Frederick M. Ausubel, Department of Genetics, Harvard Medical School, presents this Biology Division Centenary Lecture on; “Use of/ Caenorhabditis elegans/ Infection Models to Study Bacterial Virulence and Host Immunity and to Identify Antimicrobial Compounds.”
Abstract: Our laboratory uses a model pathogenesis system that involves the infection of the nematode worm/ Caenorhabditis elegans/ with a
variety of human microbial pathogens to identify virulence-related genes in bacterial and fungal pathogens, to identify host innate immunity
genes, and to identify low molecular weight compounds that function as anti-infectives or immune enhancers. Many human bacterial pathogens
including/ Pseudomonas aeruginosa and Enterococcus faecalis/ are pathogens of/ C. elegans/. To facilitate the identification of/ P.
aeruginosa/ virulence factors, we constructed a non-redundant/ P. aeruginosa/ transposon mutation library that contains ~30,000 sequenced
insertion mutants and selected a single insertion (or in some cases two insertions) to represent each annotated ORF. We have screened this
non-redundant set using the/ C. elegans/ killing assay. From the host perspective we have shown that a highly conserved PMK-1 MAPK p38
signaling cascade as well as the DAF-2/DAF-16 insulin-like signaling pathway are involved in the/ C. elegans/ innate immune response./ pmk-1/
loss-of-function mutants have enhanced sensitivity to pathogens, whereas/ daf-2/ loss-of-function mutants have enhanced resistance to
pathogens that requires upregulation of the DAF-16 transcription factor.
We used genetic epistasis analysis to show that the pathogen resistance of/ daf-2/ mutants also requires PMK-1. However, genome-wide microarray analysis indicated that there is essentially no overlap between genes positively regulated by PMK-1 and DAF-16, suggesting that they function in parallel signaling pathways to promote immunity. We propose that the PMK-1 pathway is a specific, indispensable immunity pathway that mediates expression of secreted immune response genes, while the DAF-2/DAF-16 pathway appears to regulate immunity as part of a more
general stress response. To develop a high throughput screening methodology to identify novel anti-microbial compounds, we devised a
screen to that promoted the survival of/ C. elegans/ infected with/ E. faecalis,/ which colonizes the nematode intestinal tract forming a
persistent lethal infection. We adapted the traditional agar-based/ C. elegans-E. faecalis/ infection assay so that it could be carried out in
liquid medium in standard 384-well microtiter plates. In contrast to traditional antibiotics, many compounds identified using this screen
have very little or no inhibitory activity on the growth of free-living bacteria. These findings indicate that the whole-animal/ C. elegans/
screen not only identifies traditional antibiotics, but also compounds that target bacterial virulence or stimulate host defense.