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Fungal resistance to antifungals is promoted by cell heterogeneity

Periodic Reporting for period 1 - FUNFIT (Fungal resistance to antifungals is promoted by cell heterogeneity)

Reporting period: 2015-06-01 to 2017-05-31

The main hypothesis that I will test is that fungal cell heterogeneity provides subpopulations of cells with greater fitness to resist antifungal treatment.

Importance for society
The number of people who die from fungal infections (~2 million p.a.) is greater than the numbers that die from tuberculosis or malaria (1.3 and 0.63 million p.a. respectively). Furthermore, ~25% of the world’s population at any one time suffer from superficial fungal infections of skin and nails. The lung disease aspergillosis, caused by Aspergillus fumigatus, is one of the most common invasive fungal diseases and mortality rates are nearly 100% when untreated and as high as 50% when treated. Individuals most susceptible to invasive fungal diseases are those that are immunocompromised (e.g. patients with AIDS or undergoing immunosuppressive drug treatment).
There are only 3 major classes of commercial antifungal drugs available to treat invasive fungal diseases: azoles (which inhibit ergosterol synthesis), polyenes (which target ergosterol in the plasma membrane), and echinocandins (which inhibit β-1,3-glucan synthesis in the cell wall). Resistance of A. fumigatus and other fungal pathogens against antifungal drugs is increasing. This is particularly significant with regard to the azoles that are the first-line treatment of invasive fungal diseases. A significant cause of this has been attributed to the widespread agricultural use of azole fungicides. Different resistance mechanisms against azoles and other antifungals have been reported including: exclusion or active efflux from the fungal cell; overproduction of the drug target; and structural alterations of the drug target by mutations. A mechanistic understanding of antifungal resistance and discovery of new antifungals is of critical importance to combat the very serious problem of invasive fungal diseases.

(1) Determine which features of three distinct fungal cell types contribute to cell heterogeneity;
(2) Determine which cell types and subpopulations of these cells show highest resistance or survival against antifungals; and
(3) Determine the roles of septal plugging and cell ploidy in the mechanistic basis of fungal cell heterogeneity.

(1) Cell heterogeneity can be observed in different cell types and for many different characteristics of cells (e.g. cell wall composition)
(2) Cell heterogeneity is dynamic, but persistent in successive cell generations
(3) My data suggests that cell heterogeneity impacts antifungal treatment
(4) Cell heterogeneity influences phagocytosis of spores by immune cells (macrophages)
(5) Our data provides evidence that epigenetics are involved in the emergence of cell heterogeneity
Results obtained from addressing each objective

(1) Which features of three distinct fungal cell types contribute to cell heterogeneity;
We have found that almost all cell types and markers in this study showed heterogeneity.
We tested for cell size and cell wall composition with specific antibodies and lectins.
Heterogeneity was shown in each developmental stage (i.e. dormant spores, swollen spores or germlings) analysed.
This heterogeneity within the cell population was maintained through development. For example, when single spore from a heterogeneous population was allowed to germinate and form a colony and produce spores again, these new spores had a similar level of heterogeneity as in the parental population.

(2) Which cell types and subpopulations of these cells show highest resistance or survival against antifungals
We have observed that some cells are more resistant to caspofungin treatment than others. However, I still need to perform more data analysis to be able to tell which characteristics these resistant cells possess.

(3) The roles of septal plugging and cell ploidy in the mechanistic basis of fungal cell heterogeneity.
We tried to study ploidy (i.e. the number of chromosomes per cell), but it was technically very hard to perform this analysis in a high throughput mode.

Alternatively, we tested the effect of fungal cell heterogeneity on phagocytosis (the uptake of germlings by immune cells called macrophages and assessed whether epigenetics played a role in the origin of cell heterogeneity.
We found that specific spores were not taken up by macrophages.
By studying cell heterogeneity in epigenetic mutants, we have indications that epigenetics are involved in the emergence of cell heterogeneity.

-This work will be published as 3 independent journal publications
-The work was presented at the Fungal Cell Wall conference 2015, Paris
-I was involved in public engagement during Science Spectacular 2016, Manchester Museum
The fact that fungal spore populations are heterogeneous may have a great impact on the response of the immune system, establishment of aspergillosis, and treatment of this disease. The conidia that are inhaled, and which have the potential to make an immunocompromised person ill, will be heterogeneous. First of all this will impact immune recognition. Some spores might not be recognised by the immune system and are allowed to germinate and penetrate lung epithelia. The heterogeneous nature of the infection could also impact the effectiveness of antifungal treatment. It might be that some cells are not/less sensitive to the treatment and become persister cells, making the treatment ineffective and the disease of a chronic nature. We found that epigenetics are involved in the emergence of cell heterogeneity. Companies, which produce antifungals, could exploit our results by discovering and utilising epigenetics inhibitors that reduce cell heterogeneity. We suggest a combination treatment with epigenetics inhibitors and classical antifungals, to enhance the effectiveness of antifungal therapy. During diagnosis the level and nature of heterogeneity of the infection could be identified to determine the most suitable treatment. This concept may also be applied to control fungal growth to improve food preservation or to control fungal pathogens in agriculture.

Since many fungal research studies investigate the average results or outputs of processes from entire cell populations, the importance of the behaviour and impact of single cells within the whole cell population is usually not taken into account. This project provided evidence that single cell behaviour and their heterogeneity in the cell population may be very important during antifungal treatment and immune responses. My study should increase the awareness of understanding the concept of cell heterogeneity and how it can have a significant impact on fungal biology which should be of relevance to many in the fungal scientific community in their own fields of research.