Biology subjects

Joehnk, B.

Publications and source records attributed to Joehnk, B..

4 recordsLinked to original sources

bioRxiv2024

Inferring the composition of a mixed culture of natural microbial isolates by deep sequencing

Next generation sequencing has unlocked a wealth of genotype information for microbial populations, but phenotyping remains a bottleneck for exploiting this information, particularly for pathogens that are difficult to manipulate. Here, we establish a method for high-throughput phenotyping of mixed cultures, in which the pattern of naturally occurring single-nucleotide polymorphisms in each isolate is used as intrinsic barcodes which can be read out by sequencing. We demonstrate that our method can correctly deconvolute strain proportions in simulated mixed-strain pools. As an experimental test of our method, we perform whole genome sequencing of 66 natural isolates of the thermally dimorphic pathogenic fungus Coccidioides posadasii and infer the strain compositions for large mixed pools of these strains after competition at 37{degrees}C and room temperature. We validate the results of these selection experiments by recapitulating the temperature-specific enrichment results in smaller pools. Additionally, we demonstrate that strain fitness estimated by our method can be used as a quantitative trait for genome-wide association studies. We anticipate that our method will be broadly applicable to natural populations of microbes and allow high-throughput phenotyping to match the rate of genomic data acquisition. Author summaryThe diversity of the gene pool in natural populations encodes a wealth of information about its molecular biology. This is an especially valuable resource for non-model organisms, from humans to many microbial pathogens, lacking traditional genetic approaches. An effective method for reading out this population genetic information is a genome wide association study (GWAS) which searches for genotypes correlated with a phenotype of interest. With the advent of cheap genotyping, high throughput phenotyping is the primary bottleneck for GWAS, particularly for microbes that are difficult to manipulate. Here, we take advantage of the fact that the naturally occurring genetic variation within each individual strain can be used as an intrinsic barcode, which can be used to read out relative abundance of each strain as a quantitative phenotype from a mixed culture. Coccidioides posadasii, the causative agent of Valley Fever, is a fungal pathogen that must be manipulated under biosafety level 3 conditions, precluding many high-throughput phenotyping approaches. We apply our method to pooled competitions of C. posadasii strains at environmental and host temperatures. We identify robustly growing and temperature-sensitive strains, confirm these inferences in validation pooled growth experiments, and successfully demonstrate their use in GWAS.

genetics↗

bioRxiv2023

Recyclable CRISPR/Cas9 mediated gene disruption and deletions in Histoplasma

Targeted gene disruption is challenging in the dimorphic fungal pathogen Histoplasma due to the low frequency of homologous recombination. Transformed DNA is either integrated ectopically into the genome or maintained extra chromosomally by de novo addition of telomeric sequences. Based on a system developed in Blastomyces, we adapted a CRISPR/Cas9 system to facilitate targeted gene disruption in Histoplasma with high efficiency. We express a codon-optimized version of Cas9 as well as guide RNAs from a single ectopic vector carrying a selectable marker. Once the desired mutation is verified, one can screen for isolates that have lost the Cas9 vector by simply removing the selective pressure. Multiple mutations can then be generated in the same strain by retransforming the Cas9 vector carrying different guides. We used this system to disrupt a number of target genes including RYP2 and SRE1 where loss-of-function mutations could be monitored visually by colony morphology or color, respectively. Interestingly, expression of two guide RNAs targeting the 5 and 3 ends of a gene allowed isolation of deletion mutants where the sequence between the guide RNAs was removed from the genome. Whole-genome sequencing showed that the frequency of off-target mutations associated with the Cas9 nuclease was negligible. Finally, we increased the frequency of gene disruption by using an endogenous Histoplasma regulatory sequence to drive guide RNA expression. These tools transform our ability to generate targeted mutations in Histoplasma. ImportanceHistoplasma is a primary fungal pathogen with the ability to infect otherwise healthy mammalian hosts, causing systemic and sometimes life-threatening disease. Thus far, molecular genetic manipulation of this organism has utilized RNA interference, random insertional mutagenesis, and a homologous recombination protocol that is highly variable and often inefficient. Targeted gene manipulations have been challenging due to poor rates of homologous recombination events in Histoplasma. Interrogation of the virulence strategies of this organism would be highly accelerated by a means of efficiently generating targeted mutations. We have developed a recyclable CRISPR/Cas9 system that can be used to introduce gene disruptions in Histoplasma with high efficiency, thereby allowing disruption of multiple genes.

microbiology↗

bioRxiv2022

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-{kappa}B inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more I{kappa}B expression in infected than bystander cells, but found concurrent nuclear translocation of NF-{kappa}B that I{kappa}B usually prevents. Overexpressing a nondegradable I{kappa}B mutant reduced NF-{kappa}B translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-{kappa}B feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.

microbiology↗

bioRxiv2019

Recurrent loss of abaA, a master regulator of asexual development in filamentous fungi, correlates with changes in genomic and morphological traits

Gene regulatory networks (GRNs) drive developmental and cellular differentiation, and variation in their architectures gives rise to morphological diversity. Pioneering studies in Aspergillus fungi, coupled with subsequent work in other filamentous fungi, have shown that the GRN governed by the BrlA, AbaA, and WetA proteins controls the development of the asexual fruiting body or conidiophore. A specific aspect of conidiophore development is the production of phialides, conidiophore structures that are under the developmental control of AbaA and function to repetitively generate spores. Fungal genome sequencing has revealed that some filamentous fungi lack abaA, and also produce asexual structures that lack phialides, raising the hypothesis that abaA loss is functionally linked to diversity in asexual fruiting body morphology. To examine this hypothesis, we carried out an extensive search for the abaA gene across 241 genomes of species from the fungal subphylum Pezizomycotina. We found that abaA was independently lost in four lineages of Eurotiomycetes, including from all sequenced species within the order Onygenales, and that all four lineages that have lost abaA also lack the ability to form phialides. Genetic restoration of abaA from Aspergillus nidulans into Histoplasma capsulatum, a pathogenic species from the order Onygenales that lacks an endogenous copy of abaA, did not alter Histoplasma conidiation morphology but resulted in a marked increase in spore viability. We also discovered that species lacking abaA contain fewer AbaA binding motifs in the regulatory regions of orthologs of some AbaA target genes, suggesting that the asexual fruiting body GRN of organisms that have lost abaA has been rewired. Our results provide an illustration of how repeated losses of a key regulatory transcription factor and concomitant changes in non-coding regulatory regions of the genome have contributed to the diversity of an iconic fungal morphological trait.\n\nAuthor SummaryFungi exhibit tremendous variation in their asexual fruiting bodies. For example, whereas some fungi form complex fruiting bodies whose tips repeatedly generate and release spores, others produce single spores in the absence of a specialized structure. To gain insights into the molecular differences that underpin fungal asexual fruiting body diversity, we examined the genomes of hundreds of filamentous fungi for the presence of abaA, a master regulatory gene previously shown to control the development of fungal asexual fruiting bodies. We found that abaA was repeatedly lost during fungal evolution, including in a lineage of human pathogenic fungi, and that the loss of the gene was always associated with the loss of specialized structures in fungal asexual fruiting bodies. Reintroduction of abaA into the human pathogenic fungus Histoplasma capsulatum, which normally lacks the regulator, did not result in a change in the spore-producing structure but did increase spore outgrowth. Based on these results, we hypothesize that the loss of the master regulatory gene abaA has contributed to the observed diversity of fungal fruiting body morphology. This work advances our understanding of how fungal developmental networks evolve over time and advances our understanding of how infectious spores form in pathogenic fungi.

evolutionary biology↗