Neuronal Goα and CAPS Regulate Behavioral and Immune Responses to Bacterial Pore-Forming Toxins

<div><p>Pore-forming toxins (PFTs) are abundant bacterial virulence factors that attack host cell plasma membranes. Host defense mechanisms against PFTs described to date all function in the host tissue that is directly attacked by the PFT. Here we characterize a rapid and fully penetrant cessation of feeding of <em>Caenorhabditis elegans</em> in response to PFT attack. We demonstrate via analyses of <em>C. elegans</em> mutants that inhibition of feeding by PFT requires the neuronal G protein Goα subunit <em>goa-1</em>, and that maintenance of this response requires neuronally expressed calcium activator for protein secretion (CAPS) homolog <em>unc-31</em>. Independently from their role in feeding cessation, we find that <em>goa-1</em> and <em>unc-31</em> are additionally required for immune protection against PFTs. We thus demonstrate that the behavioral and immune responses to bacterial PFT attack involve the cross-talk between the nervous system and the cells directly under attack.</p> </div

Neuronal CAPS/<i>unc-31</i> and <i>egl-21</i> function in PFT defense.

<p>(A) <i>unc-31(e928)</i> and <i>egl-21(n476)</i> mutants are qualitatively hypersensitive to <i>E. coli</i>-expressed Cry5B after 48 hr exposure. Scale bar: 500 µm. (B) <i>unc-31(e928)</i> and <i>egl-21(n476)</i> mutants show decreased survival after 8 days on various doses of purified Cry5B respectively. Expression of <i>unc-31</i> exclusively in neurons results in wild-type survival. (C) Model outlining the hypothesized roles of GOA-1 and UNC-31 in PFT defense. Cry5B damages the plasma membranes of intestinal cells, resulting in the flux or production of factors that are sensed by neurons. Neuronal signals relayed via GOA-1 and UNC-31 to the pharynx inhibit feeding. GOA-1 and UNC-31 are additionally part of neuronal pathways that activate defenses in the intestine.</p

Goα pathway components are required for PFT defense.

<p>(A) <i>goa-1(sa734)</i> and <i>eat-16(ce71)</i> mutants are qualitatively hypersensitive to <i>E. coli</i>-expressed Cry5B after 48 hr exposure. 25% Cry5B indicates a 1∶3 dilution of Cry5B-expressing bacteria with non-expressing control bacteria (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054528#s4" target="_blank">Materials and Methods</a>). Scale bar: 500 µm. (B) <i>goa-1(sa734)</i> and <i>eat-16(ce71)</i> mutants show reduced survival after 8 days on three doses of purified Cry5B. <i>goa-1(n1134)</i> and <i>goa-1(n1134) egl-30(n686)</i> mutants show hypersensitivity on two Cry5B doses. (C) <i>V. cholera</i>e-expressed VCC induces lethality in <i>goa-1(sa734)</i> and <i>eat-16(ce71)</i> mutants after 24 hr exposure. Percentages VCC indicate fraction of VCC-expressing <i>V. cholerae</i> diluted with non-expressing <i>V. cholerae</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054528#s4" target="_blank">Materials and Methods</a>).</p

PFTs inhibit feeding in <i>C. elegans</i>.

<p>(A) <i>E. coli</i>-expressed Cry5B rapidly induces cessation of feeding in wild-type <i>C. elegans</i>. (Feeding continues normally if animals are transferred to no-Cry5B-control plates instead of Cry5B-expressing plates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054528#pone.0054528.s001" target="_blank">Fig. S1B</a>).) (B) 0.5 and 2 hr after transfer to <i>E. coli</i>-Cry5B, animals are not feeding, but after 24 hr almost half of the population has resumed. (This is not due to reduced activity of Cry5B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054528#pone.0054528.s001" target="_blank">Fig. S1C</a>).) (C) <i>V. cholerae</i> expressing VCC induces cessation of feeding, following similar kinetics as Cry5B, whereas <i>V. cholerae</i> lacking VCC does not (blue line). (D) Exogenous serotonin causes constitutive feeding on Cry5B. In this and subsequent figures, graphs show mean ± standard error of three experiments unless otherwise described, and statistics indicated are: ns not significant, * p<0.05, ** p<0.01, *** p<0.001. Lack of any symbol indicates no significant difference. Here, statistics indicate significance of difference between PFT and control at the same time point. In all subsequent figures, statistics indicate the difference between mutant and wild type on the same treatment, and where applicable additional statistics are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054528#pone.0054528.s005" target="_blank">Table S2</a>.</p

Goα pathway components are required for cessation of feeding in response to PFTs.

<p>(A) <i>goa-1(sa734)</i> mutant animals constitutively feed on <i>E. coli</i>-expressed Cry5B. (B) <i>goa-1(sa734)</i> animals constitutively feed on <i>V. cholerae</i> expressing VCC. (C) 30 min after transfer to <i>E. coli</i>-Cry5B, <i>goa-1(sa734)</i> and, to a lesser extend, <i>eat-16(ce71)</i> mutant animals have significantly increased feeding rates. (The transfer process itself does not affect feeding rates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054528#pone.0054528.s002" target="_blank">Fig. S2C</a>).) Individual measurements of three combined experiments are shown; bars indicate medians. Wild type  =  <i>C. elegans</i> N2.</p

Protection and Delivery of Anthelmintic Protein Cry5B to Nematodes Using Mesoporous Silicon Particles

The ability of nano- and microparticles of partially oxidized mesoporous silicon (pSi) to sequester, protect, and deliver the anthelmintic pore-forming protein Cry5B to nematodes is assessed <i>in vitro</i> and <i>in vivo</i>. Thermally oxidized pSi particles are stable under gastric conditions and show relatively low toxicity to nematodes. Fluorescence images of rhodamine-labeled pSi particles within the nematodes Caenorhabditis elegans and Ancylostoma ceylanicum show that ingestion is dependent on particle size: particles of a 0.4 ± 0.2 μm size are noticeably ingested by both species within 2 h of introduction <i>in vitro</i>, whereas 5 ± 2 μm particles are excluded from C. elegans but enter the pharynx region of A. ceylanicum after 24 h. The anthelmintic protein Cry5B, a pore-forming crystal (Cry) protein derived from Bacillus thuringiensis, is incorporated into the pSi particles by aqueous infiltration. Feeding of Cry5B-loaded pSi particles to C. elegans leads to significant intoxication of the nematode. Protein-loaded particles of size 0.4 μm display the highest level of <i>in vitro</i> toxicity toward C. elegans on a drug-mass basis. The porous nanostructure protects Cry5B from hydrolytic and enzymatic (pepsin) degradation in simulated gastric fluid (pH 1.2) for time periods up to 2 h. <i>In vivo</i> experiments with hookworm-infected hamsters show no significant reduction in worm burden with the Cry5B-loaded particles, which is attributed to slow release of the protein from the particles and/or short residence time of the particles in the duodenum of the animal

The 1:1 Cry5B:tribendimidine combination at a higher dose eliminates hookworm infections in hamsters.

<p>Effects of treatment of higher dose Cry5B alone or higher dose tribendimidine alone or higher dose Cry5B plus higher dose tribendimidine in combination on (A) intestinal hookworm burdens and (B) fecal egg counts in hookworm-infected hamsters. None of the individual drugs eradicated the hookworm infections in hamsters, but the combination of both drugs did. TrBD = tribendimidine.</p

Dose response of tribendimidine against hookworm <i>A</i>. <i>ceylanicum</i> infections in hamsters.

<p>(A), (C), and (E): Effects of tribendimidine at indicated doses on intestinal hookworm burdens in hookworm-infected hamsters. For (A), (C), and (E) and in similar figures below, the hookworm burdens in each hamster are indicated with a separate symbol, long horizontal bars represent mean hookworm burdens per group; small bars indicate standard error. (B), (D), and (F): Effects of tribendimidine at indicated doses on parasite egg production (fecal egg counts) in hookworm-infected hamsters. For (B), (D), and (F) and in similar figures below, shown are the average eggs per gram of feces in each group on day 4 post-treatment. The fecal egg counts in each animal are indicated with a separate symbol. Long horizontal bars represent mean eggs per gram of feces (EPG) per group; small bars indicate standard error. Panel (A) & (B); (C) & (D) and (E) & (F) came from three independent <i>in vivo</i> experiments. TrBD = tribendimidine.</p

Pyrantel-resistant cyathostomins are hypersusceptible to Cry5B.

<p>Dose-dependent developmental inhibition of a non-anthelmintic resistant population of cyathostomins (Barn 10; [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006506#pntd.0006506.ref062" target="_blank">62</a>]) and of an anthelmintic-resistant population of cyathostomins (pyrantel-resistant Population S or PYR_R) to (A) pyrantel and (B) purified Cry5B. Each data point represents the % of larvae that matured to the infectious L3 stage (L3i). Error bars indicate standard error from three independent replicates. Susep = Barn 10, pyrantel susceptible; PYR_R = pyrantel-resistant population.</p

Cry5B and tribendimidine in combination are synergistic <i>in vivo</i> against hookworm infections in hamsters.

<p>Effects of individual treatment of Cry5B alone or tribendimidine alone and combination treatment of Cry5B plus tribendimidine at indicated doses on (A) intestinal hookworm burdens and (B) fecal egg counts in hookworm-infected hamsters. Brackets indicate statistical comparisons between groups, with p values shown. Data come from the combination of two independent <i>in vivo</i> experiments. TrBD = tribendimidine.</p