Periodic Reporting for period 1 - BranchOut (Filament branching during infection by a human opportunistic fungal pathogen)
Reporting period: 2021-09-01 to 2023-08-31
Human fungal pathogens are responsible for over 1.6 million deaths and approximately billion of superficial mucosal infections worldwide each year. Aging and respiratory diseases increase the risk of invasive fungal disease that can also lead to severe illness and death. Last year, the World Health Organization (WHO) released a report recommending to ”focus and drive further research and policy interventions to strengthen the global response to fungal infections and antifungal resistance”. Four Candida spp. are highlighted on the released fungal priority list with C. albicans in the most critical group. C. albicans is an opportunistic fungal pathogen. It is found as a commensal in the mouth and gastrointestinal tract in ~ 50% of healthy adults. In specific conditions, the fungus becomes pathogenic, and causes an infection, candidiasis, which results from fungal proliferation. C. albicans, a dimorphic fungus, thrives in either budding or filamentous forms due to a complex interplay of signals and polarity-regulating proteins conserved among eukaryotic cells. Both morphologies coexist in tissues during systemic infection, with filaments promoting tissue invasion and damage, and budding cells facilitating mucosal colonization, dissemination, and biofilm formation. Filamentous growth, conserved in most fungi, comprises tubular filaments that extend and explore surroundings, enabling tissue invasion. The leading filament grows apically, often accompanied by branching to maximize the number of growing filament tips. Branching may occur at cell tips or within subapical compartments, with the latter, called lateral branching, being predominantly observed in C. albicans. Although branching is implicated in fungal virulence, its exact function remains enigmatic.
In this project we aimed to understand filament branching addressing three questions:
1) Is branching distinct from the main filament?
2) Are branches responding differently to antifungal drugs?
3) Is branching important for fungal virulence?
Our results so far show that branching is a distinct developmental state that contributes to the antifungal response and plays a potential role in pathogenicity. Branching is virtually present in all filamentous fungi, including those that has a major impact on our economy. Globally, fungal pathogens cause crop losses of 10-30% (0.1-0.2 trillion €) each year and $7.5 billion of direct medical costs in the U.S.A. alone. Research conducted in BranchOut has provided a major understanding of the so far unknown functions and regulation of branching, and its implication in virulence.
In this project we aimed to understand filament branching addressing three questions:
1) Is branching distinct from the main filament?
2) Are branches responding differently to antifungal drugs?
3) Is branching important for fungal virulence?
Our results so far show that branching is a distinct developmental state that contributes to the antifungal response and plays a potential role in pathogenicity. Branching is virtually present in all filamentous fungi, including those that has a major impact on our economy. Globally, fungal pathogens cause crop losses of 10-30% (0.1-0.2 trillion €) each year and $7.5 billion of direct medical costs in the U.S.A. alone. Research conducted in BranchOut has provided a major understanding of the so far unknown functions and regulation of branching, and its implication in virulence.
1) Generation of an assay to characterize branching and quantification of branching features, such as speed growth, morphology, frequency, positioning, etc. in a wild type strain.
2) Creation of strains containing polarity, growth-associated and lipid distribution reporters, and characterization of their localization in live-cell imaging timelapses, comparing branching with main filaments.
3) Deciphering the response of branches and main filaments to antifungal drugs (Fluconazole and Caspofungin)
4) Generation of potential branching-defective mutant strains and subsequent complementation strains, perform of the branching assay for each strain plus control.
5) Mice infection assays – In progress.
In summary, our results indicate that branching is a distinct developmental state compared to main filaments. Branches grow slower and have a distinct morphology. In addition, polarity, growth-associated factors and lipid distribution are different between the two growth forms. Furthermore, when treated with commonly used antifungal drugs, these two growth states respond differently, suggesting that branches may be a niche for fungal tolerance or resistance generation. Mutants affected in branching were generated and further experiments are on progress to test whether hyphal branching is important for virulence.
These data had been presented in three international conferences by the beneficiary and the host principal investigator (USA, Austria and Brazil). In addition, we recently submitted a brief article (observation) to mBio which is under review and is currently accessible at BioRxiv. Another more manuscript is expected to be submitted by the spring of 2024. All publications will be available as open access or through a repository server.
2) Creation of strains containing polarity, growth-associated and lipid distribution reporters, and characterization of their localization in live-cell imaging timelapses, comparing branching with main filaments.
3) Deciphering the response of branches and main filaments to antifungal drugs (Fluconazole and Caspofungin)
4) Generation of potential branching-defective mutant strains and subsequent complementation strains, perform of the branching assay for each strain plus control.
5) Mice infection assays – In progress.
In summary, our results indicate that branching is a distinct developmental state compared to main filaments. Branches grow slower and have a distinct morphology. In addition, polarity, growth-associated factors and lipid distribution are different between the two growth forms. Furthermore, when treated with commonly used antifungal drugs, these two growth states respond differently, suggesting that branches may be a niche for fungal tolerance or resistance generation. Mutants affected in branching were generated and further experiments are on progress to test whether hyphal branching is important for virulence.
These data had been presented in three international conferences by the beneficiary and the host principal investigator (USA, Austria and Brazil). In addition, we recently submitted a brief article (observation) to mBio which is under review and is currently accessible at BioRxiv. Another more manuscript is expected to be submitted by the spring of 2024. All publications will be available as open access or through a repository server.
It was commonly accepted that branches were a form of filamentous growth, like main filaments. Their function and roles during any vegetative or virulence process were not studied in detail and only briefly mentioned in some studies. We have performed a first-time quantitative extensive analysis of branching, standardizing a protocol for the project, and describing the process in the reference strain and subsequent mutants generated of a human fungal pathogen. Besides, we hypothesize that branching plays a role in the antifungal drug response (to azoles or echinocandins), to potentially acquire resistance or tolerance by escaping the treated zones. This should have an important socio-economic impact, not only for health treatments but for agriculture. Azoles are currently vastly used to prevent crop losses. Investigation of branching-limiter chemicals may reduce the amount of azoles needed in the sanitary but also in the agriculture level and help to reduce the incidence of fungal infections and the appearance of antifungal resistance strains.