Biology subjects

Knowles, S. L.

Publications and source records attributed to Knowles, S. L..

7 recordsLinked to original sources

bioRxiv2023

Evolutionary origin, population diversity, and diagnostics for a cryptic hybrid pathogen

Cryptic fungal pathogens pose significant identification and disease management challenges due to their morphological resemblance to known pathogenic species while harboring genetic and (often) infection-relevant trait differences. The cryptic fungal pathogen Aspergillus latus, an allodiploid hybrid originating from Aspergillus spinulosporus and an unknown close relative of Aspergillus quadrilineatus within section Nidulantes, remains poorly understood. The absence of accurate diagnostics for A. latus has led to misidentifications, hindering epidemiological studies and the design of effective treatment plans. We conducted an in-depth investigation of the genomes and phenotypes of 44 globally distributed isolates (41 clinical isolates and three type strains) from Aspergillus section Nidulantes. We found that 21 clinical isolates were A. latus; notably, standard methods of pathogen identification misidentified all A. latus isolates. The remaining isolates were identified as A. spinulosporus (8), A. quadrilineatus (1), or A. nidulans (11). Phylogenomic analyses shed light on the origin of A. latus, indicating one or two hybridization events gave rise to the species during the Miocene, approximately 15.4 to 8.8 million years ago. Characterizing the A. latus pangenome uncovered substantial genetic diversity within gene families and biosynthetic gene clusters. Transcriptomic analysis revealed that both parental genomes are actively expressed in nearly equal proportions and respond to environmental stimuli. Further investigation into infection-relevant chemical and physiological traits, including drug resistance profiles, growth under oxidative stress conditions, and secondary metabolite biosynthesis, highlight distinct phenotypic profiles of the hybrid A. latus compared to its parental and closely related species. Leveraging our comprehensive genomic and phenotypic analyses, we propose five genomic and phenotypic markers as diagnostics for A. latus species identification. These findings provide valuable insights into the evolutionary origin, genomic outcome, and phenotypic implications of hybridization in a cryptic fungal pathogen, thus enhancing our understanding of the underlying processes contributing to fungal pathogenesis. Furthermore, our study underscores the effectiveness of extensive genomic and phenotypic analyses as a promising approach for developing diagnostics applicable to future investigations of cryptic and emerging pathogens.

evolutionary biology↗

bioRxiv2022

Genomic and phenotypic trait variation of the opportunistic human pathogen Aspergillus flavus and its non-pathogenic close relatives

Fungal diseases affect millions of humans annually, yet fungal pathogens remain understudied. The mold Aspergillus flavus is a causative agent of both aspergillosis and fungal keratitis infections, but species closely related to A. flavus are not considered clinically relevant. To study the evolution of A. flavus pathogenicity, we examined genomic and phenotypic traits of two strains of A. flavus and three closely related non- pathogenic species: Aspergillus arachidicola (two strains), Aspergillus parasiticus (two strains), and Aspergillus nomiae (one strain). We identified over 3,000 orthologous proteins unique to A. flavus, including seven biosynthetic gene clusters present in A. flavus strains and absent in the three non-pathogenic species. We chose to characterize secondary metabolite production for all seven strains under two clinically relevant conditions, temperature and salt concentration. Temperature impacted metabolite production in all species. Conversely, we found a lack of impact of salinity on secondary metabolite production. Strains of the same species produced different metabolites. Growth under stress conditions revealed additional heterogeneity within species. Using the invertebrate model of fungal disease Galleria mellonella, we found virulence of strains of the same species varied widely, and A. flavus strains were not more virulent than strains of the non-pathogenic species. In a murine model of fungal keratitis, we observed significantly lower disease severity and corneal thickness for A. arachidicola compared to other species at 48 hrs, but not at 72 hrs. Our work identifies key phenotypic, chemical, and genomic similarities and differences between the opportunistic human pathogen A. flavus and its non-pathogenic relatives.

evolutionary biology↗

bioRxiv2021

Regulation of gliotoxin biosynthesis and protection in Aspergillus species

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species. Author SummaryA. fumigatus secretes mycotoxins that are essential for its virulence and pathogenicity. Gliotoxin (GT) is a sulfur-containing mycotoxin, which is known to impair several aspects of the human immune response. GT is also toxic to different fungal species, which have evolved several GT protection strategies. To further decipher these responses, we used transcriptional profiling aiming to compare the response to GT in the GT producer A. fumigatus and the GT non-producer A. nidulans. This analysis allowed us to identify additional genes with a potential role in GT protection. We also identified 15 transcription factors (TFs) encoded in the A. fumigatus genome that are important for conferring resistance to exogenous gliotoxin. One of these TFs, KojR, which is essential for A. oryzae kojic acid production, is also important for virulence and GT protection in A. fumigatus, A. nidulans and A. oryzae. KojR regulates the expression of another TF, an oxidoreductase, previously shown to be essential for GT protection, and sulfur metabolism. Together, this work identified conserved components required for gliotoxin protection in Aspergillus species.

microbiology↗

bioRxiv2020

Genomic and phenotypic analysis of COVID-19-associated pulmonary aspergillosis isolates of Aspergillus fumigatus

The ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) first described from Wuhan, China. A subset of COVID-19 patients has been reported to have acquired secondary infections by microbial pathogens, such as fungal opportunistic pathogens from the genus Aspergillus. To gain insight into COVID-19 associated pulmonary aspergillosis (CAPA), we analyzed the genomes and characterized the phenotypic profiles of four CAPA isolates of Aspergillus fumigatus obtained from patients treated in the area of North Rhine-Westphalia, Germany. By examining the mutational spectrum of single nucleotide polymorphisms, insertion-deletion polymorphisms, and copy number variants among 206 genes known to modulate A. fumigatus virulence, we found that CAPA isolate genomes do not exhibit major differences from the genome of the Af293 reference strain. By examining virulence in an invertebrate moth model, growth in the presence of osmotic, cell wall, and oxidative stressors, and the minimum inhibitory concentration of antifungal drugs, we found that CAPA isolates were generally, but not always, similar to A. fumigatus reference strains Af293 and CEA17. Notably, CAPA isolate D had more putative loss of function mutations in genes known to increase virulence when deleted (e.g., in the FLEA gene, which encodes a lectin recognized by macrophages). Moreover, CAPA isolate D was significantly more virulent than the other three CAPA isolates and the A. fumigatus reference strains tested. These findings expand our understanding of the genomic and phenotypic characteristics of isolates that cause CAPA.

microbiology↗

bioRxiv2020

Biosynthetic gene clusters, secondary metabolite profiles, and cards of virulence in the closest nonpathogenic relatives of Aspergillus fumigatus

Aspergillus fumigatus is a major human pathogen that causes hundreds of thousands of infections yearly with high mortality rates. In contrast, Aspergillus fischeri and the recently described Aspergillus oerlinghausenensis, the two species most closely related to A. fumigatus, are not known to be pathogenic. Some of the "cards of virulence" that A. fumigatus possesses are secondary metabolites that impair the host immune system, protect from host immune cell attacks, or acquire key nutrients. Secondary metabolites and the biosynthetic gene clusters (BGCs) that typically encode them often vary within and between fungal species. To gain insight into whether secondary metabolism-associated cards of virulence vary between A. fumigatus, A. oerlinghausenensis, and A. fischeri, we conducted extensive genomic and secondary metabolite profiling analyses. By analyzing multiple A. fumigatus, one A. oerlinghausenensis, and multiple A. fischeri strains, we identified both conserved and diverged secondary metabolism-associated cards of virulence. For example, we found that all species and strains examined biosynthesized the major virulence factor gliotoxin, consistent with the conservation of the gliotoxin BGC across genomes. However, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis; these differences were reflected in sequence divergence of the intertwined fumagillin/pseurotin BGCs across genomes. These results delineate the similarities and differences in secondary metabolism-associated cards of virulence between a major fungal pathogen and its nonpathogenic closest relatives, shedding light into the genetic and phenotypic changes associated with the evolution of fungal pathogenicity. ImportanceThe major fungal pathogen Aspergillus fumigatus kills tens of thousands each year. In contrast, the two closest relatives of A. fumigatus, namely Aspergillus fischeri and Aspergillus oerlinghausenensis, are not considered pathogenic. A. fumigatus virulence stems, partly, from its ability to produce small molecules called secondary metabolites that have potent activities during infection. In this study, we examined whether A. fumigatus secondary metabolites and the metabolic pathways involved in their production are conserved in A. oerlinghausenensis and A. fischeri. We found that the nonpathogenic close relatives of A. fumigatus produce some, but not all, secondary metabolites thought to contribute to the success of A. fumigatus in causing human disease and that these similarities and differences were reflected in the underlying metabolic pathways involved in their biosynthesis. Compared to its nonpathogenic close relatives, A. fumigatus produces a distinct cocktail of secondary metabolites, which likely contributes to these organisms vastly different potentials to cause human disease. More broadly, the study of nonpathogenic organisms that have virulence-related traits, but are not currently considered agents of human disease, may facilitate the prediction of species capable of posing future threats to human health.

evolutionary biology↗

bioRxiv2019

Gliotoxin, a known virulence factor in the major human pathogen Aspergillus fumigatus, is also biosynthesized by the non-pathogenic relative A. fischeri

Aspergillus fumigatus is a major opportunistic human pathogen. Multiple traits contribute to A. fumigatus pathogenicity, including its ability to produce specific secondary metabolites, such as gliotoxin. Gliotoxin is known to inhibit the host immune response, and genetic mutants that inactivate gliotoxin biosynthesis (or secondary metabolism in general) attenuate A. fumigatus virulence. The genome of A. fischeri, a very close non-pathogenic relative of A. fumigatus, contains a biosynthetic gene cluster that exhibits high sequence similarity to the A. fumigatus gliotoxin cluster. However, A. fischeri is not known to produce gliotoxin. To gain further insight into the similarities and differences between the major pathogen A. fumigatus and the non-pathogen A. fischeri, we examined whether A. fischeri strain NRRL 181 biosynthesizes gliotoxin and whether its production, and of secondary metabolites more generally, influence its virulence profile. We found that A. fischeri biosynthesizes gliotoxin in the same conditions as A. fumigatus. However, whereas loss of laeA, a master regulator of secondary metabolite production, has been previously shown to reduce the virulence of A. fumigatus, we found that laeA loss (and loss of secondary metabolite production, including gliotoxin) in A. fischeri does not influence its virulence. These results suggest that gliotoxin and secondary metabolite production are virulence factors in the genomic and phenotypic background of the major pathogen A. fumigatus but are much less important in the background of the non-pathogen A. fischeri. We submit that understanding the observed spectrum of pathogenicity across closely related pathogenic and non-pathogenic Aspergillus species will require detailed characterization of their biological, chemical, and genomic similarities and differences. IMPORTANCEAspergillus fumigatus is a major opportunistic fungal pathogen of humans but most of its close relatives are non-pathogenic. Why is that so? This important, yet largely unanswered, question can be addressed by examining how A. fumigatus and its non-pathogenic close relatives are similar or different with respect to virulence-associated traits. We investigated whether Aspergillus fischeri, a non-pathogenic close relative of A. fumigatus, can produce gliotoxin, a mycotoxin known to contribute to A. fumigatus virulence. We discovered that the non-pathogenic A. fischeri produces gliotoxin under the same conditions as the major pathogen A. fumigatus. However, we also discovered that, in contrast to what has been previously observed in A. fumigatus, loss of secondary metabolite, including gliotoxin, production in A. fischeri does not alter its virulence. Our results are consistent with the "cards of virulence" model of opportunistic fungal disease, where the ability to cause disease stems from the combination ("hand") of individual virulence factors ("cards"), but not from individual factors per se.

microbiology↗

bioRxiv2018

Characterizing the pathogenic, genomic, and chemical traits of Aspergillus fischeri, the closest sequenced relative of the major human fungal pathogen Aspergillus fumigatus

Aspergillus fischeri is closely related to Aspergillus fumigatus, the major cause of invasive mold infections. Even though A. fischeri is commonly found in diverse environments, including hospitals, it rarely causes invasive disease; why that is so is unclear. Comparison of A. fischeri and A. fumigatus for diverse pathogenic, genomic, and secondary metabolic traits revealed multiple differences for pathogenesis-related phenotypes, including that A. fischeri is less virulent than A. fumigatus in multiple animal models of disease, grows slower in low oxygen environments, and is more sensitive to oxidative stress. In contrast, the two species exhibit high genomic similarity; ~90% of the A. fumigatus proteome is conserved in A. fischeri, including 48/49 genes known to be involved in A. fumigatus virulence. However, only 10/33 A. fumigatus biosynthetic gene clusters (BGCs) likely involved in secondary metabolite production are conserved in A. fischeri and only 13/48 A. fischeri BGCs are conserved in A. fumigatus. Detailed chemical characterization of A. fischeri cultures grown on multiple substrates identified multiple secondary metabolites, including two new compounds and one never before isolated as a natural product. Interestingly, an A. fischeri deletion mutant of laeA, a master regulator of secondary metabolism, produced fewer secondary metabolites and in lower quantities, suggesting that regulation of secondary metabolism is at least partially conserved. These results suggest that the non-pathogenic A. fischeri possesses many of the genes important for A. fumigatus pathogenicity but is divergent with respect to its ability to thrive under host-relevant conditions and its secondary metabolism.\n\nImportanceAspergillus fumigatus is the primary cause of aspergillosis, a devastating ensemble of diseases associated with severe morbidity and mortality worldwide. A. fischeri is a close relative of A. fumigatus, but is not generally observed to cause human disease. To gain insights into the underlying causes of this remarkable difference in pathogenicity, we compared two representative strains (one from each species) for a range of host-relevant biological and chemical characteristics. We found that disease progression in multiple A. fischeri mouse models was slower and caused less mortality than A. fumigatus. The two species also exhibited different growth profiles when placed in a range of stress-inducing conditions encountered during infection, such as low levels of oxygen and the presence of reactive oxygen species-inducing agents. Interestingly, we also found that the vast majority of A. fumigatus genes known to be involved in virulence are conserved in A. fischeri, whereas the two species differ significantly in their secondary metabolic pathways. These similarities and differences that we identified are the first step toward understanding the evolutionary origin of a major fungal pathogen.

microbiology↗