Biology subjects

Brewer, M. T.

Publications and source records attributed to Brewer, M. T..

8 recordsLinked to original sources

bioRxiv2025

Proline transporters balance the salicylic acid-mediated trade-off between regeneration and immunity in plants

A robust immune response and regenerative capacity are both essential for survival after injury. In plants, salicylic acid (SA) is essential for activating immunity but simultaneously suppresses regenerative capacity. The mechanisms coordinating the trade-off between immunity and regeneration remain poorly understood. Here, we identify proline transporters as key regulators of this balance. Mutations in two wound-induced proline transporters, ProT2 and ProT3, rescued exogenous proline-induced suppression of de novo root regeneration (DNRR) and enhanced DNRR. ProTs are required for SA-mediated suppression of regeneration, without affecting SA-dependent defense responses. Mechanistically, a ProT3-CPK1 complex modulates the dynamics of wound-induced reactive oxygen species (ROS), sustaining a ROS level that restricts DNRR after wounding. Notably, pharmacological inhibition of proline transport rescued SA-mediated suppression of regeneration and enhanced regeneration across multiple plant species. These findings establish proline transporter as a regulatory hub integrating stress-induced proline metabolism, SA signaling, and ROS homeostasis to balance immunity and regeneration, and highlight chemical inhibition of proline transport as a strategy to improve crop regeneration under biotic stresses without compromising disease resistance.

plant biology↗

bioRxiv2024

Transcriptional responses to in vitro macrocyclic lactone exposure in Toxocara canis larvae using RNA-seq

Toxocara canis, the causative agent of zoonotic toxocariasis in humans, is a parasitic roundworm of canids with a complex lifecycle. While macrocyclic lactones (MLs) are successful at treating adult T. canis infections when used at FDA-approved doses in dogs, they fail to kill somatic third-stage larvae. In this study, we profiled the transcriptome of third-stage larvae derived from larvated eggs and treated in vitro with 10 {micro}M of the MLs - ivermectin and moxidectin with Illumina sequencing. We analyzed transcriptional changes in comparison with untreated control larvae. In ivermectin-treated larvae, we identified 608 differentially expressed genes (DEGs), of which 453 were upregulated and 155 were downregulated. In moxidectin-treated larvae, we identified 1,413 DEGs, of which 902 were upregulated and 511 were downregulated. Notably, many DEGs were involved in critical biological processes and pathways including transcriptional regulation, energy metabolism, neuronal structure and function, physiological processes such as reproduction, excretory/secretory molecule production, host-parasite response mechanisms, and parasite elimination. We also assessed the expression of known ML targets and transporters, including glutamate-gated chloride channels (GluCls), and ATP-binding cassette (ABC) transporters, subfamily B, with a particular focus on P-glycoproteins (P-gps). We present gene names for previously uncharacterized T. canis GluCl genes using phylogenetic analysis of nematode orthologs to provide uniform gene nomenclature. Our study revealed that the expression of Tca-glc-3 and six ABCB genes, particularly four P-gps, were significantly altered in response to ML treatment. Compared to controls, Tca-glc-3, Tca-Pgp-11.2, and Tca-Pgp-13.2 were downregulated in ivermectin-treated larvae, while Tca-abcb1, Tca-abcb7, Tca-Pgp-11.2, and Tca-Pgp-13.2 were downregulated in moxidectin-treated larvae. Conversely, Tca-abcb9.1 and Tca-Pgp-11.3 were upregulated in moxidectin-treated larvae. These findings suggest that MLs broadly impact transcriptional regulation in T. canis larvae.

molecular biology↗

bioRxiv2024

Widely dispersed clonal expansion of multi-fungicide-resistant Aspergillus fumigatus limits genomic epidemiology prospects

BackgroundAspergillus fumigatus is a ubiquitous fungus that causes a range of diseases in animals, including humans. The most lethal manifestation is invasive aspergillosis for which treatment relies on triazoles. Triazole-resistant A. fumigatus can be recovered from decaying plant material and so-called hotspots containing triazole fungicide residues. Although observations have shown clonal isolates between the environment and clinical samples, a direct link between a specific environment and cases of triazole-resistant invasive aspergillus disease in an individual patient has not yet been demonstrated. MethodsTo understand where patients acquire A. fumigatus isolates causing disease, we used a genomic epidemiology approach with 157 Dutch A. fumigatus isolates, based on whole genome sequencing. Isolates were from three well-characterized environmental hotspots and two hospitals between 2016 and 2019. FindingsIn the Dutch dataset, A. fumigatus isolates from six patients showed near-identical genomes compared to five environmental isolates. One environmental isolate matched three probable cases of triazole-resistant invasive aspergillosis, including one fatal case. Patient isolates were recovered up to 34 months later than near-identical environmental isolates. Comparison to over 1{middle dot}2K global publicly available A. fumigatus genomes showed hundreds of clonal groups spread across three continents. In addition, finding variants associated with resistance to non-triazole fungicides such as benzimidazole, succinate dehydrogenase inhibitor and quinone outside inhibitor classes, strongly suggests an exposure history to multiple agricultural fungicides in these environmental hotspots. InterpretationEnvironmental hotspots represent highly selective habitats for multi-fungicide-resistant A. fumigatus, which we can now directly link to probable cases of aspergillus disease, including a triazole-resistant case. However, geographically widely dispersed clonal expansion limits the utility of genomic epidemiology to identify the source of a particular patients isolate. Furthermore, reducing a single class of fungicides in agriculture may not effectively reduce resistance selection when other classes are still in use. FundingNWO.Groen2019.002 Research in contextO_ST_ABSEvidence before this studyC_ST_ABSTriazole fungicides that exhibit activity against Aspergillus fumigatus have been shown to be a major source of resistant aspergillus disease in humans. However, the route of transmission from environmental hotspot to human remains poorly understood. Isolates of A. fumigatus can be recovered from both environmental samples and clinical specimens that harbour the same resistance signature haplotypes, e.g., TR34/L98H and TR46/Y121F/T289A, in the cyp51A-gene. We searched the literature for evidence using high resolution whole genome sequencing (WGS) to link environmental isolates to human infection. We searched PubMed for articles using the search terms Aspergillus fumigatus AND azole resistance AND whole genome sequencing on 15 April 2024. This search retrieved 32 articles describing different evolutionary routes to select for triazole-resistant A. fumigatus or population structure of whole-genome sequenced isolates. Twenty-six articles used whole-genome sequencing, but none focused on identifying clonal groups to identify direct cases of transmission between the environment and clinical cases of aspergillus disease. By using the additional search term transmission, no other records were retrieved. Added value of this studyOur study links triazole-resistant A. fumigatus isolates cultured from three environmental hotspots to cases of aspergillus disease in two hospitals in the Netherlands. Genome comparisons of isolates from environmental hotspots and patients showed multiple near-identical linked genotypes, consistent with a route of transmission from the environment to patients. Although a naive expectation may be a higher probability of matches of the hotspots located in the northwest of the Netherlands with the hospital located in the west of the country, in fact, more patient isolates from the far southeast were linked to the hotspots. Integrating the Dutch data set into a global data set showed 205 clonal groups spread across the Netherlands, Germany, the United Kingdom (UK), the United States of America (USA) and Japan. Our demonstration of a large number of geographically dispersed clonal groups suggests that current sampling is insufficient to definitively identify the source of an individual patients infection. A genetically highly diverse population combined with a wide global distribution of clones can make it impossible to definitively identify the source of an individual patients infection even with much more sampling. Implications of all the available evidenceOur study provides evidence that triazole-resistant A. fumigatus isolates with multi-fungicide resistance profiles cause aspergillus disease in at-risk patients and may contribute to treatment failure and mortality. The risk of infection due to these triazole-resistant isolates is not confined to the geographic vicinity of the environmental hotspot since clonal spread can be detected across great distances. The finding of linked cases without clear transmission routes limits epidemiological studies and underscores the need to better understand the ecology and environmental niches of this fungus. As it is highly unlikely that each patient visited the rural agricultural areas where a hotspot was located, research should address the complex and long-distance transmission routes of resistant isolates, which involves airborne dispersal of conidia or habitats of this fungus outside the agricultural environment. Furthermore, because of the multi-fungicide resistance phenotype of the triazole-resistant A. fumigatus, involving several classes of fungicides, reducing one class of fungicides in the environment may not effectively reduce resistance selection. Effective interventions should instead aim to reduce the burden of environmental resistance by modifying environments that currently favour the massive outgrowth of fungicide-resistant A. fumigatus to limit the escape of aerial spores from these environmental hotspots.

microbiology↗

bioRxiv2023

Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States

Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in 8 states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in 7 states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades and that TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow with clear recombination between two clades highlighting the potential for azole-resistance to continue spreading in the U.S. ImportanceAspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for twenty years and has recently been reported in patients in the U.S. Until this study we didnt know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus that is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.

microbiology↗

bioRxiv2023

Elevated mutation rates in the multi-azole resistant Aspergillus fumigatus cladedrives 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.

microbiology↗

bioRxiv2022

Genetic characterization of the zoonotic parasite Ancylostoma caninum in the central and eastern USA

Ancylostoma caninum is the most common nematode parasite of dogs in the United States. This study aimed to describe the molecular epidemiology of A. caninum isolates from the central and eastern states of the U.S. using the partial mitochondrial cox1 gene and to compare them with those reported globally. We isolated eggs from fecal samples of dogs and characterized each isolate based on cox1 sequences. A total of 60 samples originating from Kansas, Iowa, New York, Florida, and Massachusetts were included. 25 haplotypes were identified in the U.S. dataset with high haplotype diversity (0.904). Sequence data were compared to sequences from other world regions available in GenBank. Global haplotype analysis demonstrated 35 haplotypes with a haplotype diversity of 0.931. Phylogenetic and network analysis provide evidence for the existence of moderate geographical structuring of A. caninum haplotypes. Our results provide an updated summary of A. caninum haplotypes and data for neutral genetic markers with utility for tracking hookworm populations. Sequences have been deposited in GenBank (ON980650 - ON980674). Further studies of isolates from other regions are essential to understand the genetic diversity of this parasite.

microbiology↗

bioRxiv2022

Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across Fungi

Azole drugs target fungal sterol biosynthesis and are used to treat millions of human fungal infections each year. Resistance to azole drugs has emerged in multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and Aspergillus fumigatus. The most well-studied resistance mechanism in A. fumigatus arises from missense mutations in the coding sequence combined with a tandem repeat in the promoter of cyp51A, which encodes a cytochrome P450 enzyme in the fungal sterol biosynthesis pathway. Filamentous members of Ascomycota such as A. fumigatus have either one or two of three Cyp51 paralogs (Cyp51A, Cyp51B, and Cyp51C). Most previous research in A. fumigatus has focused on Cyp51A due to its role in azole resistance. We used the A. fumigatus Cyp51A protein sequence as the query in database searches to identify Cyp51 proteins across Fungi. We found 435 Cyp51 proteins in 301 species spanning from early-diverging fungi (Blastocladiomycota, Chytridiomycota, Zoopagomycota and Mucormycota) to late-diverging fungi (Ascomycota and Basidiomycota). We found these sequences formed 4 major Cyp51 groups: Cyp51, Cyp51A, Cyp51B, and Cyp51C. Surprisingly, we found all filamentous Ascomycota had a Cyp51B paralog, while only 50% had a Cyp51A paralog. We created maximum likelihood trees to investigate the evolution of Cyp51 in Fungi. Our results suggest Cyp51 is present in all fungi with three paralogs emerging in Pezizomycotina, including Cyp51C which appears to have diverged from the progenitor of the Cyp51A and Cyp51B groups. Author SummaryEach year millions of people are infected by a fungal pathogen and receive antifungal treatment with azole drugs. Resistance to azole drugs is becoming increasingly prevalent and is mostly caused by mutations in the azole drug target, Cyp51. Aspergillus fumigatus is an airborne fungal pathogen that causes more than 600,000 deaths every year. Azole resistance in A. fumigatus is primarily driven by a promoter repeat coupled with mutations in cyp51A. In our study, we found 435 Cyp51 proteins in 4 major groups across Fungi, with some species having multiple Cyp51 proteins (Cyp51, Cyp51A, Cyp51B, and Cyp51C). Although most research in A. fumigatus has focused on Cyp51A, we found Cyp51B in all filamentous Ascomycota fungi showing it is more conserved than Cyp51A and likely plays a vital role in these fungi. Author Summary (Shortened)Resistance to azole drugs is becoming increasingly prevalent and is mostly caused by mutations in the azole drug target, Cyp51. Azole resistance in Aspergillus fumigatus is primarily driven by a promoter repeat coupled with mutations in cyp51A. We found 435 Cyp51 proteins in 4 major groups across Fungi, with some species having multiple Cyp51 proteins (Cyp51, Cyp51A, Cyp51B, and Cyp51C). Although most research focuses on Cyp51A, we found Cyp51B in all filamentous Ascomycota fungi showing its more conserved than Cyp51A.

microbiology↗

bioRxiv2020

Evidence for the agricultural origin of antimicrobial resistance in a fungal pathogen of humans

Resistance to clinical antimicrobials is an urgent problem, reducing our ability to combat deadly pathogens of humans. Azole antimicrobials target ergosterol synthesis and are highly effective against fungal pathogens of both humans and plants leading to their widespread use in clinical and agricultural settings1,2. The fungus Aspergillus fumigatus causes 300,000 life-threatening infections in susceptible human hosts annually and azoles are the most effective treatment3. Resistance to clinical azole antifungals has become a major problem in Europe and India over the last decade, where identical mutations in cyp51A, an ergosterol biosynthetic gene, have been found in strains from both clinical and agricultural settings4. Shared cyp51A genotypes suggest that clinical azole resistance might have had an agricultural origin; however, until now, independent origins of clinical and agricultural mutations could not be ruled out. Here we show that azole-resistant isolates of A. fumigatus from clinical and agricultural settings also carry mutations conferring resistance to quinone outside inhibitor (QoI) fungicides, which are used exclusively in agricultural settings. This is the first report of a clear marker for the agricultural origin of resistance to a clinical antifungal. We anticipate that our work will increase the understanding of interactions between pathogens of plants and pathogens of humans.

microbiology↗