Resistance to a Bacterial Toxin Is Mediated by Removal of a Conserved Glycosylation Pathway Required for Toxin-Host Interactions

Abstract

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.

New Positive Regulators of lin-12 Activity in Caenorhabditis elegans Include the BRE-5/Brainiac Glycosphingolipid Biosynthesis Enzyme

“…Alleles of bre-5 in C. elegans had previously been identified in a screen for animals resistant to a Bacillus thuringiensis (Bt) toxin, a process that has not been linked to LIN-12/Notch signaling. The bre-5(ye17) allele appears likely to be a strong loss of function or null for bre-5 function, as it encodes truncated mutant protein that has no enzymatic activity in vitro (Marroquin et al 2000;Griffitts et al 2003).…”

“…Suppression of elevated lin-12 activity by reducing the activity of other genes important for glycosphingolipid synthesis: There are five bre genes that are necessary for Bt toxin susceptibility. Four of them-bre-2, bre-3, bre-4, and bre-5-encode glycosyltransferases that are believed to act in a single pathway (Griffitts et al 2003(Griffitts et al , 2005. Drosophila egghead (egh), whose mutant phenotype is very similar to that of brn, is homologous to bre-3, so it appears that this pathway has been conserved between Drosophila melanogaster and C. elegans (Goode et al 1996a;Griffitts et al 2003).…”

“…Four of them-bre-2, bre-3, bre-4, and bre-5-encode glycosyltransferases that are believed to act in a single pathway (Griffitts et al 2003(Griffitts et al , 2005. Drosophila egghead (egh), whose mutant phenotype is very similar to that of brn, is homologous to bre-3, so it appears that this pathway has been conserved between Drosophila melanogaster and C. elegans (Goode et al 1996a;Griffitts et al 2003). Drosophila BRN and EGH have been shown to catalyze successive steps in the synthesis of glycosphingolipids (Muller et al 2002;Schwientek et al 2002;Wandall et al 2003Wandall et al , 2005.…”

The Caenorhabditis elegansbus-2 Mutant Reveals a New Class of O-Glycans Affecting Bacterial Resistance

“…The effect is directly analogous to the effect of this and other Bt toxins on Lepidoptera, Diptera, and Coleoptera species, many of which are agricultural pests (4). C. elegans mutants that are resistant to crystal toxin are defective in the bre gene family (5,6), one of which is a homolog of Drosophila melanogaster egghead (egh) and encodes a GDP-Man:␤Glc-Cer-␤1,4-mannosyltransferase (bre-3), another encodes a UDP-GalNAc:␤1,4N-acetylgalactosaminyltransferase (bre-4), and a third encodes is a homolog of Drosophila brainiac (brn) and encodes a UDP-GlcNAc:␤Man N-acetylglucosaminyl transferase (bre-5). Identification of the BRE activities in C. elegans provides striking evidence of the utility of this nematode as a model organism to study the role of glycoconjugates in pathogenesis.…”

srf-3, a Mutant of Caenorhabditis elegans, Resistant to Bacterial Infection and to Biofilm Binding, Is Deficient in Glycoconjugates

“…Infection of C. elegans by the last bacterium leads to destruction of its intestine. C. elegans mutants resistant to this infection have been isolated and are defective in the bre gene family, one of which is a homolog of Drosophila melanogaster egghead (egh) and encodes a GDPMan:␤Glc-Cer ␤1,4-mannosyltransferase (bre-3); another encodes a UDP-GalNAc:␤1,4N-acetylgalactosaminyltransferase (bre-4); and a third is a homolog of Drosophila brainiac (brn) and encodes a UDP-GlcNAc:Man N-acetylglucosaminyltransferase (bre-5) (12,13). Identification of these BRE activities of C. elegans provides striking evidence for the importance of carbohydrates in host-pathogen interactions of this nematode.…”