Combatting resistance to combinatorial stress and macrophage killing in Candida glabrata.

The processes of life are dynamic and it is change on a molecular level that enables organisms to grow but to also adapt to and survive in different environments, such as the ability to cause disease within a human host. My research focuses on the human fungal pathogen, Candida glabrata, which can cause illness in humans ranging from allergic reactions, infections such as thrush which affects ~75% of women at least once, to serious disease in patients that have impaired immune systems. These fungi are increasing in incidence and the reason for this increase is not understood. However, it is clear that the fungus can defend itself against high levels of stress and antifungal drugs used in treatment regimes. My hypothesis is that C. glabrata has evolved the capabilities to withstand a challenge from the combination of environmental and imposed drug stresses.

Firstly, to look at C. glabrata, I will take advantage of my recent discovery of the sexual cycle in this fungus which offers novel methods to test hypotheses about evolution and pathogenesis. Pathogens of humans (microbes that can cause infection), such as C. glabrata, are successful because they adapt effectively to environmental stresses encountered within the host body. Upon recognition by host immune cells, C. glabrata is engulfed and exposed to a combination of stresses. In contrast to other pathogenic fungi, C. glabrata is highly resistant to stress allowing it to survive the host immune defences. This suggests that resistance to both antifungal drugs (a stress brought on by medical intervention) and natural host-induced stresses are essential for establishment and progression of infection. The molecular mechanisms underpinning antifungal resistance and the response to individual stresses, have been investigated in isolation, however little is known about how C. glabrata adapts to combinatorial stresses. The mechanistic explanation of stress adaptation will yield new insights into Candida infection.

Using my newly discovered sexual cycle in C. glabrata, I have generated a series of related strains of the same fungal pathogen that have increased resistance to combinatorial and drug stresses. I will sequence their genomes (a process for analysing DNA) to identify the critical genes involved in stress resistance and characterise the mechanisms of C. glabrata stress responses. My preliminary data and publications demonstrate that the C. glabrata response to in vitro (performed in the lab outside of the human body) combinatorial stress is similar to that observed upon phagocyte engulfment (when immune cells recognise pathogens and try to remove them). At the level of gene expression, there is an up-regulation of genes (the process by which information encoded in a gene is used to make more proteins) encoding functions related to stress adaptation and nutrient recycling overlap. Understanding this regulatory network and the role that selected components (different genes) play in stress resistance, is essential to the development of future drug regimes.

Stress resistance is an underlying trait of importance in the understanding the virulence of major fungal pathogens of humans. The incidence of Candida infections caused by species other than C. albicans has been rapidly growing over the last 20 years. Candida glabrata is now the causative fungal pathogen of up to 30% of infections. This increase in incidence is thought to be due to its high innate tolerance to stress and ability to rapidly acquire drug resistance. C. glabrata can survive and multiply inside human phagocytic cells and defend itself against the combinatorial stresses encountered within the host. The molecular mechanisms that underpin this response cannot be predicted by looking at each stress individually. Therefore with this fellowship I will determine the mechanistic basis of combinatorial stress resistance and its impact on virulence and the development of infection.

The objectives of my fellowship are:
1: To identify and characterise the genes that underpin Candida glabrata combinatorial stress resistance phenotypes using bulk segregant analysis sequencing on a series of 400 C. glabrata isolates categorised by their resistance phenotypes.
2: The characterisation of the genes regulated during adaptation to stress through long-term RNA-seq studies. My preliminary data has shown that cells respond different to stress applied singly vs in combination and it is not an additive effect. In addition I have shown that C. glabrata cells are still adapting to the stress up 640mins post exposure.
3: Determining the contribution of CS genes to immune evasion and the progression of infection, through in vivo, ex vivo and in vitro suite of tools I have developed in the lab and my network of collaborators.