Protist-type lysozymes of the nematode Caenorhabditis elegans contribute to resistance against pathogenic Bacillus thuringiensis

Abstract

Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role of several protist-type lysozyme genes in defence against a nematocidal strain of the Gram-positive bacterium Bacillus thuringiensis. Based on microarray and subsequent qRT-PCR gene expression analysis, we identified protist-type lysozyme genes as one of the differentially transcribed gene classes after infection. A functional genetic analysis was performed for three of these genes, each belonging to a distinct evolutionary lineage within the protist-type lysozymes (lys-2, lys-5, and lys-7). Their knock-out led to decreased pathogen resistance in all three cases, while an increase in resistance was observed when two out of three tested genes were overexpressed in transgenic lines (lys-5, lys-7, but not lys-2). We conclude that the lysozyme genes lys-5, lys-7, and possibly lys-2 contribute to resistance against B. thuringiensis, thus highlighting the particular role of lysozymes in the nematode's defence against pathogens.

Figure 1. Information on lysozymes including their phylogenetic relationships and Bt-induced expression profiles.

The phylogeny shown in (A) is derived from previously published phylogenetic analysis . Expression profiles are given in (B) and were established from quantitative realtime PCR. The three C. elegans strains (MY15, MY18, N2) were confronted either with pathogenic or non-pathogenic Bt for 8 h. Lysozyme induction is given as the normalized expression difference between pathogen and non-pathogen treatment, such that positive values indicate upregulation and negative values downregulation after pathogen exposure. Relative expression levels were calculated from crossing points following the 2^−ΔΔCT method (see methods section). Reddish/yellowish bar colours refer to the lysozymes from chromosome V (lys-1, lys-2, lys-3, and lys-7), bluish colours to those from chromosome IV (lys-4, lys-5, lys-6, and lys-10), and green to that from chromosome II (lys-8). An expression difference of 2 or −2 is indicated by dashed vertical lines. Stars highlight groups that are significantly different from 0 according to a t test and false-discovery-rate adjusted significance levels (all groups with the exception of the three lys-8 groups and lys-2 from MY15).

Figure 2. Survival of C. elegans knock-out strains.

Survival rate was studied on (A) the nematocidal Bt strain B-18247 or (B) the non-nematocidal Bt strain DSM-350. It was checked daily for a period of 7 days. Every other day worms were transferred to fresh treatment plates. The C. elegans wildtype N2 was compared to the mutants with lysozyme knock-out alleles lys-2(tm2398), lys-5(tm2439), and lys-7(ok1384).

Figure 3. Variation among knock-out strains in infection load and body size.

Infection load (A) and body size (B) were compared after 8 h exposure among the wildtype N2 and the mutants with lysozyme knock-out alleles lys-2(tm2398), lys-5(tm2439), and lys-7(ok1384). Nematodes were confronted with the pathogenic Bt strain B-18247 (panel A) or both the pathogenic strain and the non-pathogenic strain DSM-350 (panel B). The results for infection load are shown as box–plots, where the horizontal black line gives the median and the boxes the interquartile range (25% of the data above and below the median). Nomarski images show examples of body sizes for the different strains and treatments. Detailed statistics are given in Table S3.

Figure 4. Variation among knock-out strains in feeding rate and population size.

Feeding rate (A) and population size (B) were studied for the wildtype N2 and the mutants with lysozyme knock-out alleles lys-2(tm2398), lys-5(tm2439), and lys-7(ok1384). Nematodes were confronted with either the pathogenic Bt strain B-18247 (results are found in the bottom part of each panel) or the non-pathogenic strain DSM-350 (top part of each panel). Feeding rate was determined by counting grinder movements within a 20 sec period after 8 h exposure. Population size assays were initiated with ten L4 larvae and the total number of worms were scored after five days. Results are shown as box–plots, where the horizontal black line gives the median and the boxes the interquartile range (25% of the data above and below the median). Population size is shown in logarithmic scale.

Figure 5. Survival of knock-out strains and corresponding transgenic lines with lysozyme overexpression.

Survival was evaluated in the presence of (A) the nematocidal Bt strain B-18247 or (B) the non-nematocidal Bt strain DSM-350. Survival was checked daily for a period of 7 days. Every other day worms were transferred to fresh treatment plates. The transgenic lines overexpressed either lys-2, lys-5, or lys-7 in the N2 genomic background. The knock-out mutants had the following alleles: lys-2(tm2398), lys-5(tm2439), lys-7(ok1384).