Biology subjects

Fortwendel, J. R.

Publications and source records attributed to Fortwendel, J. R..

3 recordsLinked to original sources

bioRxiv2023

The Aspergillus fumigatus maiA gene contributes to cell wall homeostasis and fungal virulence.

In this study, two distinct in vitro infection models of Aspergillus fumigatus, using murine macrophages (RAW264.7) and human lung epithelial cells (A549), were employed to identify the genes important for fungal adaptation during infection. Transcriptomic analyses of co-incubated Aspergillus uncovered 140 fungal genes up-regulated in common between both models that, when compared with a previously published in vivo transcriptomic study, allowed the identification of 13 genes consistently up-regulated in all three infection conditions. Among them, the maiA gene, responsible for a critical step in the L-phenylalanine degradation pathway, was identified. Disruption of maiA resulted in a mutant strain unable to complete the Phe degradation pathway, leading to an excessive production of pyomelanin when this amino acid served as the sole carbon source. Moreover, the disruption mutant exhibited noticeable cell wall abnormalities, with reduced levels of {beta}-glucans within the cell wall. the maiA-1 mutant strain induced reduced inflammation in primary macrophages and displayed significantly lower virulence in a neutropenic mouse model of infection. This is the first study linking the A. fumigatus maiA gene to fungal cell wall homeostasis and virulence.

microbiology↗

bioRxiv2023

Identification and functional characterization of the putative members of the CTDK-1 kinase complex as regulators of growth and development in the genus Aspergillus.

The genus Aspergillus includes industrially, medically and agriculturally important species. All of them, as do fungi in general, disperse to new niches principally by means of asexual spores. Regarding the genetic/molecular control of asexual development, Aspergillus nidulans is the main reference. In this species, two pathways control the production of conidiophores, the structures bearing asexual spores (conidia). The Upstream Developmental Activation (UDA) pathway transduces environmental signals, determining whether the Central Developmental Pathway (CDP) and the required morphological changes are induced. The transcriptional regulator BrlA links both pathways as loss-of-function mutations in flb (UDA) genes block brlA transcription and, consequently, conidiation. However, the aconidial phenotype of specific flb mutants is reverted under salt-stress conditions. Previously, we generated a collection of {Delta}flbB mutants unable to conidiate on culture medium supplemented with NaH2PO4 (0.65M). Here, we identified a Gly347Stop mutation within flpA as responsible for the FLIP57 phenotype. The putative cyclin FlpA and the remaining putative components of the C-terminal domain kinase-1 (CTDK-1) complex are necessary for proper germination, growth and developmental patterns in both A. nidulans and A. fumigatus. Cellular localization and functional interdependencies of the three proteins are also analyzed. Overall, this work links the putative CTDK-1 complex of aspergilli with growth and developmental control. One-sentence summaryIdentification of a mutation in flpA as inhibitor of conidiation in A. nidulans and functional characterization of FlpA, Stk47 and FlpB as putative members of the C-terminal domain kinase complex CTDK-1 in the genus Aspergillus.

microbiology↗

bioRxiv2020

Genome-Wide Association for Itraconazole Sensitivity in Non-Resistant Clinical Isolates of Aspergillus fumigatus

Aspergillus fumigatus is a potentially lethal opportunistic pathogen that infects over [~]200,000 people and causes [~]100,000 deaths per year globally. Treating A. fumigatus infections is particularly challenging because of the recent emergence of azole-resistance. The majority of studies focusing on the molecular mechanisms underlying azole resistance have examined azole-resistant isolates. However, isolates that are susceptible to azoles also display variation in their sensitivity, presenting a unique opportunity to identify genes contributing to azole sensitivity. Here, we used genome-wide association (GWA) analysis to identify loci involved in azole sensitivity by analyzing the association between 68,853 SNPs and itraconazole (ITCZ) minimum inhibitory concentration (MIC) in 76 clinical isolates of A. fumigatus from Japan. Population structure analysis suggests the presence of four distinct populations, with ITCZ MICs distributed relatively evenly across populations. We independently conducted GWA when treating ITCZ MIC as a quantitative trait and a binary trait and identified two SNPs with strong associations that were identified in both analyses. These SNPs fell within the coding regions of Afu2g02220 and Afu2g02140. We functionally validated Afu2g02220 by knocking it out using a CRISPR/Cas-9 approach, because orthologs of this gene are involved in sterol modification and ITCZ targets the ergosterol pathway. Knockout strains displayed no difference in growth compared to the parent strain in minimal media, yet a minor but consistent inhibition of growth in the presence of 0.15 ug/ml ITCZ. Our results suggest that GWA paired with efficient gene deletion is a powerful and unbiased strategy for identifying the genetic basis of complex traits in A. fumigatus. ImportanceAspergillus fumigatus is a pathogenic mold that can infect and kill individuals with compromised immune systems. The azole class of drugs provide antifungal activity against A. fumigatus infections and have become an essential treatment strategy. Unfortunately, A. fumigatus azole resistance has recently emerged and rapidly risen in frequency making treatment more challenging. Our understanding of the molecular basis of azole sensitivity has been shaped mainly through candidate gene studies. Unbiased approaches are necessary to understand the full repertoire of genes and genetic variants underlying azole resistance and sensitivity. Here, we provide the first application of genome-wide association analysis in A. fumigatus in the identification of a gene (Afu2g02220) that contributes to itraconazole susceptibility. Our approach, which combines association mapping and CRISPR/Cas-9 for functional validation of candidate genes, has broad application for investigating the genetic basis of complex traits in fungal systems.

genomics↗