Intravital imaging-based genetic screen reveals the transcriptional network governing Candida albicans filamentation during mammalian infection

Abstract

Candida albicans is one of the most common human fungal pathogens. C. albicans pathogenesis is tightly linked to its ability to under a morphogenetic transition from typically budding yeast to filamentous forms of hyphae and pseudohyphae. Filamentous morphogenesis is the most intensively studied C. albicans virulence traits; however, nearly all of these studies have been based on in vitro induction of filamentation. Using an intravital imaging assay of filamentation during mammalian (mouse) infection, we have screened a library of transcription factor mutants to identify those that modulate both the initiation and maintenance of filamentation in vivo. We coupled this initial screen with genetic interaction analysis and in vivo transcription profiling to characterize the transcription factor network governing filamentation in infected mammalian tissue. Three core positive (Efg1, Brg1, and Rob1) and two core negative regulators (Nrg1 and Tup1) of filament initiation were identified. No previous systematic analysis of genes affecting the elongation step has been reported and we found that large set of transcription factors affect filament elongation in vivo including four (Hms1, Lys14, War1, Dal81) with no effect on in vitro elongation. We also show that the gene targets of initiation and elongation regulators are distinct. Genetic interaction analysis of the core positive and negative regulators revealed that the master regulator Efg1 primarily functions to mediate relief of Nrg1 repression and is dispensable for expression of hypha-associated genes in vitro and in vivo. Thus, our analysis not only provide the first characterization of the transcriptional network governing C. albicans filamentation in vivo but also revealed a fundamentally new mode of function for Efg1, one of the most widely studied C. albicans transcription factors.

The role of theC. albicanstranscriptional repressorNRG1during filamentation and disseminated candidiasis is strain-dependent

“…In agreement with its inability to filament in vitro, we previously reported that 94015 also failed to undergo filamentation in vivo (15). In contrast, 50755 forms filaments in vivo to a similar extent as SN250 and SC5314 despite having reduced filamentation under a variety of in vitro conditions (Fig.…”

“…6B. We previously found that deletion of NRG1 in an efg1∆∆ mutant (SN250 background) restored expression of hypha-associated genes in vivo and in vitro (15).…”

“…NanoString analysis was carried out as described previously (15). Briefly, in total, 80 ng of in vitro or 400 ng of in vivo RNA was added to a NanoString codeset mix and incubated at 65 0 C for 18 hours.…”

“…The inoculation and imaging of mice ear were carried out as described previously (9,15). Acquired multiple Z stacks (minimum 15) were used to score the yeast vs filamentous ratio.…”

Temporal dynamics ofCandida albicansmorphogenesis and gene expression reveals distinctions between in vitro and in vivo filamentation

“…In vivo, we observed very little evidence of the hyphae-to-yeast transition during the time course examined. Furthermore, we have observed very few lateral yeasts in vivo up to 24hr post-infection and the filament-to-yeast ratios are stable over the same time period (7). Once again, these morphological observations can be correlated with the expression patterns of positive regulators of hyphae and the best characterized positive regulator of lateral yeast formation, PES1 .…”