Protist-Type Lysozymes of the Nematode Caenorhabditis elegans Contribute to Resistance against Pathogenic Bacillus thuringiensis

et al. (2011) Protist-Type Lysozymes of the Nematode Caenorhabditis elegans Contribute to Resistance against Pathogenic Bacillus thuringiensis. PLoS ONE 6(9): e24619. doi:10.1371/journal.pone.0024619 Protist-Type Lysozymes of the Nematode Caenorhabditis elegans Contribute to Resistance against Pathogenic Bacillus thuringiensis Claudia Boehnisch 0 Daniel Wong 0 Michael Habig 0 Kerstin Isermann 0 Nicolaas K. Michiels 0 Thomas Roeder 0 Robin C. May 0 Hinrich Schulenburg 0 Thierry Soldati, Universite de Gene`ve, Switzerland 0 1 Institute for Evolution and Biodiversity, University of Muenster , Muenster, Germany , 2 Department of Animal Evolutionary Ecology, University of Tuebingen , Tuebingen, Germany , 3 School of Biosciences, University of Birmingham , Birmingham , United Kingdom , 4 Centre d'Immunologie de Marseille-Luminy, Universite de la Me diterrane e , Marseille, France, 5 INSERM, Marseille, France, 6 CNRS, Marseille , France , 7 Wellcome Trust Centre for Human Genetics, University of Oxford , Oxford , United Kingdom , 8 Department of Evolutionary Ecology and Genetics, University of Kiel , Kiel, Germany , 9 Department of Zoophysiology, University of Kiel , Kiel , Germany 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. - Funding: RCM acknowledges financial support from the BBSRC (grant BB/F000138/1); HS from the German Science Foundation (grant SCHU 1415/3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Lysozymes are small enzymes, which can cleave peptidoglycan, an essential component of bacterial cell walls. They are found in almost all groups of organisms and play important roles in both immunity and digestion [16]. In several organisms including the Caenorhabditis taxon the evolution of lysozymes is characterized by gene duplication and adaptive sequence evolution, leading to substantial intra-specific enzyme diversification [710]. C. elegans is of particular interest in this context, because its genome contains 15 phylogenetically diverse lysozyme genes, the largest number recorded to date [10]. C. elegans lysozymes are of two distinct types, the invertebrate- (ilys genes) and the protist- or Entamoeba-type (lys genes). The latter group further diverges into two distinct clades [10]. Representative members of the two main types are known from other organisms to act as functional antimicrobial enzymes [11,12], suggesting that they have a similar function in C. elegans. In the nematode, the encountered genetic diversity may reflect functional diversity [10], in a similar way to that demonstrated for the antimicrobial nlp genes [13]. To date, only little information is available on the exact function of lysozymes in C. elegans immunity. All available data is based on genetic analysis, whereas none of the lysozymes have been characterized at the protein level. In particular, four lysozyme genes were directly shown by overexpression and mutant or RNAi-knock down analysis to contribute to the nematodes defence against pathogens: lys-1 against Serratia marcescens and Staphylococcus aureus [14,15]; lys-2 against Pseudomonas aeruginosa [16]; lys-7 against Microbacterium nematophilum, P. aeruginosa, Salmonella Typhimurium, the pathogenic Escherichia coli strain LF82, and Cryptococcus neoformans [1620], and ilys-3 against M. nematophilum [17]. In addition, seven other lysozymes have been implicated in immunity because exposure of C. elegans to various pathogens leads to changes in their transcription patterns [10,2125]. In the present study we focused on the role of protist-type lysozymes in C. elegans defence against the Gram-positive bacterium B. thuringiensis (Bt). Bt infects invertebrate hosts in a highly specialized process. The bacteriums infectious stages are spores associated with crystal toxins (Cry and Cyt toxins). After oral uptake of the spore-toxin mixture by a suitable host organism such as insects or nematodes, toxin solubilisation occurs inside the gut. The solubilised toxins specifically bind to glycolipids of intestinal cells [2629], followed by formation of membrane pores and subsequent cellular disintegration [30]. Cell destruction appears to lead to a change in milieu (e.g., change of gut pH in insects) that triggers germination of spores and vegetative proliferation of bacteria [30,31]. Most Bt strains express several different toxin genes [31,32]. Overall, Bt produces an enormously diverse array of toxins and hence the taxon includes strains with high specificity towards different hosts including free-living nematodes such as C. elegans [3335]. The nematode-specific Bt strains can establish persistent infections in C. elegans under laboratory conditions, even if the environmental medium does not support bacterial growth [3336]. Some Bt strains are able to produce highly specific interactions with different natural C. elegans isolates [37,38], suggesting that the two coexist in nature. Previous studies characterized in much detail the nematodes defence against one of the nematocidal Bt toxins, namely Cry5B. The toxin binds to glycolipids on membranes of the epithelial cells in the intestine. Thus, alteration of these glycolipids and competitive binding of galectins to these glycolipids mediates resistance [2629,39,40]. Resistance is also influenced by plasma membrane repair, as mediated by RAB-5- and RAB-11dependent vesicle trafficking pathways [41]. Moreover, wholegenome microarray-based gene expression analyses and a recent RNAi knock-down screen revealed the invol (...truncated)