Aspergillus fumigatus Can Display Persistence to the Fungicidal Drug Voriconazole

Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first-line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non-mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A subpopulation of the persister isolates can survive for extended periods and even grow at low rates in the presence of supra-MIC of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seemed to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. Importance: The phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge of antifungal tolerance and, in particular, persistence is still very limited. In this study, we have characterized the response of the prominent fungal pathogen Aspergillus fumigatus to the first-line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and propose various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus.J.A. is funded by an Atracción de Talento Modalidad 1 (020-T1/BMD-200) contract of the Madrid Regional Government. J.S. has been funded by a BSAC Scholarship (bsac-2016-0049). C.V. was funded by FAPESP (2108/00715-3 and 2020/01131-5). G.H.G. hasbeen funded by FAPESP (2016/07870-9 and 2021/04977-5), CNPq (301058/2019-9 and404735/2018-5) and by the NIH/NIAID (grant R01AI153356). S.G. was cofunded by the NIHR Manchester Research Centre and the Fungal Infection Trust.S

A comparison of machine learning algorithms for the prediction of Hepatitis C NS3 protease cleavage sites

Hepatitis is a global disease that is on the rise and is currently the cause of more deaths than the human immunodeficiency virus each year. As a result, there is an increasing need for antivirals. Previously, effective antivirals have been found in the form of substrate-mimetic antiviral protease inhibitors. The application of machine learning has been used to predict cleavage patterns of viral proteases to provide information for future drug design. This study has successfully applied and compared several machine learning algorithms to hepatitis C viral NS3 serine protease cleavage data. Results have found that differences in sequence-extraction methods can outweigh differences in algorithm choice. Models produced from pseudo-coded datasets all performed with high accuracy and outperformed models created with orthogonal-coded datasets. However, no single pseudo-model performed significantly better than any other. Evaluation of performance measures also show that the correct choice of model scoring system is essential for unbiased model assessment

A comparison of machine learning algorithms for the prediction of Hepatitis C NS3 protease cleavage sites

Hepatitis is a global disease that is on the rise and is currently the cause of more deaths than the human immunodeficiency virus each year. As a result, there is an increasing need for antivirals. Previously, effective antivirals have been found in the form of substrate-mimetic antiviral protease inhibitors. The application of machine learning has been used to predict cleavage patterns of viral proteases to provide information for future drug design. This study has successfully applied and compared several machine learning algorithms to hepatitis C viral NS3 serine protease cleavage data. Results have found that differences in sequence-extraction methods can outweigh differences in algorithm choice. Models produced from pseudo-coded datasets all performed with high accuracy and outperformed models created with orthogonal-coded datasets. However, no single pseudo-model performed significantly better than any other. Evaluation of performance measures also show that the correct choice of model scoring system is essential for unbiased model assessment

Elevated mutation rates in the multi-azole resistant Aspergillus fumigatus clade drives rapid evolution of antifungal resistance

The evolution of antifungal resistance is an emerging global threat. Particularly concerning is the widespread occurrence of azole resistance within Aspergillus fumigatus, a globally ubiquitous environmental mould that causes over 1 million life-threatening invasive infections in humans each year. It is increasingly evident that the environmental use of azoles has led to selective sweeps across multiple genomic loci resulting in the rapid expansion of a genetically distinct cluster of genotypes (clade A) that results in resistance to clinically deployed azoles. Isolates within this cluster are more likely to be cross resistant to agricultural antifungals with unrelated modes of action suggesting they may be adapting rapidly to antifungal challenge. Here we show that this cluster is not only multi-azole resistant but has increased propensity to develop resistance to new antifungals because of variants in the DNA mismatch repair system. A variant in msh6 is found almost exclusively within clade A, occurs in 88% of multi-azole resistant isolates harbouring the canonical cyp51A azole resistance allelic variant TR34/L98H, and is globally distributed. Naturally occurring isolates with this msh6 variant display a 4 to 9-times higher rate of mutation, leading to an increased propensity to evolve resistance to current and next generation antifungals. We argue that pervasive environmental use of fungicides creates selective arenas whereby genotypes of A. fumigatus with increased adaptive capability thrive in the face of strong directional selection, leading to the genesis and amplification of antifungal resistance. These results help explain the pronounced clustering of multiple independent resistance mechanisms within the mutable clade A. Our findings further suggest that resistance to next generation antifungals is more likely to emerge within organisms that are already multi-azole resistant, posing a major problem due to the prospect of dual use of novel antifungals in clinical and agricultural settings

Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. The azole class of antifungals represent first line therapeutics for most of these infections however resistance is rising. Identification of novel antifungal targets that, when inhibited, synergise with the azoles will aid the development of agents that can improve therapeutic outcomes and supress the emergence of resistance. As part of the A. fumigatus genome-wide knockout program (COFUN), we have completed the generation of a library that consists of 120 genetically barcoded null mutants in genes that encode the protein kinase cohort of A. fumigatus. We have employed a competitive fitness profiling approach (Bar-Seq), to identify targets which when deleted result in hypersensitivity to the azoles and fitness defects in a murine host. The most promising candidate from our screen is a previously uncharacterised DYRK kinase orthologous to Yak1 of Candida albicans, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. Here we show that the orthologue YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress via phosphorylation of the Woronin body tethering protein Lah. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and impacts growth in murine lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit Yak1 in C. albicans prevents stress mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.</p

Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections