Transcript C. albicans
YJC Laboratory, NTHU-Medical Science Zebrafish as a model host of Candida albicans infection Chung-Feng 1 Jen 1Department (任中鳳), Chun-Cheih 1 Chao (趙俊傑), Po-Chen 2 Hsu (許博琛), Chung-Yu 2 Lan (藍忠昱), Yung-Jen 1 Chuang (莊永仁) of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, R.O.C. 2Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan, R.O.C Introduction C.albicans is an opportunistic human pathogen that colonizes various body locations asymptomatically. However, once immune dysfunction occurred, often found in hospitalized patients, anticancer drug treated patients and AIDS patients, C.albicans can proliferate and cause infections termed candidiasis. In the United States, excess cost attributes to candidemia is about $ 1 billion per year. Model organisms have been proven to be powerful tools to study infectious pathophysiology. Mammalian animal models have been developed to elucidate the complex relationship between C.albicans and its hosts. However, the drawback of mammalian models, such as mice and rats, is too expensive for the average labs . Therefore, scientists have exploited other species including fruit fly (Drosophila melanogaster) and the nematode (Caenorhabditis elegans) to substitute mammals in deciphering various host-pathogen interactions. Several studies of host-C.albicans interactions in Drosophila and C.elegans have been also reported recently. Nevertheless , They only possess the innate immune systems but lack of the adaptive ones. Along these lines, we want to test the possibility of zebrafish as a new infectious disease model host of C.albicans. Because being a vertebrate, zebrafish are evolutionarily closer to humans than flies and C.elegans. Fig. 6. Yeast to hyphae transition is important in C.albicans virulence in zebrafish model The arrow head indicates (A) colonized HLC54 C.albicans in yeast form, and (B) SC5314 C.albicans in hyphae form at 93hr post-infection. (A) (B) ?? V.S Fig. 7. C.albicans can induce specific inflammatory genes response in zebrafish (A) zTNFa and (B) zIL-1b were (C) up-regulated; whereas (C) ziNOS was downregulated at 2 and 15 hpi. Fig. 1. Zebrafish have both an innate and adaptive immune system (Astrid M. el. 2004) Material s and Methods 1. 2. 3. 4. 5. 6. Fig. 8. C.albicans can infect embryonic zebrafish The arrow head indicates C.albicans hyphae, which protrudes out from zebrafish hindbrain. Conclusion AB strain/TL strain adult zebrafish Candida albicans strains Zebrafish intra-peritoneal injection and CFU counting Histology and PAS staining RT qPCR Live cell image 1. The survival rate assay showed that wild type C. albicans can infect and kill zebrafish in a dose-dependent manner, and the fatality was not caused by endotoxic shock. 2. The number of C. albicans injected by intra-peritoneal approach was determined by CFU counting experiment, and the result indicated that C. albicans can successfully colonize and proliferate within the zebrafish gut as expected. 3. The distinctive yeast-to-hyphae transition of C. albicans in infected zebrafish was observed by PAS staining and histology analysis. 4. Specific inflammatory genes responses (i.e. up-regulation of IL-1b and TNFa, down-regulation of iNOS) during C. albicans infection in zebrafish was detected by RT-qPCR. 5. C.albicans can also infect embryonic zebrafish elucidated by live cell image. Results * References 1. Astrid M. van der Sar, Ben J. Appelmelk, Christina M.J.E. Vandenbroucke-Grauls, and Wilbert Bitter. A star with stripes: zebrafish as an infection model. TRENDS in Microbiology Vol.12 No.10 October (2004). 2. Con Sullivan, Carol H. Kim. Zebrafish as a model for infectious disease and immune function. Fish & Shellfish Immunology (2008). 3. Meagan E. Pressley, Peter E. Phelan III, P. Eckhard Witten, Mark T. Mellon, Carol H. Kim. Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish. Developmental and Comparative Immunology 29 (2005). Fig. 3. C.albicans can proliferate in zebrafish in a dose-dependent manner The hollow/black Fig. 2. C.albicans can kill zebrafish in a dosedependent manner. 10 zebrafish were injected with 108, 109, 1010 CFU C.albicans intra-peritoneally, we found that the fatality of zebrafish was 0%, 50%, and 100% respectively. We also injected 109 CFU heat-killed C.albicans into zebrafish to exclude the endotoxic shock effect of C.albicans infection . ( A ) circle stands for 109/108 CFU C.albicans injection respectively, and the horizontal line represents the CFU mean value. As the diagram shows, C.albicans could colonize on zebrafish gut and proliferate in it. Acknowledgement We thank Dr. Bon-chu Chung, and Chieh-Hui Wang for experimental support. Funding for this project was provided by the National Science Concil of Taiwan, R.O.C. ( B ) Contact Information Chung-Feng Jen epartment of Life Science & Institute of Bioinformatics and Structural Biology National Tsing Hua University Address: NO.101, Sec 2,Kuang Fu Road, Hsinchu, 30013, Taiwan, R.O.C. Office: LS-2, Room 408 Tel: 886-3-5715131#33451/Fax: 886-3-5715934 E-mail: [email protected] Fig. 4. C.albicans can undergo yeast to hyphae transition within zebrafish internal organs After PAS staining, we observed that (A) C.albicans transformed into hyphae form to invade liver, adipose, muscle, and (B) C.albicans invaded gut of zebrafish. Fig. 5. Yeast to hyphae transition is important in C.albicans virulence in zebrafish model Kaplan-Meier survival test showed that zebrafish infected by HLC54 and SC5314 resulted in different survival rate significantly (P<0.05). Yung-Jen Chuang Ph.D. Department of Life Science & Institute of Bioinformatics and Structural Biology National Tsing Hua University Address: NO.101, Sec 2,Kuang Fu Road, Hsinchu, 30013, Taiwan, R.O.C. Office: LS-2, Room 410 Tel: 886-3-5715131#42764/Fax: 886-3-5715934 E-mail: [email protected]