The Type VI Secretion System Spike Protein VgrG5 Mediates Membrane Fusion during Intercellular Spread by Pseudomallei Group Burkholderia Species

Abstract

Pseudomallei group Burkholderia species are facultative intracellular parasites that spread efficiently from cell to cell by a mechanism involving the fusion of adjacent cell membranes. Intercellular fusion requires the function of the cluster 5 type VI secretion system (T6SS-5) and its associated valine-glycine repeat protein, VgrG5. Here we show that VgrG5 alleles are conserved and functionally interchangeable between Burkholderia pseudomallei and its relatives B. mallei, B. oklahomensis, and B. thailandensis. We also demonstrate that the integrity of the VgrG5 C-terminal domain is required for fusogenic activity, and we identify sequence motifs, including two hydrophobic segments, that are important for fusion. Mutagenesis and secretion experiments using B. pseudomallei strains engineered to express T6SS-5 in vitro show that the VgrG5 C-terminal domain is dispensable for T6SS-mediated secretion of Hcp5, demonstrating that the ability of VgrG5 to mediate membrane fusion can be uncoupled from its essential role in type VI secretion. We propose a model in which a unique fusogenic activity at the C terminus of VgrG5 facilitates intercellular spread by B. pseudomallei and related species following injection across the plasma membranes of infected cells.

Pathogens Penetrating the Central Nervous System: Infection Pathways and the Cellular and Molecular Mechanisms of Invasion

“…In an epithelial cell line or a murine macrophage-like cell line, cell-to-cell spread then occurs by cell fusion, with the formation of multinucleated giant cells, in a type VI secretion-dependent process. BimA, which is necessary for actin-mediated motility, and Fla2, which is thought to mediate intracellular motility, are also required and are considered to mediate cell-to-cell contact prior to cell fusion (223)(224)(225)(226)(227). To our knowledge, the ability of B. pseudomallei to directly invade HBMECs has not been investigated, but it would be reasonable to hypothesize that the intracellular lifestyle of B. pseudomallei may contribute to the pathogenesis of CNS invasion.…”

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

“…In some cases, the same effector can function in bacterial antagonism and also alter cell-signalling pathways in eukaryotic cells (Jiang et al, 2014). Also, 'evolved' VgrGs have been described that contain various C-terminal extensions leading, for instance, to actin cross-linking or actin-ADP ribosylation in eukaryotic cells (Brooks et al, 2013;Pukatzki et al, 2007;Suarez et al, 2010), and host cell fusion presumably to facilitate intercellular bacterial spreading (Schwarz et al, 2014;Toesca et al, 2014).…”

pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells

“…Similar to the findings for T3SS-3 with BopC and BopE, the different T6SS-1 effectors likely receive different and multiple signals to fine-tune the regulation of the secretory system in a temporal and local manner, allowing Burkholderia to take full advantage of its intracellular niche. Our finding that expression of Hcp1, an essential component of the T6SS apparatus, is upregulated at acid pH likely affects the secretion of many T6SS-1-dependent effectors, including the bacterial fusogen VgrG5 (also known as VgrG1) (25). Because Hcp1 expression is also dependent on VirAG, it is also possible that pH might regulate VirAG, thus influencing the expression of the 60-plus VirAG-regulated genes (20), many of which are also T6SS-1 effectors.…”

“…This model is disfavored because few, if any, double-membraned vacuoles containing Burkholderia have been observed and MNGC formation is not part of the pathogenesis of any other actin tail-forming bacteria. In the more favored model, intercellular spread occurs by cell-cell fusion, with a fusogen being inserted in two adjacent and tightly apposed cell membranes (18,25). Vgr5 of T6SS-5 (also known as T6SS-1) has been identified to be a fusogen (25).…”

“…In the more favored model, intercellular spread occurs by cell-cell fusion, with a fusogen being inserted in two adjacent and tightly apposed cell membranes (18,25). Vgr5 of T6SS-5 (also known as T6SS-1) has been identified to be a fusogen (25).…”