Neuronal Goα and CAPS regulate behavioral and immune responses to bacterial pore-forming toxins

Abstract

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 Caenorhabditis elegans in response to PFT attack. We demonstrate via analyses of C. elegans mutants that inhibition of feeding by PFT requires the neuronal G protein Goα subunit goa-1, and that maintenance of this response requires neuronally expressed calcium activator for protein secretion (CAPS) homolog unc-31. Independently from their role in feeding cessation, we find that goa-1 and unc-31 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.

Figure 1. PFTs inhibit feeding in C. elegans.

(A) E. coli-expressed Cry5B rapidly induces cessation of feeding in wild-type C. elegans. (Feeding continues normally if animals are transferred to no-Cry5B-control plates instead of Cry5B-expressing plates (Fig. S1B).) (B) 0.5 and 2 hr after transfer to E. coli-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 (Fig. S1C).) (C) V. cholerae expressing VCC induces cessation of feeding, following similar kinetics as Cry5B, whereas V. cholerae 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 Table S2.

Figure 2. Goα pathway components are required for cessation of feeding in response to PFTs.

(A) goa-1(sa734) mutant animals constitutively feed on E. coli-expressed Cry5B. (B) goa-1(sa734) animals constitutively feed on V. cholerae expressing VCC. (C) 30 min after transfer to E. coli-Cry5B, goa-1(sa734) and, to a lesser extend, eat-16(ce71) mutant animals have significantly increased feeding rates. (The transfer process itself does not affect feeding rates (Fig. S2C).) Individual measurements of three combined experiments are shown; bars indicate medians. Wild type  =  C. elegans N2.

Figure 3. Goα pathway components are required for PFT defense.

(A) goa-1(sa734) and eat-16(ce71) mutants are qualitatively hypersensitive to E. coli-expressed Cry5B after 48 hr exposure. 25% Cry5B indicates a 1∶3 dilution of Cry5B-expressing bacteria with non-expressing control bacteria (see Materials and Methods). Scale bar: 500 µm. (B) goa-1(sa734) and eat-16(ce71) mutants show reduced survival after 8 days on three doses of purified Cry5B. goa-1(n1134) and goa-1(n1134) egl-30(n686) mutants show hypersensitivity on two Cry5B doses. (C) V. cholerae-expressed VCC induces lethality in goa-1(sa734) and eat-16(ce71) mutants after 24 hr exposure. Percentages VCC indicate fraction of VCC-expressing V. cholerae diluted with non-expressing V. cholerae (see Materials and Methods).

Figure 4. Neuronal CAPS/unc-31 and egl-21 function in PFT defense.

(A) unc-31(e928) and egl-21(n476) mutants are qualitatively hypersensitive to E. coli-expressed Cry5B after 48 hr exposure. Scale bar: 500 µm. (B) unc-31(e928) and egl-21(n476) mutants show decreased survival after 8 days on various doses of purified Cry5B respectively. Expression of unc-31 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.