Re-engineering of fungal sulphur metabolism to limit mould viability and virulence.

On a world-wide basis, A. fumigatus is the most prominent fungal pathogen of the human lung. In Europe, the all-cause burden of Aspergillus-related lung disease exceeds 2 million cases per annum, including up to 50,000 potentially fatal cases of invasive aspergillosis. The mortality rate associated with this disease is very high, and antifungal drugs currently in use have suboptimal efficacy and result in antifungal resistance. In order to develop more successful therapies, it is necessary to better understand why A. fumigatus is able to grow in mammalian tissues. Critical to this process is metabolic versatility and the capacity to obtain nutrients from the host.

I have previously shown that regulation of sulphur (S) assimilation is essential for virulence, however the exact S-source exploited in lung tissue and the in vivo fungal sulphur-related metabolic status remain unknown. Therefore, in this project I aim to clarify these two important issues, which may derive in the identification of suitable targets for the development of novel antifungal therapy.

First, I will utilize state-of-the-art gene expression analysis technology to investigate the sulphur-related metabolic status during infection. This approach has the potential to identify novel pathogenic processes which will aid the development of novel antifungal strategy. Based on my preliminary data, I will concentrate on two specific S-containing molecules which are strongly suggested to be relevant for virulence: methionine and sulphide (H2S).

Methionine is an amino acid whose biosynthesis appears to be essential for A. fumigatus, in contrast to closely related fungal species but in line with other pathogenic fungal species. I will determine what aspect of the biosynthetic pathway is essential, as well as the functional relevance of this activity for fungal survival.

In addition, I will look for compounds that specifically inhibit the activity of the fungal cobalamin-independent methionine synthase in vitro, and test their suitability as anti-aspergillosis drugs in animal models of disease. I will also ask whether A. fumigatus exploits H2S as sulphur source during infection. Sulphide is a gas produced in the human tissues, including the lungs, as signalling molecule. It has been described that H2S modifies proteins inside the cells, to regulate their activity. This protein alteration, termed sulfhydration, has been observed in bacteria and mammalian cells, demonstrating its conservation throughout evolution, and therefore, highlighting its importance. Nothing is known about sulfhydration in fungal species, I will therefore investigate whether this protein alteration occurs in A. fumigatus and whether it is important for the fungus to grow within the lungs.

I will use the gathered results to look for correlates between fungal and/or mammalian sulphur metabolism, and risk for aspergillosis. This will be achieved by interrogation of the human and fungal genome data curated by the University of Manchester. Importantly, these analyses may underscore relevant epidemiologic information which may aid patient management.

I will seek a mechanistic understanding of A. fumigatus in vivo S-requirements using state-of-the-art NanoString gene expression technology of fungal RNA isolated from infected mice. I have demonstrated that methionine (met) synthase is essential in A. fumigatus. I will next attempt to construct a met auxotroph in which met synthase activity is maintained (metG cysD double mutant), to ascertain whether met is a vital in vivo S-source and met biosynthesis per se is essential for A. fumigatus.
I will seek specific inhibitors of fungal cobalamin-independent met synthases, and assess their relevance as antifungal therapeutics. I will test polyglutamated folate analogues and I will also screen a MRCT library of 9000 compounds held at the Manchester Fungal Infection Group (MFIG), using an in vitro, spectrophotometric method. Drug candidates will then be tested to confirm their impact upon fungal growth using a luciferase expressing A. fumigatus strain. Drugs having an inhibitory effect will be further tested in animals to evaluate their suitability for treatment.
I will investigate the relevance of H2S, a gas produced in mammalian tissues, for A. fumigatus growth and virulence. I will test virulence of a cysD cysB double mutant, which should not incorporate H2S, to determine if H2S is exploited as S-source. Since H2S has been described to target cysteines, a protein modification termed sulfhydration, I will also ascertain whether this occurs in A. fumigatus. I will adapt two previously described methods to compare protein extracts from wt and a mecA mecB double mutant (which I hypothesise would be a sulfhydration-defective strain). Furthermore, I will utilize a mouse CSE-/- line, defective in H2S production in peripheral tissues, to confirm the relevance of this gas in vivo. Finally, I will seek correlations between fungal S- assimilation and/or human S-status, and the occurrence of disease using the database of genome sequences for aspergilloses curated at the MFIG.

1. Individuals suffering, or at risk from, Aspergillus-related disease: On a world-wide basis, A. fumigatus is the most prominent fungal pathogen of the human lung, and persistence in human airways of inhaled A. fumigatus spores is a major cause of acute and chronic lung disease. In Europe, the all-cause burden of Aspergillus-related lung disease exceeds 2 million cases per annum, including up to 50,000 potentially fatal cases of invasive aspergillosis. Due to the low efficiency of current therapy, the mortality rate associated with aspergilloses remains unacceptably high; encouragingly, the use of the knowledge derived from our investigations to improve treatment has the potential to be beneficial for the prognosis and survival of patients. Therefore, our results will have a positive impact on public health.

2. Pharmaceutical companies seeking novel antifungal drugs. The pipeline for new antifungal compounds is sparse and antifungal resistance is advancing. Given the proximity of multiple spin-out antifungal drug discovery companies (F2G, Blueberry Therapeutics and Evotech) to the Manchester Fungal Infection Group and the resultant connectivity of academic and pharmaceutical research ongoing in this geographical domain it is certain that new and critical inter-sectorial collaborations will be founded to seek for novel antifungal therapies directed against the disclosed molecular targets. Therefore, my results will be exploitable by the commercial private sector, having potential to give an economic benefit to UK based companies, and fostering the competitiveness of the UK. The global market for clinical antifungals was estimated to be $9.4 billion US in 2010 (GBI research) and is anticipated to exceed $12 billion by 2016.

3. Healthcare Service Providers: The cost of invasive infections caused by Aspergillus sp. in the US alone was estimated at $633 million in 1996. This cost included the expense of failed chemotherapy, or bone marrow or organ transplantation [Dasbach EJ, Davies GM, Teutsch SM (2000) Burden of aspergillosis-related hospitalizations in the United States. Clin Infect Dis 31:1524-8]. The utilization of our identified targets to develop novel therapies and the implementation of such therapies in medical practise would give medical doctors an alternative to the low efficient antifungal treatments currently in use.

4. Scientists seeking novel antibacterial therapies. Sulphur metabolism has been extensively studied both in bacteria and fungi. However, although it is also well established that metabolism is an essential trait of pathogenic species, the relevance of sulphur metabolism for pathogenicity has been neglected. Just in the last few years it has started to receive attention in some bacterial species, as Mycobacterium tuberculosis and Neisseria meningitides. This understudied field has the potential to undercover novel traits of virulence which can be exploited to fight many types of infections. For instance, many sulphur-related pathways and enzymatic reactions performed by microbes are different from those in mammals, highlighting the potential to use some of these processes for treatment. Sulphate reducing bacteria produce H2S as an end product of the dissimilatory reduction of sulphate used to obtain energy. These bacteria are present in the digestive tract, where play a role in the onset and perpetuation of inflammatory diseases, and in the oral cavity, where a correlation with other pathogens has been demonstrated. This suggests a possible connection between the presence of external sources of H2S and virulence. Therefore, the production of H2S itself and/or the putatively triggered sulfhydration of other pathogenic species in the human body may constitute novel virulence traits to be targeted to fight infections.

5. Scientists and industries pursuing the advancement of host-pathogen interaction technologies. This study will improve systems approaches in Aspergilli.