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.2014 Apr;82(4):1436-44.
doi: 10.1128/IAI.01367-13. Epub 2014 Jan 13.

The Type VI secretion system spike protein VgrG5 mediates membrane fusion during intercellular spread by pseudomallei group Burkholderia species

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The Type VI secretion system spike protein VgrG5 mediates membrane fusion during intercellular spread by pseudomallei group Burkholderia species

Isabelle J Toesca et al. Infect Immun.2014 Apr.

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.

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Figures

FIG 1
FIG 1
VgrG5 is required for membrane fusion. (A and B) MNGCs were visualized 18 h after infection of HEK293-eGFP or -msRFP cells mixed in 1:1 ratios with WT, ΔvgrG5 mutant, or complemented ΔvgrG5 mutant (carrying a cognatevgrG5 allele expressed intrans on a replicating plasmid) Bp340 (A) or B. thailandensis E264 (B). Green and red, individual cells; yellow, fused MNGCs. Representative MNGCs are shown for WT and complemented strains. No MNGCs were observed in multiple fields for ΔvgrG5 mutants. The numbers of MNGCs per bacterial CFU are reported below the images as the means ± SD for a minimum of 3 independent experiments. ND, not detected (<1 × 10−4 MNGC/CFU). (C) Immunofluorescence microscopy showing actin tails (arrows) formed by B. thailandensis E264 or its ΔbimA or ΔvgrG5 derivative. Blue, DAPI-stained nuclei;, red, immunostained bacteria; green, polymerized actin stained with Alexa Fluor 488-labeled phalloidin. Images in the lower panel are close-ups of areas boxed in red in the upper panel.
FIG 2
FIG 2
vgrG5 alleles from Pseudomallei group Burkholderia species are functionally interchangeable. (A) Alignment of VgrG proteins by BLAST. Gray bars, amino-terminal VgrG “core” region with homology to the gp27/gp5 T4 bacteriophage tail spike complex proteins; red bars, conserved CTD; striped bars, protein regions with no similarities. The boundary of the CTD was assigned based on the alignment of the Burkholderia VgrG5 sequence to the sequences of VgrG proteins in other species. The percentage of identity with the B. pseudomallei 1026b VgrG5 coding sequence is given for each sequence diagramed here.Bp, B. pseudomallei;Bm, B. mallei;Bt, B. thailandensis;Bo, B. oklahomensis. (B) Phylogenetic tree based on comparisons of sequences of VgrG5 proteins from an expanded set of Burkholderia isolates and of VgrG and VgrG1 proteins from V. cholerae and P. aeruginosa, respectively. Analysis was based on the JTT matrix-based model (62) and was conducted in MEGA5 (63). (C) MNGC assays. HEK293 cells were infected with a Bp340 ΔvgrG5 mutant complemented with a plasmid expressingvgrG5 from the Australian B. pseudomallei strain MSHR668 (Bp Aus), B. mallei ATCC 23344, B. thailandensis E264, or B. oklahomensis C6786. (D and E) Numbers of MNGCs per CFU 18 h after infection of HEK293 cells with B. pseudomallei Bp340 (D) or B. thailandensis E264 (E) containing ΔvgrG5 deletions and complementing plasmids as indicated. No fusion activity was observed in ΔvgrG5 strains containing the empty vector. Values are means ± SD for 3 independent experiments (*,P < 0.05).
FIG 3
FIG 3
Mutational analysis of the VgrG5 CTD. (A) Bp340vgrG5 and derivatives with truncation and deletion mutations. “HA” stands for a C-terminal HA epitope tag. The abilities of plasmids carrying wild-type or mutantvgrG5 alleles to complement MNGC formation by Bp340 ΔvgrG5 are shown on the right. TM, transmembrane domain; yellow box, sequence similarity to the enzymatic domain of E. coli hydroxymyristoyl glucosamine-N-transferase; Ab, epitope for antibody against the VgrG5 CTD. (B) Numbers of MNGCs per CFU obtained 18 h after infection of HEK293 cells with Bp340, Bp340 ΔvgrG5, or Bp340 ΔvgrG5 mutants complemented with a wild-type or mutantvgrG5 allele expressed on a replicating plasmid intrans. No fusion activity was observed in ΔvgrG5 strains containing the empty vector. Values are means ± SD for 3 independent experiments. *,P < 0.05. (C) Alignment of VgrG5 C-terminal domains of B. mallei ATCC 23344 (Bm), B. thailandensis E264 (Bt), and B. oklahomensis C6786 (Bo) to that of B. pseudomallei 1026b (Bp) and bioinformatic predictions of internal features. Cons., consensus. Red letters indicate amino acid identity; residues shown in blue/black are nonidentical but generally conserved. Potential regions likely to exhibit TM topology are highlighted in blue. The TM1 and TM2 regions were highly predicted using the TMHMM or TMpred algorithm (92% or 88% confidence, respectively). The TM3 region was identified only by TMpred, with a lower probability (24%). A domain with moderate similarity (30% confidence; 15% aa identity) to E. coli hydroxymyristoyl glucosamine-N-transferase (Enz.) (yellow highlight) was identified using the Phyre2 homology recognition engine. The epitope used to raise polyclonal antisera to the VgrG5 C-terminal domain is highlighted in pink.
FIG 4
FIG 4
Differential roles of the VgrG5 CTD in T6SS-associated membrane damage and T6SS-5 activity. (A) Assays of intracellular replication of Bp340 ΔclpV5 or Bp340 ΔvgrG5 complemented with an empty plasmid vector (pVec) or with plasmid-expressedvgrG5 orvgrG5-C850. Values are means ± SD for 3 independent experiments (*,P < 0.05). (B) Western blot analysis of bacterial supernatant (S) or pellet (P) fractions from Bp340, Bp340virAG(Con), Bp340virAG(Con) ΔvgrG5, or Bp340virAG(Con) ΔvgrG5 expressing WT or mutantvgrG5 from a replicating plasmid. Blots were probed with an antibody against Hcp5 (α-Hcp) or against the VgrG5 CTD (α-CTD).virAG(Con),virAG constitutively expressed. Although T6SS apparatuses contain numerous Hcp monomers, which are released upon disengagement, they are predicted to include only one VgrG trimer, at the tip (30). VgrG5 derivatives that retain the α-CTD antibody epitope (Fig. 3A) can be detected in cell pellets but are not exported in sufficient quantities to be reproducibly detected in the supernatant fractions of broth-grown cells.
FIG 5
FIG 5
VgrG5-induced membrane fusion during Burkholderia infection: a hypothesis. Lateral flagella or actin polymerization provides motility, facilitating contact between bacteria and the plasma membrane (PM), bringing adjacent membranes into close apposition (stage 1). After contact with the membrane, T6SS-5 is deployed, and VgrG5 is inserted across the infected and adjacent cell surfaces, inducing a region of localized disturbance of lipid bilayers (stage 2) that ultimately leads to membrane fusion (stage 3). See the text for details.
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