Biology subjects

Bastos, R.

Publications and source records attributed to Bastos, R..

5 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

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi

A. fumigatus is the main etiological agent of a group of heterogeneous diseases called aspergillosis of which the most lethal form is the invasive pulmonary aspergillosis (IPA). Fungicidal azoles and amphotericin B are the first line defense against A. fumigatus, but fungistatic echinocandins, such as caspofungin (CAS), can be used as salvage therapy for IPA. Here, we screened repurposing libraries and identified several compounds that potentiate CAS activity against A. fumigatus, including the host defense peptide mimetic, brilacidin (BRI). BRI converts CAS into a fungicidal drug and potentiates voriconazole (VOR) against A. fumigatus. BRI increases the ability of both CAS and VOR to control A. fumigatus biofilm growth. BRI depolarizes the A. fumigatus cell membrane leading to disruption of membrane potential. By using a combination of protein kinase inhibitors and screening of a catalytic subunit null mutant library, we identified the mitogen activated protein kinase (MAPK) MpkA and the phosphatase calcineurin as mediators of the synergistic action of BRI. These results suggest the most likely BRI mechanism of action for CAS potentiation is the inhibition of A. fumigatus cell wall integrity (CWI) pathway. BRI potentiates CAS activity against C. albicans, C. auris, and C. neoformans. Interestingly, BRI overcomes the CAS-acquired resistance in both A. fumigatus and C. albicans and the CAS-intrinsic resistance in C. neoformans. BRI also has an additive effect on the activity of posaconazole (POSA) against several Mucorales fungi. Cell toxicity assays and fungal burden studies in an immunosuppressed murine model of IPA showed that BRI combined with CAS is not toxic to the cells and significantly clears A. fumigatus lung infection, respectively. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of IPA and other fungal infections.

microbiology↗

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↗

bioRxiv2022

Secondary metabolites produced during Aspergillus fumigatus and Pseudomonas aeruginosa biofilm formation

In Cystic Fibrosis (CF), mucus plaques are formed in the patients lung, creating a hypoxic condition and a propitious environment for colonization and persistence of many microorganisms. There is clinical evidence showing that Aspergillus fumigatus can co-colonize CF patients with Pseudomonas aeruginosa, which has been associated with lung function decline. P. aeruginosa produces several compounds with inhibitory and anti-biofilm effects against A. fumigatus in vitro; however, little is known about the fungal compounds produced in counterattack. Here, we annotated fungal and bacterial secondary metabolites (SM) produced in mixed biofilms in normoxia and hypoxia conditions. We detected nine SMs produced by P. aeruginosa. Phenazines and different analogs of pyoverdin were the main compounds produced by P. aeruginosa, and their secretion were increased by the fungal presence. The roles of the two operons responsible for phenazines production (phzA1 and phzA2) were also investigated showing both mutants are able to produce partial sets of phenazines. We detected a total of 20 SMs secreted by A. fumigatus either in monoculture or in co-culture with P. aeruginosa. All these compounds are secreted during biofilm formation either in normoxia or hypoxia. However, only eight compounds (demethoxyfumitremorgin C, fumitremorgin, ferrichrome, ferricrocin, tricetylfusigen, gliotoxin, gliotoxin E, and pyripyropene A) were detected during the biofilm formation by the co-culture of A. fumigatus and P. aeruginosa upon both normoxia and hypoxia conditions. Overall, we showed how diverse is SM secretion during A. fumigatus and P. aeruginosa mixed culture and how this can affect biofilm formation both in normoxia and hypoxia. Author SummaryThe interaction between Pseudomonas aeruginosa and Aspergillus fumigatus has been well-characterized in vitro. In this scenario, the bacterium exerts a strong inhibitory effect against the fungus. However, little is known about the metabolites produced by the fungus to counterattack the bacteria. Our work aimed to annotate secondary metabolites (SM) secreted during co-culture between P. aeruginosa and A. fumigatus during biofilm formation in both normoxia and hypoxia. The bacterium produces several different types of phenazines and pyoverdins, in response to the fungus presence. In contrast, we were able to annotate 29 metabolites produced during A. fumigatus biofilm formation but only eight compounds were detected during biofilm formation by the co-culture of A. fumigatus and P. aeruginosa upon both normoxia and hypoxia. In conclusion, we have detected many SMs secreted during A. fumigatus and P. aeruginosa biofilm formation. This analysis can provide several opportunities to understand the interaction between these two species.

microbiology↗

bioRxiv2021

Screening of chemical libraries for new antifungal drugs against Aspergillus fumigatus reveals the potential mechanism of action of miltefosine

Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation called invasive pulmonary aspergillosis (IPA) in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over the last decades. Therefore, new strategies are necessary to combat aspergillosis and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was more susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the rmiA (resistant to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and the {Delta}rmiA strains and the Chromatin Immunoprecipitation coupled to next generation sequencing (ChIP-Seq) of a RmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipids pathway that are directly or indirectly regulated by RmiA. Sphingolipids analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of RmiA represents the first genetic element described and characterized which plays a direct role in miltefosine response in fungi. Author summaryThe filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over the last decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungals agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown and aiming to get more insights about it, we identified a transcription factor RmiA (Resistant to miltefosine) important for miltefosine resistance. Our results suggest that miltefosine plays antifungal activity against A. fumigatus interfering in the sphingolipids biosynthesis.

microbiology↗