Intravital imaging-based genetic screen reveals the transcriptional network governing Candida albicans filamentation during mammalian infection
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eLife assessment
Candida morphogenesis is important for virulence. This study provides important new information as to how C. albicans regulates the switch from budding to hyphal morphology. Their results identify transcription factors involved in the process of hyphal morphogenesis in the host. The results are convincing and will be interesting to scientists in the fields of medical mycology and cell biology.
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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.
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eLife assessment
Candida morphogenesis is important for virulence. This study provides important new information as to how C. albicans regulates the switch from budding to hyphal morphology. Their results identify transcription factors involved in the process of hyphal morphogenesis in the host. The results are convincing and will be interesting to scientists in the fields of medical mycology and cell biology.
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Reviewer #1 (Public Review):
Many previous studies have examined the regulation of hyphal growth in vitro, and have identified about 1,000 genes capable of influencing this process. However, a weakness is that most of these genes have weak effects and are not important in vivo. Therefore, it is very significant that this is the first large-scale study to examine the regulation of hyphal growth in vivo by analyzing a set of 156 transcription factor mutants in mice. A strength of these innovative studies is that mutant strains were injected into a mouse ear, which permitted the use of high-resolution microscopy to quantify the fraction of cells forming hyphae and the rate of hyphal elongation. Furthermore, wild-type cells were co-injected to serve as an internal control, which enhanced the rigor of these studies.
One major conclusion is …
Reviewer #1 (Public Review):
Many previous studies have examined the regulation of hyphal growth in vitro, and have identified about 1,000 genes capable of influencing this process. However, a weakness is that most of these genes have weak effects and are not important in vivo. Therefore, it is very significant that this is the first large-scale study to examine the regulation of hyphal growth in vivo by analyzing a set of 156 transcription factor mutants in mice. A strength of these innovative studies is that mutant strains were injected into a mouse ear, which permitted the use of high-resolution microscopy to quantify the fraction of cells forming hyphae and the rate of hyphal elongation. Furthermore, wild-type cells were co-injected to serve as an internal control, which enhanced the rigor of these studies.
One major conclusion is that three core transcription factors were identified as being important in vivo (Rob1, Brg1, and Efg1) and two negative regulators (Tup1 and Efg1). Previously, many transcription factors were found to be important in vitro, so this is important for focusing future studies on the key regulators. Nanostring gene expression studies verified that these core factors regulate overlapping but distinct sets of genes in vitro and in vivo, which reinforces the importance of carrying out studies in vivo. Additional mutants were discovered to have minor defects in filamentous growth and were considered to be ancillary factors that act in concert with the core regulators.
Another innovative aspect of the manuscript is that they examined the rate of hyphal elongation in vivo. This is an understudied area both in vitro and in vivo. Transcription factors UME6, LYS14, and HMS1 were shown to regulate the elongation rate, which opens up new opportunities to study the mechanisms. Consistent with this, these transcription factors were shown to regulate a set of genes that is distinct from those regulated by transcription factors that control the initiation of hyphal growth.
Genetic approaches (complex haploinsufficiency) were used to examine the relationship between the core factors and the ancillary factor TEC1. Interestingly, these results revealed genetic interactions between TEC1 and the core factors EFG1 and BRG1, including their ability to regulate other transcription factors. This shows how these complex networks are functioning in vivo.
Another major advance was that the in vivo analysis of the two negative regulators of hyphal growth (Nrg1 and Tup1) revealed a new model for how they interact with the master transcriptional regulator Efg1. The results indicate that the major function of Efg1 in vivo is to mediate relief of Nrg1 repression. It was not needed to regulate the expression of hypha-induced genes.
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Reviewer #2 (Public Review):
This manuscript is focused on the identification and characterization of transcriptional networks that control the major Candida albicans virulence property of filamentation during infection in vivo. Using an intravital imaging assay, the authors have screened a C. albicans transcription factor mutant library to identify factors important for controlling both filament initiation and elongation in vivo. They also perform Nanostring experiments to identify the in vivo transcriptional profiles of genes controlled by specific key factors in the network. Overall, the authors identify three positive and two negative core factors important for the initiation of filamentation and several factors specifically important for filament elongation (including 4 factors whose mutants have no in vitro elongation phenotypes). …
Reviewer #2 (Public Review):
This manuscript is focused on the identification and characterization of transcriptional networks that control the major Candida albicans virulence property of filamentation during infection in vivo. Using an intravital imaging assay, the authors have screened a C. albicans transcription factor mutant library to identify factors important for controlling both filament initiation and elongation in vivo. They also perform Nanostring experiments to identify the in vivo transcriptional profiles of genes controlled by specific key factors in the network. Overall, the authors identify three positive and two negative core factors important for the initiation of filamentation and several factors specifically important for filament elongation (including 4 factors whose mutants have no in vitro elongation phenotypes). Target genes associated with filament initiation and elongation were shown to be mostly distinct. Unexpectedly, the authors also show that the main role of Efg1, a major positive regulator of filamentation, is to mediate relief of repression by Nrg1.
Overall, the manuscript is well-written and the data are clearly presented. In addition, the authors clearly appear to have achieved their Aim of identifying and characterizing transcriptional networks that regulate C. albicans morphogenesis during infection in vivo. In general, the conclusions of this paper are well-supported by the results. The results of this study are likely to have a significant impact on the field for several reasons: 1) new and valuable information will be provided about transcriptional networks that control C. albicans filamentation in vivo, 2) this study describes an important distinction between genes associated with filament initiation and elongation and will be the first to systematically analyze C. albicans genes associated with filament elongation, 3) while there are similarities, the authors also observe several important differences between transcriptional networks that control C. albicans filamentation in vivo vs. in vitro, which will help to clarify regulation that actually occurs during infection, 4) as indicated above, a new and surprising role for the C. albicans master regulator of filamentation, Efg1, is reported, 5) because filamentation is an important C. albicans virulence property, several of the target genes of transcription factor networks identified by this study (and the factors themselves) could serve as potential targets for new antifungals. As a consequence, this study is likely to provide information that opens up new and useful lines of research for the field.
Strengths:
1. Intravital imaging allows for the identification of transcription factors specifically important for C. albicans filamentation during infection.
2. Distinct sets of C. albicans genes and factors associated with filament initiation vs. elongation are identified.
3. Key differences between in vivo and in vitro transcriptional regulation of C. albicans filamentation are demonstrated, which in some cases challenge current paradigms. This also highlights the effect of the environment in determining target genes.
4. Evidence is presented to suggest that Efg1 promotes C. albicans filamentation primarily through relief of Nrg1 repression.Weaknesses:
1. Nanostring does not profile the complete set of C. albicans genes, but rather a subset that is pre-selected. Therefore, defining proportions of genes and gene classes controlled by specific transcription factors may not give the complete picture and may not be accurate with respect to the transcriptome as a whole.
2. As the authors have noticed, transcription factors and target genes associated with C. albicans filamentation may vary significantly depending on the environment. It is therefore unclear whether the in vivo gene expression patterns observed in this study apply to other host niches besides the ear.
3. Similarly, variations in filamentation-associated transcription factors and target genes may occur in the "in vitro" conditions used by the authors. RPMI + 10% serum is the main "in vitro" condition but many other conditions are known to drive C. albicans filamentation.
4. Lines 361-366: A clear rationale for additional TFs to study in more detail was not provided.
5. Post-translational mechanisms, particularly septin phosphorylation, are likely to have an important effect on filament elongation (see work from Yue Wang's lab), which was not discussed.
6. Many Nrg1 targets are known to also be Tup1 targets (Kadosh & Johnson, 2005), which counters the argument that this corepressor and DNA-binding protein function separately.
7. While useful, examining genetic interactions using haploinsufficiency has several limitations and certain interactions may escape detection. -
