The PAAR5 Protein of a Polarly-Localized Type VI Secretion System (T6SS-5) is Required for the Formation of Multinucleated Cells (MNCs) and Virulence by Burkholderia pseudomallei.

The Burkholderia pseudomallei complex (Bpc) includes B. pseudomallei, B. mallei and B. thailandensis. These species share conserved virulence determinants that facilitate survival in mammalian cells and can spread from cell to cell by a unique mechanism involving fusion of plasma membranes. The activity of a contractile type VI secretion system, T6SS-5, is a central requirement. Using fluorescence confocal microscopy, we found localization and dynamic turnover of fluorescently-labeled T6SS-5 components at the forward pole of Burkholderia residing at the ends of actin protrusions. We identified the proline-alanine-alanine-arginine repeat protein of T6SS-5 (PAAR5), which forms the heteromeric tip of the T6SS-5 apparatus along with VgrG5. Mutational analysis revealed a unique N-terminal extension (NTE) of PAAR5 that is indispensable for cell fusion. Deletion of paar5 allowed us to uncouple fusogenic activity from the functionality of T6SS-5 for exploring the role of cell fusion in pathogenesis. B. pseudomallei {Delta}paar5 deletion mutants retained a functional T6SS-5 apparatus and the ability to secrete the Hcp5 protein. In cellular and animal infection models, {Delta}paar5 mutants mirrored the phenotype of a T6SS-5-defective {Delta}vgrG5 strain, being defective for cell fusion and avirulent in hamsters. These results demonstrate concordance between the fusogenic and in vivo virulence phenotypes, suggesting that T6SS-5-mediated cell fusion may be a central feature of B. pseudomallei pathogenesis and not an in vitro artifact.