Effectors of tissue invasion in Aspergillus fumigatus, the major fungal pathogen of human lungs

The often-fatal fungal lung disease invasive aspergillosis (IA) has claimed more than 8 million human lives since its emergence ~ 40 years ago. IA-related mortality is unacceptably high (50- 90%) and usually results from respiratory failure due to haemorrhage occurring at sites of fungal invasion. Given the life-saving potential of anti-invasion therapies, a high premium has been placed upon identifying effectors of tissue invasion, against which drugs and vaccines might be engineered. However, the quest for causal associations between tissue-invasive phenotypes and secreted fungal products has repeatedly failed, presumably due to likely functional redundancy amongst the effector repertoire and insufficiency of genetic approaches in A. fumigatus with which to drive high throughput analyses of secreted fungal products. Using mouse models of IA, in vitro infections of cultured human lung cells, functional genomics and a high throughput A. fumigatus gene deletion methodology we have discovered a first-in-field cohort of 42 effectors of tissue invasion (eTINs) which govern lytic death of human lung cells. We will now combine state-of-the-art genetic, single cell imaging and effector-trap approaches with our established methodological workflow to conclusively define the invasive A. fumigatus secretome and to characterise the modes of entry and/or lytic activity of the individual effectors. This work will reveal for the first time the molecular basis of tissue invasive IA pathologies and define the genomic cohort of targets against which anti-invasion therapies and novel vaccines and diagnostics can be designed.

To identify A. fumigatus effectors of tissue invasion (eTINs) we used a HTP fusion PCR approach to construct null A. fumigatus mutants for all 253 of the SignalP proteins whose expression, relative to laboratory culture, is significantly upregulated during mouse colonisation. We then screened the mutant collection in a 384 well format, epithelial lysis assay to identify mutants having lytic defects in host co-culture. The starting point for this proposal is a collection of n = 42 signalP protein-encoding genes which are specifically upregulated in response to the mammalian host environment and whose encoded gene products are required for lysis of human respiratory epithelia. This collection is a first-in-field collection of A. fumigatus effectors of tissue invasion (eTINs).

The aims of this study are to:
1. Screen the collection of eTIN null mutants in neutropenic mice to identify dominant effectors of tissue invasion
2. Sort eTINs according to modes of host activation using established in vitro infection models
3. Define spatiotemporal localisation profiles of eTINs during A. fumigatus co-culture with human respiratory epithelia
4. Combine high throughput gene deletion and classical genetics to define the minimal invasive secretome of A. fumigatus
5. Define eTIN modes of translocation and identify host binding partners

1. Scientists, clinicians and industries pursuing novel anti-fungal diagnostics, and vaccines. A single serodiagnostic marker of invasive aspergillosis has been the mainstay of clinical diagnosis for the past quarter century but sensitivity and specificity vary by centre and user. Secreted microbial proteins are of major importance as diagnostic agents and for vaccination strategy. We are already working with protein biochemists, clinicians and antibody technologists to identify the most immunogenic of our novel proteins with a view to producing reagents with which to detect and treat infections due to Aspergillus fumigatus.

2. Pharmaceutical companies seeking novel antifungal agents. The pipeline for new antifungal compounds is sparse and antifungal resistance is advancing. The use of agricultural azoles is prompting emergence of clinically significant resistant isolates. Three new triazoles (albaconazole, isavuconazole and ravuconazole) are in clinical trials however, as with extant triazoles, they target lanosterol 14-demethylase and are likely to be dogged by similar resistance issues. The glucan synthase inhibitor in development by Merck has been stalled in Phase I since 2009, as have the novel compounds corifungin (Acea biotech), T-2307 (Toyama) and the HDAc inhibitor of Methylgene, thereby indicating problems in development. The global market for clinical antifungals was estimated to be $9.4 billion US in 2010 (GBI research) and is anticipated to exceed $18 billion by 2018. The advancement of novel target-based strategies currently falls outside the remit of many large drug discovery companies and it will therefore fall to academic partners to progress research in this area. This deficiency will require pharmaceutical companies to interact with academics that have expertise in drug design and toxicology. This work will have direct applicability to these efforts, and will help us to forge links with pharmaceutical collaborators that will provide tangible and valuable outputs far into the future.

3. Individuals suffering, or at risk from, Aspergillus-related disease: The health burden of diseases caused by fungi is considerable. Where severe invasive infections are concerned, deep-organ infection occurs as a negative corollary to otherwise successful, but nonetheless expensive, treatments for serious illness. In 2002 almost 10,000 patients in England were estimated to have suffered a deep-organ fungal infection. Almost half of them died from their fungal disease (HPA).

4. 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].