Benjamin P. Knox

Molecular Genetic Characterization of a Cluster in A. terreus for Biosynthesis of the Meroterpenoid Terretonin

Meroterpenoids are natural products produced from polyketide and terpenoid precursors. A gene targeting system for A. terreus NIH2624 was developed, and a gene cluster for terretonin biosynthesis was characterized. The intermediates and shunt products were isolated from the mutant strains, and a pathway for terretonin biosynthesis is proposed. Analysis of two meroterpenoid pathways corresponding to terretonin in A. terreus and austinol in A. nidulans reveals that they are closely related evolutionarily.

Distinct Innate Immune Phagocyte Responses to Aspergillus fumigatus Conidia and Hyphae in Zebrafish Larvae

ABSTRACT Aspergillus fumigatus is the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense against A. fumigatus; however, innate phagocyte responses to A. fumigatus in an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafish A. fumigatus infection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection with A. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain of A. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses to A. fumigatus that reveals distinct roles for neutrophils and macrophages in mediating host defense against IA.

Application of an Efficient Gene Targeting System Linking Secondary Metabolites to their Biosynthetic Genes in Aspergillus terreus

Nonribosomal peptides (NRPs) are natural products biosynthesized by NRP synthetases. A kusA-, pyrG- mutant strain of Aspergillus terreus NIH 2624 was developed that greatly facilitated the gene targeting efficiency in this organism. Application of this tool allowed us to link four major types of NRP-related secondary metabolites to their responsible genes in A. terreus. In addition, an NRP affecting melanin synthesis was also identified in this species.

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

ABSTRACT The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin’s carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique. IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin. IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.

Characterization of Aspergillus fumigatus Isolates from Air and Surfaces of the International Space Station

As durations of manned space missions increase, it is imperative to understand the long-term consequence of microbial exposure on human health in a closed human habitat. To date, studies aimed at bacterial and fungal contamination of space vessels have highlighted species compositions biased toward hardy, persistent organisms capable of withstanding harsh conditions. In the current study, we assessed traits of two independent Aspergillus fumigatus strains isolated from the International Space Station. Ubiquitously found in terrestrial soil and atmospheric environments, A. fumigatus is a significant opportunistic fungal threat to human health, particularly among the immunocompromised. Using two well-known clinical isolates of A. fumigatus as comparators, we found that both ISS isolates exhibited normal in vitro growth and chemical stress tolerance yet caused higher lethality in a vertebrate model of invasive disease. These findings substantiate the need for additional studies of physical traits and biological activities of microbes adapted to microgravity and other extreme extraterrestrial conditions. ABSTRACT One mission of the Microbial Observatory Experiments on the International Space Station (ISS) is to examine the traits and diversity of fungal isolates to gain a better understanding of how fungi may adapt to microgravity environments and how this may affect interactions with humans in a closed habitat. Here, we report an initial characterization of two isolates, ISSFT-021 and IF1SW-F4, of Aspergillus fumigatus collected from the ISS and a comparison to the experimentally established clinical isolates Af293 and CEA10. Whole-genome sequencing of ISSFT-021 and IF1SW-F4 showed 54,960 and 52,129 single nucleotide polymorphisms, respectively, compared to Af293, which is consistent with observed genetic heterogeneity among sequenced A. fumigatus isolates from diverse clinical and environmental sources. Assessment of in vitro growth characteristics, secondary metabolite production, and susceptibility to chemical stresses revealed no outstanding differences between ISS and clinical strains that would suggest special adaptation to life aboard the ISS. Virulence assessment in a neutrophil-deficient larval zebrafish model of invasive aspergillosis revealed that both ISSFT-021 and IF1SW-F4 were significantly more lethal than Af293 and CEA10. Taken together, these genomic, in vitro, and in vivo analyses of two A. fumigatus strains isolated from the ISS provide a benchmark for future investigations of these strains and for continuing research on specific microbial isolates from manned space environments. IMPORTANCE As durations of manned space missions increase, it is imperative to understand the long-term consequence of microbial exposure on human health in a closed human habitat. To date, studies aimed at bacterial and fungal contamination of space vessels have highlighted species compositions biased toward hardy, persistent organisms capable of withstanding harsh conditions. In the current study, we assessed traits of two independent Aspergillus fumigatus strains isolated from the International Space Station. Ubiquitously found in terrestrial soil and atmospheric environments, A. fumigatus is a significant opportunistic fungal threat to human health, particularly among the immunocompromised. Using two well-known clinical isolates of A. fumigatus as comparators, we found that both ISS isolates exhibited normal in vitro growth and chemical stress tolerance yet caused higher lethality in a vertebrate model of invasive disease. These findings substantiate the need for additional studies of physical traits and biological activities of microbes adapted to microgravity and other extreme extraterrestrial conditions.

Key Players in the Regulation of Fungal Secondary Metabolism

Secondary metabolites (SMs) produced by filamentous fungi are well known for having a broad range of bioactivities that have detrimental, as well as beneficial, impacts on human affairs. With the grouping of SM biosynthetic genes into clusters it is possible for regulatory elements, such as individual transcription factors or multiprotein complexes, to modulate metabolite expression individually or at a larger, more global scale. In this chapter, we survey some key transcriptional players in the regulation of secondary metabolism including cluster-specific Zn(II)2Cys6, Cys2His2, and winged helix proteins, bZIP factors associated with stress and SM, environmental monitoring Cys2His2 proteins (AreA, CreA, and PacC) and two global regulatory complexes—the velvet complex and the CCAAT-binding complex.

Real-time visualization of immune cell clearance of Aspergillus fumigatus spores and hyphae

Invasive aspergillosis (IA) is a disease of the immunocompromised host and generally caused by the opportunistic fungal pathogen Aspergillus fumigatus. While both host and fungal factors contribute to disease severity and outcome, there are fundamental features of IA development including fungal morphological transition from infectious conidia to tissue-penetrating hyphae as well as host defenses rooted in mechanisms of innate phagocyte function. Here we address recent advances in the field and use real-time in vivo imaging in the larval zebrafish to visually highlight conserved vertebrate innate immune behaviors including macrophage phagocytosis of conidia and neutrophil responses post-germination.

Macrophages inhibit Aspergillus fumigatus germination and neutrophil-mediated fungal killing

In immunocompromised individuals, Aspergillus fumigatus causes invasive fungal disease that is often difficult to treat. Exactly how immune mechanisms control A. fumigatus in immunocompetent individuals remains unclear. Here, we use transparent zebrafish larvae to visualize and quantify neutrophil and macrophage behaviors in response to different A. fumigatus strains. We find that macrophages form dense clusters around spores, establishing a protective niche for fungal survival. Macrophages exert these protective effects by inhibiting fungal germination, thereby inhibiting subsequent neutrophil recruitment and neutrophil-mediated killing. Germination directly drives fungal clearance as faster-growing CEA10-derived strains are killed better in vivo than slower-growing Af293-derived strains. Additionally, a CEA10 pyrG-deficient strain with impaired germination is cleared less effectively by neutrophils. Host inflammatory activation through Myd88 is required for killing of a CEA10-derived strain but not sufficient for killing of an Af293-derived strain, further demonstrating the role of fungal-intrinsic differences in the ability of a host to clear an infection. Altogether, we describe a new role for macrophages in the persistence of A. fumigatus and highlight the ability of different A. fumigatus strains to adopt diverse modes of virulence.

Selenate sensitivity of a laeA mutant is restored by overexpression of the bZIP protein MetR in Aspergillus fumigatus

LaeA is a conserved global regulator of secondary metabolism and development in filamentous fungi. Examination of Aspergillus fumigatus transcriptome data of laeA deletion mutants have been fruitful in identifying genes and molecules contributing to the laeA mutant phenotype. One of the genes significantly down regulated in A. fumigatus ΔlaeA is metR, encoding a bZIP DNA binding protein required for sulfur and methionine metabolism in fungi. LaeA and MetR deletion mutants exhibit several similarities including down regulation of sulfur assimilation and methionine metabolism genes and ability to grow on the toxic sulfur analog, sodium selenate. However, unlike ΔmetR, ΔlaeA strains are able to grow on sulfur, sulfite, and cysteine. To examine if any parameter of the ΔlaeA phenotype is due to decreased metR expression, an over-expression allele (OE::metR) was placed in a ΔlaeA background. The OE::metR allele could not significantly restore expression of MetR regulated genes in ΔlaeA but did restore sensitivity to sodium selenate. In A. nidulans a second bZIP protein, MetZ, also regulates sulfur and methionine metabolism genes. However, addition of an OE::metZ construct to the A. fumigatus ΔlaeA OE::metR strain still was unable to rescue the ΔlaeA phenotype to wildtype with regards gliotoxin synthesis and virulence in a zebrafish aspergillosis model.