Genomic clusters, putative pathogen recognition molecules, and antimicrobial genes are induced by infection of C. elegans with M. nematophilum

Abstract

The interaction between the nematode Caenorhabditis elegans and a Gram-positive bacterial pathogen, Microbacterium nematophilum, provides a model for an innate immune response in nematodes. This pathogen adheres to the rectal and post-anal cuticle of the worm, causing slowed growth, constipation, and a defensive swelling response of rectal hypodermal cells. To explore the genomic responses that the worm activates after pathogenic attack we used microarray analysis of transcriptional changes induced after 6-h infection, comparing virulent with avirulent infection. We defined 89 genes with statistically significant expression changes of at least twofold, of which 68 were up-regulated and 21 were down-regulated. Among the former, those encoding C-type lectin domains were the most abundant class. Many of the 89 genes exhibit genomic clustering, and we identified one large cluster of 62 genes, of which most were induced in response to infection. We tested 41 of the induced genes for involvement in immunity using mutants or RNAi, finding that six of these are required for the swelling response and five are required more generally for defense. Our results indicate that C-type lectins and other putative pathogen-recognition molecules are important for innate immune defense in C. elegans. We also found significant induction of genes encoding lysozymes, proteases, and defense-related proteins, as well as various domains of unknown function. The genes induced during infection by M. nematophilum appear largely distinct from genes induced by other pathogens, suggesting that C. elegans mounts pathogen-specific responses to infection.

Figure 1.

Infection of N2 worms in liquid culture. Synchronized L1 worms were grown in liquid culture with E. coli HB101 for 24 h at 25°C before addition of M. nematophilum; either virulent CBX102 (A) or avirulent UV336 (B) was added. Infection was allowed to proceed for 6 h before worms were harvested for RNA extraction and staining adherent bacteria with the nucleic acid dye SYTO 13 (Molecular Probes). Infection (A) is apparent from the bacterial staining in the rectum and the associated tail swelling. Arrow indicates anus.

Figure 2.

Expression of the C-type lectin gene clec-60. A promoter fusion, clec-60p::GFP, was expressed in adults in the posterior intestine cells, int-8 (i8) and int-9 (i9) on both OP50 (A) and MBL plates (B,C). The variation in the intensity and extent of the GFP signal within infected worms is evident in B and C. (D) RT–PCR experiment showing F21C10.10 and clec-60 (top bands) expression compared with ama-1 (control, bottom band a) in uninfected (U) and infected (I) worms.

Figure 3.

Lys-7 worms grown on MBL are severely constipated and developmentally delayed. Two mutants of lys-7, RB1286 (ok1385) and RB1285 (ok1384) were grown on MBL plates. Both lys-7 alleles were severely constipated, had a strong Dar response, and a developmental delay, such that most of the population arrested at the L3 larval stage. (A) RB1285 on OP50; (B) RB1285 on MBL; (C) RB1286 on OP50; (D) RB1286 on MBL.

Figure 4.

Clusters of genes whose transcript levels are altered following infection with M.nematophilum. Chromosomes are represented as black bars and the position and number of up-regulated (above chromosome) or down-regulated genes (below chromosome) are illustrated.