DEALING WITH THERAPY-RESISTANT CRYPTOCOCCOSIS BY TARGETING INTRACELLULAR PATHOGENS

Cryptococcosis is a life-threatening fungal disease that kills more than 600 000 patients per annum worldwide. The majority of these individuals are newly diagnosed HIV-positive patients in the developing world, for whom the prognosis is relatively good, PROVIDED that they survive the cryptococcal infection. In addition, there is a globally increasing incidence of cryptococcal infection in otherwise healthy people, which appears to be due to the occurrence of localized, hypervirulent populations of the fungus. Thus understanding more about the biology of this pathogen, and developing ways to improve treatment of it, is critical.
One major problem is that the 'best practice' antifungal treatment available still fails in more than 10% of patients. One reason for this is that, during an infection, some cryptococcal cells 'hide' within white blood cells of the patient. These intracellular fungi are not exposed to high enough levels of antifungal drugs and can thus act as 'reservoirs' of disease.

To try and address this problem, our proposal aims to identify drugs that can be used to stimulate the expulsion of intracellular cryptococci from white blood cells, thus exposing them to lethal levels of antifungal drugs in the circulation. Our group has recently discovered just such an expulsion process, which we call vomocytosis, and shown that it can be stimulated by applying different agents, such as the anti-malarial drug chloroquine.

In order to move this finding into the clinic, we need to follow two lines of investigation. Firstly, we need to understand the molecular events that happen within infected white blood cells and lead to fungal expulsion. Such information is essential if we are to devise methods of manipulating that process without causing unwanted 'collateral damage' (for instance, dramatically impairing the body's ability to tackle other pathogens that may be infecting at the same time as Cryptococcus). Secondly, we plan to undertake a high-throughput screen to identify new, more effective, compounds that stimulate cryptococcal expulsion. Importantly, we will take a 'new tricks for old drugs' approach, focusing our search on the list of (>1000) compounds that have already been approved by the Food and Drug Administration. These drugs therefore meet minimum safety standards and have already been used to treat other disease conditions. Therefore compounds that we identify can be moved into clinical practice more rapidly and more cheaply than totally new molecules.

At the end of the project, we will thus have provided both important new information about a fatal human disease and revealed a set of compounds that might, in the near future, be utilized to help treat that disease.

The etiological agents of cryptococcosis, Cryptococcus neoformans and C. gattii , are both facultative intracellular pathogens that can survive and proliferate within macrophages. Intracellular yeast represent a reservoir of disease that is difficult to eradicate with current antifungal regimes. We have recently identified a novel expulsion process, termed vomocytosis, by which intracellular cryptococci exit macrophages. In addition, we have shown that vomocytosis can be enhanced by the application of specific cytokine subsets, gaseous stimuli or chemical agents.

Here we propose a programme of work to both investigate the intracellular behaviour of cryptococci and identify agents that manipulate this behaviour. Specifically, we will:
a) identify the molecular steps that lead to cryptococcal expulsion
b) determine how differing execution of these steps leads to inter-strain variation in these infectious species
c) identify compounds that induce vomocytosis, and
d) reveal the mode of action of such compounds.

In following this line of investigation, our intention is to develop a new therapeutic strategy to accelerate fungal clearance in patients. This strategy is based around combining existing antifungal regimes with new compounds, identified from our screen, which will induce the expulsion of intracellular cryptococci, rendering them susceptible to the antifungal agents. Our approach, focusing on a drug-redeployment screen within current FDA-approved compounds, is likely to identify agents that are safe in patients and can be rapidly deployed into clinical settings. In addition, we are mindful of the fact that analogous pathogen expulsion processes have recently been identified in a range of infectious organisms. Thus findings we make as part of this proposal are likely to have far-reaching consequences both in terms of fundamental microbiology and translational approaches for dealing with intracellular infections.

The major beneficiaries of our research will ultimately be cryptococcosis patients, particularly those for whom best-practice antifungal treatment does not lead to a rapid rate of fungal clearance and who are thus at the highest risk of death. As with any novel therapeutic approach, it will take time for our findings to be adopted in clinical practice. However, by focusing our drug-screening approach on existing FDA-approved compounds, we aim to identify lead molecules that can be rapidly and cheaply 'rolled out'; certainly on a much shorter timescale than the 17 years that MRC has previously identified as the average time taken for research findings to enter the clinic.

More broadly, a substantial fraction of this proposal is dedicated to a detailed molecular investigation of intracellular parasitism by cryptococci. The information arising from this section impacts not only on cryptococcal disease, but also provides detailed information on intracellular parasitism and phagocyte biology that will be of value to immunologists, cell biologists and microbiologists. Specifically, non-lytic expulsion processes, similar to the one we propose to study, have now been identified in a diverse range of human pathogens. Hence both clinical and non-clinical colleagues working on other infections (and thus, ultimately, their patients) will benefit from the data we produce.

Finally, the processes we propose to investigate are tightly linked to host cell vesicle trafficking and exocytosis. Thus we will be generating both data and reagents of value to a wide range of individuals, ranging from academic cell biologists to commercial microscope companies.