Caenorhabditis elegans as a model for Candida albicans-Pseudomonas aeruginosa co-infection and infection induced prostaglandin production

[摘要] The discovery of substantial commonality between microbial pathogenesis in mammals andinvertebrate model hosts, such as the nematode Caenorhabditis elegans, has provided thefoundation for genetic analysis of microbial virulence factors in live animal models. In mostcases, Candida albicans yeast cells inhabit the human intestines, yet this opportunisticpathogen can led to host tissues invasion, causing life-threatening infections inimmunocompromised hosts. Given the importance of this fungus to human health and its coexistencewith other pathogenic microbes, particularly bacteria, such as emerging GramnegativePseudomonas aeruginosa, thus we used C. elegans as an infection model to studyinteractions between C. albicans and P. aeruginosa. Our goal was to firstly, successfullypropagate and monitor the life cycle of C. elegans at 15 °C. Secondly, for this reason, weestablished a liquid medium assay using C. elegans model for C. albicans or P. aeruginosamonomicrobial infections. We demonstrate that the C. albicans yeast form establishes anintestinal infection in C. elegans, while the hyphal form is not required to efficiently kill thenematode. Furthermore, investigating mutants and genetically engineered C. albicans strains,we proved that hyphal formation is indeed not required for full virulence in this system. Thirdlywe demonstrated that polymicrobial interactions are more virulent to C. elegans thanmonomicrobial species. We also aimed to understand the genetic mechanisms of virulenceobserved in C. elegans in vitro, since it was shown that not only does C. albicans and P.aeruginosa kill the nematode C. elegans, but also that C. albicans and P. aeruginosa virulencefactors required for mammalian pathogenesis might also be required for efficient killing of C.elegans. Here our in vitro results suggested that there are multiple virulence factors of P.aeruginosa that may cause virulence, including pyoverdine, pyocyanin and swarming motility.Another factor that contributes to virulence is the hydrolytic enzyme production, known tofacilitate pathogenicity of bacteria, protozoa, and pathogenic yeasts. Our results demonstratedthat although C. albicans and P. aeruginosa possess a wide range of hydrolytic enzymes,proteinases are more predominantly associated with virulence. Furthermore, when comparingthe effect of infection on the microbial burden of specific pathogens, from monomicrobialinfections, it is clear that the virulence observed in killing of nematodes was not due to numberof cells but rather specific virulence factors of the different strains. Surprisingly, frompolymicrobial infections, we see that for both P. aeruginosa strains, co-infection resulted in anincreased microbial burden. This is due to the fact that virulence of co-infection is stronglyinfluenced by microbial burden and that this is dependent on the specific strains in the coinfection.For further understanding of the influence of virulence that underlie susceptibility tothis pathogens, we used this pathogen model system to further evaluate the influence of infection towards the nematodes fatty acid composition. Total lipids of C. elegans wereextracted using chloroform and methanol [2:1 ratio (v/v)]. Fatty acids composition of theextracted total lipids was converted to their corresponding fatty acids methyl esters (FAMEs)and analysed by gas chromatography (GC). From the nematodes feeding on control E. coliOP50, we identified twenty-three different fatty acids ranging from 12 to 22 carbons in length,with 35 % being saturated, while 65 % being unsaturated. We then only focused on majorunsubstituted long chain fatty acids (LCFAs), with margaric acid (17:0) being the predominantsaturated fatty acid, comprising of an average of 24 % total major fatty acids. Through thisprocess, after C. albicans and P. aeruginosa infection, we identified three fatty acids that havevarying degrees of influence in C. elegans, namely linoleic acid (18:2n6), eicosapentaenoicacid (20:5n3) and docosenoic acid (22:1n9). Therefore, we observed changes in fatty acidprofile being strain dependent, however there is no clear correlation between production offatty acid and virulence. We further extended the usage of C. elegans infection models toinvestigate the influence of signalling molecules called prostaglandins, on infection. Here weshow that only the co-infection synthesize prostaglandin E2. Together, these results expandthe use of C. elegans in the field of polymicrobial pathogenesis and provide further evidenceof the likely importance of polymicrobial interactions. Since there is an urgent need fordevelopment of new antimicrobial agents, C. elegans which is known to evaluate differentchemical compounds libraries could be used to solve some of the main obstacles in currentantimicrobial discovery.

[发布机构] University of the Free State