Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Abstract

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level. The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique. The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

Control of Mucosal Candidiasis in the Zebrafish Swim Bladder Depends on Neutrophils That Block Filament Invasion and Drive Extracellular-Trap Production

“…The infection is initiated by injecting C. albicans directly in the swim bladder lumen (Fig. 1A), and immunocompetent fish control a moderate infection inoculum (up to 50 organisms) so that mortality is rare over the first 3 days of infection (28).…”

“…The water temperature was kept at 28°C. Collected eggs were reared to 4 days postfertilization (dpf) at 33°C as described previously (28). The fish lines used were wild-type (WT) AB from ZIRC, Tg(BACmpo:gfp) 114 as described previously (59) and referred to here as mpx:GFP, Tg(mpo:mCherry-A2-Rac2D57N) as described previously (38) and referred to here as mpo:Rac2-D57N, as well as mpeg1:Gal4 (60) crossed with UAS:NfsB-mCherry (52) and referred to here as mpeg1-NTR and mpeg1:EGFP (60).…”

“…At 4 dpf, larvae reared at 33°C were screened for an inflated swim bladder and injected in the swim bladder with 4 nl of Candida albicans in PVP or PVP only as described previously (28). For ear injection, at 3 dpf larvae reared at 33°C were injected in the otic vesicle with 1 nl of Candida albicans (2 ϫ 10 7 CFU/ml) or Pseudomonas aeruginosa (10 9 CFU/ml) in 5% PVP or PVP only.…”

“…We recently developed the first microinjection mucosal infection model in the zebrafish larva, and it has been used to study sterile inflammation and infection with both viral and fungal pathogens (27)(28)(29). In this study, we leveraged this infection model to achieve a high spatiotemporal resolution of neutrophil-C. albicans dynamics at the mucosal level.…”

Yeast and Filaments Have Specialized, Independent Activities in a Zebrafish Model of Candida albicans Infection

“…To visualize the functions of different C. albicans morphotypes during infection, we developed a zebrafish yolk infection route in which a localized tissue infection can spread to the bloodstream. At the normal temperature for zebrafish husbandry, 28°C, wild-type C. albicans yeast rapidly germinates in the zebrafish host unless contained by phagocytes (8,43). Therefore, to test the roles of different morphological forms, we infected fish at this temperature with either the filamentdominant wild-type strain (green fluorescent protein-labeled SC5314 [SC5314-GFP]) ( Fig.…”