomerase activity, but complements DsbA activity in an E. coli dsbAmutant.

    Methods: A cis-proline mutant of DsbA2N (P198T) was expressed in Lpn AA100 strain to assess the effect on virulence. Intracellular growth in Acanthamoeba castellanii, attachment and invasion of HeLa cells and T4SS contact-dependent hemolysis of erythrocytes was compared to empty vector control. The interacting DSB partners of DsbA2N were identified in an E. colibackground using a soft agar motility assay.

    Results: A measurable defect in intracellular growth by expressing DsbA2NP198T in A. castellanii was seen at 48 and 72 hours, and attachment and invasion of HeLa cells was significantly reduced (p=0.005); suggesting DsbA2 is important for pathogenesis. Erythrocyte hemolysis was significantly attenuated by expression of DsbA2NP198T (p<0.0001), demonstrating the importance of DsbA2 for T4SS function. Soft agar motility assay determined DsbA2N interacts with both DsbBs of Lpn(DsbB1 20% and DsbB2 (LidJ) 40% complementation).

    Conclusion: DsbA2NP198T produces a dominant negative effect on DsbA2 function, resulting in a decrease in infectivity, intracellular replication and function of the Dot/Icm T4SS. DsbA2N now interacts with DsbB2 (LidJ). These results suggest that the oxidized DsbA2N out competes DsbA2 for substrates, affecting T4SS assembly and function and Lpn virulence.

    Zegbeh Z. Kpadeh, Micobiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA, Shandra R. Day, MD, Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville, VA and Paul S. Hoffman, PhD, Division of Infectious Disease and International Health, University of Virginia, Charlottesville, VA


    Z. Z. Kpadeh, None

    S. R. Day, None

    P. S. Hoffman, None

    Findings in the abstracts are embargoed until 12:01 a.m. PST, Oct. 2nd with the exception of research findings presented at the IDWeek press conferences.