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Are Heat Shock Protein 90 (Hsp90) Co-Chaperones Virulence Factors in the Human Fungal Pathogen Candida albicans?

Final Report Summary - HSP90COCHAPERONES (Are Heat Shock Protein 90 (Hsp90) Co-Chaperones Virulence Factors in the Human Fungal Pathogen Candida albicans?)

HSP90COCHAPERONES - Final Report

Are Heat Shock Protein 90 Co-chaperones Virulence Factors in the Human Fungal Pathogen Candida albicans?

Background
Each year, fungi kill >1.6 million patients world-wide (Bongomin et al., 2017. JoF). This is comparable to tuberculosis, which is widely recognised as one of the leading causes of death from infectious diseases. One of the leading fungal pathogens of humans, Candida albicans, causes ~700,000 invasive and life-threatening infections (Bongomin et al., 2017. JoF) with mortality rates of up to 75% (Brown et al., 2012. Sci Transl Med). Fungal infections are difficult to treat and prevent due to lack of efficacious antifungal therapies, paucity of suitable antifungal drug targets and Candida's status as a common inhabitant of the human oral cavity and gastrointestinal tract. It is thus imperative to understand the genetic and molecular underpinnings of fungal virulence to aid in the development of more efficacious treatment strategies.

The essential chaperone heat shock protein 90 (Hsp90) regulates several Candida virulence factors, such as cellular morphology, drug resistance, and the survival of high temperatures (Alaalm et al., 2020. submitted), all of which are critical for the yeast's success as a human pathogen. Yet, due to high sequence conservation, Hsp90 cannot be targeted specifically. Hence, we focussed on Hsp90's co-chaperones, which facilitate Hsp90 function and specificity and are much less similar to their human homologs. Candida has ten Hsp90 co-chaperones and we investigated five non-essential candidates. The advantage of exploiting non-essential drug targets over essential ones would be the reduced selective pressure for emergence of antifungal resistance. With this in mind, we asked "Are Hsp90 co-chaperones C. albicans non-essential virulence factors that could be further exploited as drug targets?''. To address this question, we deployed a suite of basic and advanced cell and molecular biological techniques including whole genome sequencing, ddRAD sequencing, Western blotting, in vitro virulence testing and in vivo modelling of virulence.

Results
We set out to investigate the: (i) impact of Hsp90 co-chaperones on fungal virulence, (ii) effect of Loss-of-Heterozygosity (LoH) on co-chaperone gene location, and (iii) transcriptional regulation of co-chaperone gene expression. As part of objective (i), we also aimed to develop the nematode Caenorhabditis elegans as a model host for the study of fungal virulence.

To assess the impact of Hsp90 co-chaperones on Candida virulence, we deleted both alleles of five non-essential co-chaperones. Growth assays validated that none of the selected genes was essential for growth under different conditions. Next, we measured the mutants' response to oxidative and heat stress as well as their ability to survive antifungal drug treatment, form biofilms, produce hyphae and to kill an invertebrate host. We found that co-chaperones play a complex role in the regulation of Candida virulence, different co-chaperones modulate different aspects thereof. For example, Sti1 is a positive regulator of biofilm formation but Ssa2 is a negative regulator of drug resistance and survival of oxidative stress (Lyons et al., manuscript in preparation). To be able to test the mutants’ virulence potential in a host model, we developed caterpillars of the Tobacco Hornworm (Manduca sexta) as a novel invertebrate model (Lyons et al., 2020. Virulence). Unlike the nematode, these caterpillars can be maintained at 37˚C, can be injected with a defined amount of yeast inoculum, are large enough for host transcriptomic profiling of specific tissues in a single animal, and facilitate tracking of fungal burden in the faeces or haemolymph of a single animal throughout the course of infection. This unique combination of advantages recommends Manduca caterpillars as a host model for fungal disease.

Based on our observation that Candida co-chaperones aggregate in the sub-telomeric region, a locale that is prone to LoH events affecting genetic diversity and gene regulation, we hypothesised that LoH would affect their localisation. We measured the effect of Hsp90 disturbances on LoH events across the genome, showing that LoH is abundant but different between high temperature stress and Hsp90 pharmacological inhibition (Figure 1.pdf). LoH events range from short gene conversions to large whole chromosome events, including aneuploidy (Dong et al., manuscript in preparation). As a potential mechanism, we showed that disturbing Hsp90 function results in reduced stability of clients involved in chromosome segregation and DNA repair.

Lastly, to elucidate the impact of heterochromatin, a feature of the sub-telomeric region, on co-chaperone gene regulation, we measured co-chaperone gene expression in genetic backgrounds prone to heterochromatisation. Indeed, histone deacetylation affects expression of specific Hsp90 co-chaperones (Lyons et al., manuscript in preparation).

Conclusions
We achieved our scientific objective of assessing the virulence potential of Hsp90 co-chaperones. Our research recommends them for further exploitation as antifungal drug targets. Our scientific achievements together with our knowledge transfer and public engagement activities as well as the project's socio-economic impact (training the next generation of scientists, novel invertebrate host model) made this research programme successful.

Socio-economic impact
I consider training the next generation of scientists an essential part of my profession as an academic. Throughout this project, two graduate students and two postdoctoral researchers received training in cellular and molecular mycology. Both postdoctoral researchers have since moved on to their next career stages, one as an NHS Genetic Technologist and the other as a postdoctoral fellow at Brown University. The graduate student contributing the majority of work to two of the three publications resulting from this project moved on to conduct postdoctoral training at the University of Tel Aviv.

Studying fungal virulence necessitates the use of host models. Here, we developed an alternative invertebrate model host for the study of fungal diseases that addresses several shortcomings of already existing invertebrate models. The caterpillars can be easily obtained by Researchers in Europe and North America, are low-maintenance and provide reliable and reproducible insights into fungal virulence. While impossible to measure now, the paper has just been published, our caterpillar model is expected to contribute to a reduction of the usage of mammalian models for the study of fungal disease.

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