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. 2011 Feb 17;9(2):147-57.
doi: 10.1016/j.chom.2011.01.005.

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin

Ferdinand C O Los  1 , Cheng-Yuan Kao, Jane Smitham, Kent L McDonald, Christine Ha, Christina A Peixoto, Raffi V Aroian
Affiliations

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin

Ferdinand C O Los et al. Cell Host Microbe. .

Abstract

Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics.

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Figures

Figure 1
Figure 1. PFTs induce uptake of apical plasma membrane markers
(A) Differential interference contrast (DIC) image of anterior half of C. elegans, with intestine false-colored in blue and pharynx in green; arrowhead indicates the transition from pharynx to intestine, and arrow indicates the posterior bulb of the pharynx (left). Fluorescence image (middle) and overlay over DIC image (right), showing PGP-1::GFP marks the apical plasma membrane of C. elegans intestinal cells. Scale bar: 50 μm. (B) Confocal images of PGP-1::GFP after 0 (left) or 2 hr (middle) exposure to E. coli-expressed Cry5B. Indicated area in the middle panel was magnified 3x (right), with arrows indicating intracellular PGP-1::GFP-positive vesicular structures. Scale bar: 25 μm left (same for middle); 10 μm right. (C) Confocal images showing intracellular PGP-1::GFP-positive vesicular structures after 5 minutes exposure to E. coli-expressed Cry5B, which are absent from untreated animals. Panels and scales as (B). (D) Deconvolved images showing V. cholerae expressing VCC induces PGP-1::GFP relocalization to intracellular vesicular structures after 2 hr exposure (middle and right), whereas V. cholera lacking VCC does not (left). Occasionally, vesicles are visible in control images; these were confirmed to be autofluorescent gut granules (see Experimental Procedures). Scale bar: 25 μm left (middle same scale); 10 μm right.
Figure 2
Figure 2. PFTs induce plasma membrane uptake into early endosomes and increased rates of endocytosis
(A) PGP-1::GFP-positive vesicles induced by 2 hr exposure to E. coli-expressed Cry5B PFT and vesicles positive for mCherry::RAB-5 overlap (indicated by arrows). Due to intensity differences, overlapping signals do not always appear yellow in merged image. Autofluorescence shown in blue in the merged image. Scale bar: 10 μm. (B) Two hr exposure to V. cholerae VCC induces PGP-1::GFP-positive vesicles that show similar overlap with RAB-5::mCherry. Scale bar: 10 μm. (C) After 2 hr exposure to TRITC-labeled BSA in absence of toxin, the dye is confined to the intestinal lumen. After simultaneous exposure to 1 μg/mL purified Cry5B PFT, TRITC-BSA is abundantly found inside intestinal cells. Scale bar: 25 μm. (D) Quantification of TRITC-BSA fluorescence, in absence or presence of 1 or 15 μg/mL purified Cry5B PFT, demonstrates PFT attack leads to increased signal intensity, consistent with increased endocytosis. Means of three experiments. Error bars are standard error of the mean. Statistics indicated here and elsewhere: ns: not significant, ***: P < 0.001, *: P < 0.05.
Figure 3
Figure 3. RAB-5 and RAB-11 are required for defense against Cry5B PFT
(A) rrf-3(pk1426) animals after rab-5 or rab-11.1 RNAi are qualitatively hypersensitive to a low (10%) dose of E. coli-expressed Cry5B PFT after 48 hr exposure. Scale bar: 0.5 mm. (B) VP303 animals grown in liquid on rab-5, rab-11.1, or sek-1 RNAi bacteria show significantly decreased survival rates on 15 μg/mL purified Cry5B, whereas pgp-1 and empty vector (L4440) controls do not. (C) In simultaneously performed assays, RNAi treatments did not cause significant hypersensitivity to CuSO4. (B) and (C) show means of at least three independent experiments. Statistics indicate difference between toxin treatment and its accompanying no-toxin control for each RNAi treatment. Error bars are standard error of the mean.
Figure 4
Figure 4. RAB-5 and RAB-11 are required for PFT-induced endocytosis
(A) PGP-1::GFP animals show a diminished endocytic response to E. coli-expressed Cry5B PFT when grown on rab-5 or rab-11.1 RNAi. Images show anterior half of intestine. Scale bar: 25 μm. (B) Fractions of population showing PGP-1::GFP on endocytic vesicles after RNAi against rab-5, rab-11.1, or sek-1, and subsequent exposure to E. coli-expressed Cry5B. L4440 is empty vector control. Means of at least six independent experiments. Error bars are standard error of the mean.
Figure 5
Figure 5. RAB-5 and RAB-11 are required for removal of PFT from the plasma membrane
(A) PI is restricted to the intestinal lumen when fed to C. elegans. PGP-1::GFP marks the apical boundary of the intestinal cells. Scale bar: 25 μm. (B) PI stains the cytosol of the intestinal cells when animals are simultaneously exposed to purified Cry5B PFT for 2 hr. Scale as (A). (C) Fractions of population showing cytosolic PI staining after 0.5 or 24 hr recovery after a 15 minute pulse with E. coli-expressed Cry5B. After 0.5 hr recovery all RNAi treatments resulted in statistically equal fractions of animals with compromised integrity (internalized PI) of the intestinal cells. The majority of control (L4440) animals and animals on pgp-1 RNAi have repaired their intestinal cells after 24 hr recovery. RNAi against rab-5 or rab-11.1 causes significantly impaired repair after 24 hr. Means of at least three independent experiments. Error bars are standard error of the mean.
Figure 6
Figure 6. Cry5B PFT induces expulsion of plasma membrane into the intestinal lumen
(A) EM images showing extensive damage to the microvilli of the intestinal cells following 3 hr E. coli-expressed Cry5B treatment. Unintoxicated controls (receptor-negative bre-5(ye17) mutant) have healthy microvilli (left). Middle and right panels: intoxicated wild type animals. Intoxicated wild-type animals show microvilli deficiency (middle, arrow), and dislodged microvilli in intestinal lumen (right, arrows). Each panel shows a single focal plane from a different animal. All focal planes were analyzed to confirm the lack of microvilli or disconnection of membranous material. Scale bars: 0.5 μM. (B) Deconvolved images showing PGP-1::GFP positive material in the intestinal lumen (arrows) after 2 hr exposure to E. coli-expressed Cry5B. Scale bar: 10 μm. (C) Confocal images showing debris in the lumen after 5 minutes exposure to E. coli-expressed Cry5B. Scale bar: 10 μm. (D) Fluorescence image showing PGP-1::GFP-labeled material in the posterior bulb of the pharynx (indicated by arrow) after exposure to E. coli-expressed Cry5B. Scale bar: 10 μm. (E) Fractions of animals containing luminal PGP-1::GFP-positive material after E. coli-expressed Cry5B PFT treatment. Error bars are standard error of the mean.
Figure 7
Figure 7. Model for PFT pore removal by RAB-5 and RAB-11-dependent endo- and exocytosis
Microvilli containing PFT pores (red) are expelled from the apical cell surface through RAB-11-dependent vertex fusion (indicated with “V”) or some other mechanism. RAB-5-dependent endocytosis supports this process by maintaining membrane homeostasis through increased endocytosis (indicated by green arrow). Alternatively or in addition, pores are taken up from the plasma membrane into the cells by RAB-5-controlled endocytosis, and potentially transported to lysosomes. RAB-11-controlled exocytosis may balance increased endocytosis (indicated by green arrow).
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