Multiple genes affect sensitivity of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum

Abstract

Interactions with bacteria play a major role in immune responses, ecology, and evolution of all animals, but they have been neglected until recently in the case of C. elegans. We report a genetic investigation of the interaction of C. elegans with the nematode-specific pathogen Microbacterium nematophilum, which colonizes the rectum and causes distinctive tail swelling in its host. A total of 121 mutants with altered response to infection were isolated from selections or screens for a bacterially unswollen (Bus) phenotype, using both chemical and transposon mutagenesis. Some of these correspond to known genes, affecting either bacterial adhesion or colonization (srf-2, srf-3, srf-5) or host swelling response (sur-2, egl-5). Most mutants define 15 new genes (bus-1-bus-6, bus-8, bus-10, bus-12-bus-18). The majority of these mutants exhibit little or no rectal infection when challenged with the pathogen and are probably altered in surface properties such that the bacteria can no longer infect worms. A number have corresponding alterations in lectin staining and cuticle fragility. Most of the uninfectable mutants grow better than wild type in the presence of the pathogen, but the sur-2 mutant is hypersensitive, indicating that the tail-swelling response is associated with a specific defense mechanism against this pathogen.

Figure 1.

Infection of wild-type and resistant worms. A–F show the tail region of adult hermaphrodites. (A) Uninfected wild type. (B) Infected wild type with weak Dar phenotype. Adherent bacteria are visible (arrow). (C) Infected wild type with strong Dar phenotype. (D) Infected bus-1, Bus phenotype. (E) Infected bus-12, Bus phenotype. (F) Infected bus-12 with weak Dar phenotype, seen in a minority of animals.

Figure 2.

Genetic map. Linkage groups (autosomes I–V and X chromosome) are shown in standard vertical format. The zero coordinate for each linkage group is shown as a bar to the left of each vertical line. Approximate positions of genes studied in this article (5 previously mapped, 15 new) are as shown.

Figure 3.

Tests for colonization: SYTO 13 staining. A–E show the tail region of adult hermaphrodites after infection, extensive washing to remove E. coli cells, and brief staining with SYTO 13 to reveal infecting bacteria (green fluorescence). (A) Wild type: extensive rectal colonization and strong Dar phenotype. (B) bus-13: trace infection and very weak Dar response. (C) bus-4: no colonization and no Dar response. (D) bus-14: strong colonization and no Dar response. (E) sur-2: strong colonization and no Dar response. (F) Head region of adult bus-16 hermaphrodite, showing patchy adherence of bacteria to the cuticle surface.

Figure 4.

Lectin staining. (A and B) Wild-type worms after staining with SBA and WGA, respectively; long exposure. No surface staining is seen, only faint background autofluorescence from the gut. (C) SBA-stained srf-2, vulval region: strong surface staining, especially of vulva and alae. (D) WGA-stained srf-5: strong staining. (E) SBA-stained bus-15: specific staining of nose. (F) WGA-stained bus-8: patchy surface staining. (G) SBA-stained bus-16 showing disintegration of outer cuticle. (H) WGA-stained bus-16: strong patchy surface staining. (I) TPA-stained bus-13: alae staining. (J) CON-stained bus-13: alae staining. In C, I, and J, alae are indicated with an arrow.