GATA transcription factor required for immunity to bacterial and fungal pathogens

Abstract

In the past decade, Caenorhabditis elegans has been used to dissect several genetic pathways involved in immunity; however, little is known about transcription factors that regulate the expression of immune effectors. C. elegans does not appear to have a functional homolog of the key immune transcription factor NF-kappaB. Here we show that that the intestinal GATA transcription factor ELT-2 is required for both immunity to Salmonella enterica and expression of a C-type lectin gene, clec-67, which is expressed in the intestinal cells and is a good marker of S. enterica infection. We also found that ELT-2 is required for immunity to Pseudomonas aeruginosa, Enterococcus faecalis, and Cryptococcus neoformans. Lack of immune inhibition by DAF-2, which negatively regulates the FOXO transcription factor DAF-16, rescues the hypersusceptibility to pathogens phenotype of elt-2(RNAi) animals. Our results indicate that ELT-2 is part of a multi-pathogen defense pathway that regulates innate immunity independently of the DAF-2/DAF-16 signaling pathway.

Figure 1. elt-2(RNAi) animals are hypersusceptible to S. enterica-mediated killing and are colonized by E. coli.

(A) Wild-type nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were exposed to S. enterica SL1344 (P<0.0001). 60 nematodes were used for each condition. Results are representative of at least 3 independent experiments. (B) Wild-type nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were placed on FUdR containing plates with lawns of heat-killed E. coli OP50 (P = 0.0011). 20 nematodes were used for each condition. Results are representative of at least 3 independent experiments. (C) and (D) Wild-type nematodes grown on E. coli carrying a vector control (C) or on E. coli expressing elt-2 double-stranded RNA (D) were exposed to E. coli expressing DSred for 24 hours, and then visualized using a Leica TCS SL spectral confocal microscope (bar = 50 µm). (E) Wild-type nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were transferred to a clean LB plate either immediately or after 24 hours exposure to E. coli OP50 (+24 hrs), where they were allowed to defecate for 2 hours. Plates then were placed at 37°C overnight and colonies counted. The combined data from 10 individual animals are shown, and data were normalized to the median colony count for the vector control at each timepoint. Error bars represent SEM. Results are representative of at least 3 independent experiments.

Figure 2. Expression of clec-67 is controlled by ELT-2.

(A) Pclec-67::gfp nematodes were grown on E. coli OP50 and were visualized using a Leica MZ FLIII fluorescence stereomicroscope. (B) Microarray (white) and qRT-PCR (light grey) comparing expression of clec-67 in wild-type nematodes grown on S. enterica SL1344 versus wild-type nematodes grown on E. coli OP50. qRT-PCR comparing expression of clec-67 in wild-type nematodes grown on E. coli carrying a vector control versus wild-type nematodes grown on E. coli expressing elt-2 double-stranded RNA (dark grey). Results are the average of 3-7 independent experiments. Error bars represent SEM. (C) qRT-PCR comparing expression of a variety of intestinal housekeeping genes in wild-type nematodes grown on E. coli carrying a vector control versus wild-type nematodes grown on E. coli expressing elt-2 double-stranded RNA. Results are the average of 5 independent experiments. Error bars represent SEM.

Figure 3. elt-2(RNAi) animals are more susceptible than wild-type nematodes to a variety of pathogens.

(A)Wild-type nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were exposed to P. aeruginosa PA14 (P = 0.0001). (B) Wild-type nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were exposed to E. faecalis OG1RF (P<0.0001). (C) Wild-type nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were exposed to C. neoformans H99 (P<0.0001). 60–120 nematodes were used for each condition. Results are representative of at least 3 independent experiments.

Figure 4. The daf-2(e1370) mutation rescues the increased susceptibility to pathogens phenotype of elt-2(RNAi) animals.

(A) Wild-type and daf2(e1370) nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were exposed to S. enterica SL1344. Significant differences were found when wild-type was compared to daf-2(e1370) (P<0.0001), when elt-2(RNAi) was compared to daf-2(e1370);elt-2(RNAi) (P<0.0001), and when daf-2(e1370) was compared to daf-2(e1370);elt-2(RNAi) (P<0.0001). (B) Wild-type and daf2(e1370) nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were exposed to E. faecalis OG1RF. Significant differences were found when wild-type was compared to daf-2(e1370) (P = 0.0056), when elt-2(RNAi) was compared to daf-2(e1370);elt-2(RNAi) (P<0.0001), and when daf-2(e1370) was compared to daf-2(e1370);elt-2(RNAi) (P<0.0001). (C) Wild-type and daf2(e1370) nematodes grown on E. coli carrying a vector control or on E. coli expressing elt-2 double-stranded RNA were exposed to C. neoformans H99 (P<0.0001). Significant differences were found when wild-type was compared to daf-2(e1370) (P = 0.0174) and when elt-2(RNAi) was compared to daf-2(e1370);elt-2(RNAi) (P<0.0001). No significant difference was found when daf-2(e1370) was compared to daf-2(e1370);elt-2(RNAi) (P = 0.1666). (D) Wild-type and daf2(e1370) nematodes grown on E. coli carrying a vector control or on E. coli expressing daf-16 double-stranded RNA were exposed to C. neoformans H99. A significant difference was found when daf-2(e1370) was compared to daf-2(e1370);daf-16(RNAi) (P<0.0001), but no significant difference was found when wild-type was compared to daf-16(RNAi) (P = 0.9375). 60-240 nematodes were used for each condition. Results are representative of at least 3 independent experiments.