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. 2018 Apr 23;86(5):e00872-17.
doi: 10.1128/IAI.00872-17. Print 2018 May.

Determinants of Raft Partitioning of the Helicobacter pylori Pore-Forming Toxin VacA

Krishnan Raghunathan #  1 , Nora J Foegeding #  2 , Anne M Campbell  3 , Timothy L Cover  3   4   5 , Melanie D Ohi  6   7 , Anne K Kenworthy  8   2
Affiliations

Determinants of Raft Partitioning of the Helicobacter pylori Pore-Forming Toxin VacA

Krishnan Raghunathan et al. Infect Immun. .

Abstract

Helicobacter pylori, a Gram-negative bacterium, is a well-known risk factor for gastric cancer. H. pylori vacuolating cytotoxin A (VacA) is a secreted pore-forming toxin that induces a wide range of cellular responses. Like many other bacterial toxins, VacA has been hypothesized to utilize lipid rafts to gain entry into host cells. Here, we used giant plasma membrane vesicles (GPMVs) as a model system to understand the preferential partitioning of VacA into lipid rafts. We show that a wild-type (WT) toxin predominantly associates with the raft phase. Acid activation of VacA enhances binding of the toxin to GPMVs but is not required for raft partitioning. VacA mutant proteins with alterations at the amino terminus (resulting in impaired membrane channel formation) and a nonoligomerizing VacA mutant protein retain the ability to preferentially associate with lipid rafts. Consistent with these results, the isolated VacA p55 domain was capable of binding to lipid rafts. We conclude that the affinity of VacA for rafts is independent of its capacity to oligomerize or form membrane channels.

Keywords: GPMV; Helicobacter pylori; VacA; lipid rafts; oligomerization; ordered phase; phase separation; pore formation; pore-forming toxins.

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Figures

FIG 1
FIG 1
VacA localizes to the liquid ordered phase of GPMVs. (A) Representative images of GPMVs isolated from HeLa and AGS cells using the DTT method or from HeLa cells using the NEM method. GPMVs were incubated with DiI-C12 (red) and acid-activated Alexa Fluor 488-VacA (green) and imaged within 2 h at 2°C (HeLa cells with the NEM method), 5°C (AGS cells with the DTT method), or 10°C (HeLa cells with the DTT method). The temperature differences in imaging relate to the differences in transition temperature between the different samples. DiI-C12 marks the liquid disordered phase (ld). Scale bar, 5 μm. (B) Quantification of the phase preference of VacA for each condition represented in panel A. Each data point represents a raft partitioning coefficient measured from a single GPMV. VacA preferentially partitioned into the ordered phase (gray-shaded area) in the majority but not all of the GPMVs across the conditions tested. Nonshaded area represents the liquid disordered phase. Bars and whiskers represent mean values ± standard deviations (SD).
FIG 2
FIG 2
Acid activation is not required for VacA lipid raft association. (A) Representative images of GPMVs, isolated from HeLa cells using the DTT method, after incubation with either acid-activated (top) or nonactivated (bottom) Alexa Fluor 488-VacA. Note that the extent of binding of non-acid-activated VacA to GPMVs was markedly lower than that of acid-activated VacA and the VacA signal in the medium surrounding the GPMVs was higher for non-acid-activated VacA than for acid-activated VacA (see Fig. S3 in the supplemental material). Scale bar, 5 μm. (B) Quantification of the phase preferences of activated and nonactivated VacA. Each data point corresponds to a raft partitioning coefficient measured from a single GPMV. Bars and whiskers represent mean values ± SD. lo, liquid ordered phase (gray-shaded area); ld, liquid disordered phase (nonshaded area).
FIG 3
FIG 3
Oligomerization is not required for VacA lipid raft association. (A) A representative image of GPMVs incubated with Alexa Fluor 488-VacA Δ346–7 demonstrates that the nonoligomerizing mutant localizes to the ordered phase. Scale bar, 5 μm. ld, liquid disordered phase. (B) Quantification of the phase preference of VacA Δ346–7. Each data point corresponds to a raft partitioning coefficient measured from a single GPMV. Bars and whiskers represent mean values ± SD. Gray-shaded area represents the liquid ordered phase, and nonshaded area represents the liquid disordered phase.
FIG 4
FIG 4
Disruptions to the VacA amino-terminal pore-forming region do not abrogate VacA lipid raft association. (A) Representative images of GPMVs after incubation with either acid-activated Alexa Fluor 488-VacA Δ6–27 (top) or acid-activated Alexa Fluor 488-VacA s2m1 (bottom). Scale bar, 5 μm. ld, liquid disordered phase. (B) Quantification of the phase preferences of VacA Δ6–27 and VacA s2m1. Each data point corresponds to a raft partitioning coefficient measured from a single GPMV. Bars and whiskers represent mean values ± SD. Gray-shaded area represents the liquid ordered phase, and nonshaded area represents the liquid disordered phase.
FIG 5
FIG 5
The p55 domain is sufficient for VacA partitioning into lipid rafts. (A) Representative images of GPMVs incubated with Alexa Fluor 488-VacA p55. Scale bar, 5 μm. ld, liquid disordered phase. (B) Quantification of the phase preference of the VacA p55 domain. Each data point corresponds to a raft partitioning coefficient measured from a single GPMV. Bars and whiskers represent mean values ± SD. Gray-shaded area represents the liquid ordered phase, and nonshaded area represents the liquid disordered phase.
FIG 6
FIG 6
Working model of VacA partitioning into lipid rafts. Initially, acid activation promotes disassembly of VacA oligomers (1) into monomeric VacA (2). Monomeric VacA primarily binds raft domains in cell membranes. Given that the p55 domain alone partitions into lipid rafts, we expect the p55 domain to play a large role in targeting VacA into the raft phase (3). VacA oligomerizes and potentially forms a pore in the raft phase. It is not known whether these two steps occur simultaneously or sequentially (4). In the absence of acid activation, VacA binds more weakly to cell membranes (in either an oligomeric or monomeric form) but is still targeted to lipid rafts.
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