The Role of Autotransporter proteins in Burkholderia pseudomallei Pathogenesis Public Deposited

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  • March 22, 2019
  • Campos, Cristine Gomes
    • Affiliation: School of Medicine, Department of Microbiology and Immunology
  • Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease that ranges from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the Type V Secretion System family, and many have been shown to play crucial roles in pathogenesis in other bacterial species. The genome of B. pseudomallei strain 1026b encodes two putative classical ATs, and nine putative trimeric AT proteins. The open reading frame bcaA in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with a passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is bcaB, which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in- frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB to wild type B. pseudomallei in vitro demonstrated similar adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, a similar bacterial burden was observed after 48 hours in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB and wild type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting a role for this AT in dissemination or in survival from the site of infection to the spleen. Using a bioinformatics approach, we annotated eight putative domains within each trimeric AT protein, excluding the well-studied BimA, and found short repeated sequences unique to Burkholderia species, as well as an unexpectedly high proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence, invasion, and plaque formation in A549 cells. Five of the ATs, BoaA, BoaB, BpaA, BpaC, and BpaD, contribute to adherence, and four of the ATs, BpaA, BpaC, BpaE, and BpaF, are necessary for efficient internalization in A549 cells. Using a BALB/c mouse model of infection, we then determined the contribution of each AT to bacterial burden in lungs, liver, and spleen. At 48 hours post-inoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model.
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  • Cotter, Peggy
  • Doctor of Philosophy
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  • 2013

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