Genomic and Phenotypic Trait Variation of the Opportunistic Human Pathogen Aspergillus flavus and Its Close Relatives.

Fungal diseases affect millions of humans annually, yet fungal pathogens remain understudied. The mold Aspergillus flavus can cause both aspergillosis and fungal keratitis infections, but closely related species 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 species, Aspergillus arachidicola (two strains), Aspergillus parasiticus (two strains), and Aspergillus nomiae (one strain). We identified >3,000 orthologous proteins unique to A. flavus, including seven biosynthetic gene clusters present in A. flavus strains and absent in the three nonpathogens. We characterized secondary metabolite production for all seven strains under two clinically relevant conditions, temperature and salt concentration. Temperature impacted metabolite production in all species, whereas salinity did not affect production of any species. Strains of the same species produced different metabolites. Growth under stress conditions revealed additional heterogeneity within species. Using the invertebrate fungal disease model Galleria mellonella, we found virulence of strains of the same species varied widely; A. flavus strains were not more virulent than strains of the nonpathogens. 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 h postinfection, but not at 72 h. Our work identifies variations in key phenotypic, chemical, and genomic attributes between A. flavus and its nonpathogenic relatives and reveals extensive strain heterogeneity in virulence that does not correspond to the currently established clinical relevance of these species. IMPORTANCE Aspergillus flavus is a filamentous fungus that causes opportunistic human infections, such as aspergillosis and fungal keratitis, but its close relatives are considered nonpathogenic. To begin understanding how this difference in pathogenicity evolved, we characterized variation in infection-relevant genomic, chemical, and phenotypic traits between strains of A. flavus and its relatives. We found extensive variation (or strain heterogeneity) within the pathogenic A. flavus as well as within its close relatives, suggesting that strain-level differences may play a major role in the ability of these fungi to cause disease. Surprisingly, we also found that the virulence of strains from species not considered to be pathogens was similar to that of A. flavus in both invertebrate and murine models of disease. These results contrast with previous studies on Aspergillus fumigatus, another major pathogen in the genus, for which significant differences in infection-relevant chemical and phenotypic traits are observed between closely related pathogenic and nonpathogenic species.

Metabolomics-Guided Comparison of Pollen and Microalgae-Based Artificial Diets in Honey Bees.

Managed honey bee colonies used for crop pollination are fed artificial diets to offset nutritional deficiencies related to land-use intensification and climate change. In this study, we formulated novel microalgae diets using Chlorella vulgaris and Arthrospira platensis (spirulina) biomass and fed them to young adult honey bee workers. Diet-induced changes in bee metabolite profiles were studied relative to a natural pollen diet using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) metabolomics. Untargeted analyses of pollen- and microalgae-fed bees revealed significant overlap, with 248 shared features determined by LC-MS and 87 shared features determined by GC-MS. Further metabolomic commonalities were evident upon subtraction of unique diet features. Twenty-five identified metabolites were influenced by diet, which included complex lipids, essential fatty acids, vitamins, and phytochemicals. The metabolomics results are useful to understand mechanisms underlying favorable growth performance as well as increased antioxidant and heat shock protein gene expression in bees fed the microalgae diets. We conclude that the tested microalgae have potential as sustainable feed additives and as a source of bee health-modulating natural products. Metabolomics-guided diet development could eventually help tailor feed interventions to achieve precision nutrition in honey bees and other livestock animals.

Polychlorinated cyclopentenes from a marine derived Periconia sp. (strain G1144).

Studies on an organic extract of a marine fungus, Periconia sp. (strain G1144), led to the isolation of three halogenated cyclopentenes along with the known and recently reported rhytidhyester D; a series of spectrometric and spectroscopic techniques were used to elucidate these structures. Interestingly, two of these compounds represent tri-halogenated cyclopentene derivatives, which have been observed only rarely from Nature. The relative and absolute configurations of the compounds were established via mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, Mosher's esters method, optical rotation and GIAO NMR calculations, including correlation coefficient calculations and the use of both DP4+ and dJ DP4 analyses. Several of the isolated compounds were tested for activity in anti-parasitic, antimicrobial, quorum sensing inhibition, and cytotoxicity assays and were shown to be inactive.

Fungal-fungal co-culture: a primer for generating chemical diversity.

Covering: 2002 to 2020In their natural environment, fungi must compete for resources. It has been hypothesized that this competition likely induces the biosynthesis of secondary metabolites for defence. In a quest to discover new chemical diversity from fungal cultures, a growing trend has been to recapitulate this competitive environment in the laboratory, essentially growing fungi in co-culture. This review covers fungal-fungal co-culture studies beginning with the first literature report in 2002. Since then, there has been a growing number of new secondary metabolites reported as a result of fungal co-culture studies. Specifically, this review discusses and provides insights into (1) rationale for pairing fungal strains, (2) ways to grow fungi for co-culture, (3) different approaches to screening fungal co-cultures for chemical diversity, (4) determining the secondary metabolite-producing strain, and (5) final thoughts regarding the fungal-fungal co-culture approach. Our goal is to provide a set of practical strategies for fungal co-culture studies to generate unique chemical diversity that the natural products research community can utilize.

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.

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 coronavirus disease 2019 (COVID-19), first described in Wuhan, China. A subset of COVID-19 patients has been reported to have acquired secondary infections by microbial pathogens, such as opportunistic fungal 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 significant differences from the genome of the Af293 reference strain. By examining a number of factors, including virulence in an invertebrate moth model, growth in the presence of osmotic, cell wall, and oxidative stressors, secondary metabolite biosynthesis, and the MIC 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. Moreover, CAPA isolate D was significantly more virulent than the other three CAPA isolates and the A. fumigatus reference strains Af293 and CEA17, but similarly virulent to two other clinical strains of A. fumigatus. These findings expand our understanding of the genomic and phenotypic characteristics of isolates that cause CAPA. IMPORTANCE The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has already killed millions of people. COVID-19 patient outcome can be further complicated by secondary infections, such as COVID-19-associated pulmonary aspergillosis (CAPA). CAPA is caused by Aspergillus fungal pathogens, but there is little information about the genomic and phenotypic characteristics of CAPA isolates. We conducted genome sequencing and extensive phenotyping of four CAPA isolates of Aspergillus fumigatus from Germany. We found that CAPA isolates were often, but not always, similar to other reference strains of A. fumigatus across 206 genetic determinants of infection-relevant phenotypes, including virulence. For example, CAPA isolate D was more virulent than other CAPA isolates and reference strains in an invertebrate model of fungal disease, but similarly virulent to two other clinical strains. These results expand our understanding of COVID-19-associated pulmonary aspergillosis.

Opportunities and Limitations for Assigning Relative Configurations of Antibacterial Bislactones using GIAO NMR Shift Calculations.

Four new bislactones, dihydroacremonol (1), clonostachyone (2), acremodiol B (3), and acremodiol C (4), along with one known compound, hymeglusin (5), were isolated from cultures of two fungal strains (MSX59876 and MSX59260). Both strains were identified based on phylogenetic analysis of molecular data as Clonostachys spp.; yet, they biosynthesized a suite of related, but different, secondary metabolites. Given the challenges associated with elucidating the structures and configurations of bislactones, GIAO NMR calculations were tested as a complement to traditional NMR and HRESIMS experiments. Fortuitously, the enantiomer of the new natural product (4) was known as a synthetic compound, and the predicted configuration from GIAO NMR calculations (i.e., for the relative configuration) and optical rotation calculations (i.e., for the absolute configuration) matched those of the synthesis product. These results engendered confidence in using similar procedures, particularly the mixture of GIAO NMR shift calculations coupled with an orthogonal technique, to predict the configuration of 1-3; however, there were important limitations, which are discussed for each of these. The metabolites displayed antimicrobial activities, with compounds 1 and 4 being the most potent against Staphylococcus aureus with MICs of 1 and 4 µg/mL, respectively.

Capturing the antimicrobial profile of Rosmarinus officinalis against methicillin-resistant Staphylococcus aureus (MRSA) with bioassay-guided fractionation and bioinformatics.

Natural products have been a primary source of medicines throughout the history of human existence. It is estimated that close to 70 % of small molecule pharmaceuticals on the market are derived from natural products. With increasing antibiotic resistance, natural products remain an important source for the discovery of novel antimicrobial compounds. The plant rosemary (Rosmarinus officinalis), has been widely and commonly used as a food preservative due to its antimicrobial potential. To evaluate the antimicrobial profile of this plant, we used bioassay-guided fractionation and bioinformatics approaches. Through bioassay-guided fractionation, we tested in vitro activities of a R. officinalis extract and fractions thereof, as well as pure compounds micromeric acid (1), oleanolic acid (2), and ursolic acid (3) against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 1 and 3 showed complete inhibition of MRSA (with MIC values of 32 µg/mL and 8 µg/mL, respectively) while compound 2 displayed only partial inhibition (MIC > 64 µg/mL). In addition, we utilized orthogonal partial least square-discriminant analysis (OPLS-DA) and selectivity ratio (SR) analysis to correlate the isolated compounds 1-3 with the observed antimicrobial activity, as well as to identify antimicrobials present in trace quantities. For mass spectrometry (MS) data collected in the negative ionization mode, compound 1 was the most positively correlated with activity, while for MS data collected in the positive ion mode, compounds 2-3 had the highest positive correlation. Using the bioinformatics approaches, we highlighted additional antimicrobials associated with the antimicrobial activity of R. officinalis, including genkwanin (4), rosmadial (5a) and/or 16-hydroxyrosmadial (5b), rosmanol (6), and hesperetin (7). Compounds 1-3 resulting from the bioassay-guided fractionation were identified by MS-MS fragmentation patterns and 1H NMR spectra. Among the compounds highlighted by the biochemical analysis, compound 6 was identified by comparison with its commercial standard by employed ultra-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS), while 4, 5a-b and 7 were putatively identified based on MS data and in comparison with the literature. This is the first reported antimicrobial activity of micromeric acid (1) against MRSA.

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.

Bioactive diterpenoid metabolism and cytotoxic activities of genetically transformed Euphorbia lathyris roots.

Plants in the genus Euphorbia produce a wide variety of pharmacologically active diterpenoids with anticancer, multidrug resistance reversal, and antiviral properties. Some are the primary industrial source of ingenol mebutate, which is approved for treatment of the precancerous skin condition actinic keratosis. Similar to other high value phytochemicals, Euphorbia diterpenoids accumulate at low concentrations in planta and chemical synthesis produces similarly low yields. We established genetically transformed root cultures of Euphorbia lathryis as a strategy to gain greater access to diterpenoids from this genus. Transformed roots produced via stem explant infection with Agrobacterium rhizogenes strain 15834 recapitulated the metabolite profiles of field-grown plant roots and aerial tissues. Several putative diterpenoids were present in transformed roots, including ingenol and closely related structures, indicating that root cultures are a promising approach to Euphorbia-specific diterpenoid production. Treatment with methyl jasmonate led to a significant, albeit transient increase in mRNA levels of early diterpenoid biosynthetic enzymes (farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and casbene synthase), suggesting that elicitation could prove useful in future pathway characterization and metabolic engineering efforts. We also show the potential of transformed E. lathyris root cultures for natural product drug discovery applications by measuring their cytotoxic activities using a panel of human carcinoma cell lines derived from prostate, cervix, breast, and lung.

Variation Among Biosynthetic Gene Clusters, Secondary Metabolite Profiles, and Cards of Virulence Across Aspergillus Species.

Aspergillus fumigatus is a major human pathogen. 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 genetic determinants of virulence (or "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. To examine whether secondary metabolism-associated cards of virulence vary between these species, we conducted extensive genomic and secondary metabolite profiling analyses of multiple A. fumigatus, one A. oerlinghausenensis, and multiple A. fischeri strains. We identified two cards of virulence (gliotoxin and fumitremorgin) shared by all three species and three cards of virulence (trypacidin, pseurotin, and fumagillin) that are variable. For example, we found that all species and strains examined biosynthesized gliotoxin, which is known to contribute to virulence, consistent with the conservation of the gliotoxin biosynthetic gene cluster (BGC) across genomes. For other secondary metabolites, such as fumitremorgin, a modulator of host biology, we found that all species produced the metabolite but that there was strain heterogeneity in its production within species. Finally, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis. A. fumigatus biosynthesized fumagillin and pseurotin, while A. oerlinghausenensis biosynthesized fumagillin and A. fischeri biosynthesized neither. These biochemical 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 onto the genetic and phenotypic changes associated with the evolution of fungal pathogenicity.

Pathogenic Allodiploid Hybrids of Aspergillus Fungi.

Interspecific hybridization substantially alters genotypes and phenotypes and can give rise to new lineages. Hybrid isolates that differ from their parental species in infection-relevant traits have been observed in several human-pathogenic yeasts and plant-pathogenic filamentous fungi but have yet to be found in human-pathogenic filamentous fungi. We discovered 6 clinical isolates from patients with aspergillosis originally identified as Aspergillus nidulans (section Nidulantes) that are actually allodiploid hybrids formed by the fusion of Aspergillus spinulosporus with an unknown close relative of Aspergillus quadrilineatus, both in section Nidulantes. Evolutionary genomic analyses revealed that these isolates belong to Aspergillus latus, an allodiploid hybrid species. Characterization of diverse infection-relevant traits further showed that A. latus hybrid isolates are genomically and phenotypically heterogeneous but also differ from A. nidulans, A. spinulosporus, and A. quadrilineatus. These results suggest that allodiploid hybridization contributes to the genomic and phenotypic diversity of filamentous fungal pathogens of humans.

Wheldone: Characterization of a Unique Scaffold from the Coculture of Aspergillus fischeri and Xylaria flabelliformis.

Wheldone (1) was isolated and elucidated from a coculture of Aspergillus fischeri (NRRL 181) and Xylaria flabelliformis (G536), where secondary metabolite biosynthesis was stimulated by antagonism between these fungi. First observed via in situ analysis between these competing fungal cultures, the conditions were scaled to reproducibly generate 1, whose novel structure was elucidated by one- and two-dimensional NMR and mass spectrometry. Compound 1 displayed cytotoxic activity against breast, ovarian, and melanoma cancer cell lines.

Gliotoxin, a Known Virulence Factor in the Major Human Pathogen Aspergillus fumigatus, Is Also Biosynthesized by Its Nonpathogenic Relative Aspergillus 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 Aspergillus fischeri, a very close nonpathogenic relative of A. fumigatus, contains a biosynthetic gene cluster that is homologous 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 nonpathogen A. fischeri, we examined whether A. fischeri strain NRRL 181 biosynthesizes gliotoxin and whether the production of secondary metabolites influences the virulence profile of A. fischeri We found that A. fischeri biosynthesizes gliotoxin under the same conditions as A. fumigatus However, whereas loss of laeA, a master regulator of secondary metabolite production (including gliotoxin biosynthesis), has previously been shown to reduce A. fumigatus virulence, we found that laeA loss (and loss of secondary metabolite production) in A. fischeri does not influence its virulence. These results suggest that LaeA-regulated secondary metabolites 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 nonpathogen A. fischeri Understanding the observed spectrum of pathogenicity across closely related pathogenic and nonpathogenic 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 nonpathogenic. Why is that so? This important, yet largely unanswered, question can be addressed by examining how A. fumigatus and its close nonpathogenic relatives are similar or different with respect to virulence-associated traits. We investigated whether Aspergillus fischeri, a nonpathogenic close relative of A. fumigatus, can produce gliotoxin, a mycotoxin known to contribute to A. fumigatus virulence. We discovered that the nonpathogenic A. fischeri produces gliotoxin under the same conditions as those of the major pathogen A. fumigatus However, we also discovered that, in contrast to what has previously been observed in A. fumigatus, the loss of secondary metabolite production in A. fischeri does not alter its virulence. Our results are consistent with the "cards of virulence" model of opportunistic fungal disease, in which the ability to cause disease stems from the combination ("hand") of virulence factors ("cards") but not from individual factors per se.

Orthogonal Method for Double-Bond Placement via Ozone-Induced Dissociation Mass Spectrometry (OzID-MS).

Most often, the structures of secondary metabolites are solved using a suite of NMR techniques. However, there are times when it can be challenging to position double bonds, particularly those that are fully substituted or when there are multiple double bonds in similar chemical environments. Ozone-induced dissociation mass spectrometry (OzID-MS) serves as an orthogonal structure elucidation tool, using predictable fragmentation patterns that are generated after ozonolysis across a carbon-carbon double bond. This technique is finding growing use in the lipidomics community, suggestive of its potential value for secondary metabolites. This methodology was evaluated by confirming the double-bond positions in five fungal secondary metabolites, specifically, ent-sartorypyrone E (1), sartorypyrone A (2), sorbicillin (3), trichodermic acid A (4), and AA03390 (5). This demonstrated its potential with a variety of chemotypes, ranging from polyketides to terpenoids and including those in both conjugated and nonconjugated polyenes. In addition, the potential of using this methodology in the context of a mixture was piloted by studying Aspergillus fischeri, first examining a traditional extract and then sampling a live fungal culture in situ. While the intensity of signals varied from pure compound to extract to in situ, the utility of the technique was preserved.

Draft Genome Sequence of the Griseofulvin-Producing Fungus Xylaria flabelliformis Strain G536.

The draft genome of the ascomycete fungus Xylaria flabelliformis (previously known as Xylaria cubensis) was sequenced using Illumina paired-end technology. The assembled genome is 41.2 Mb long and contains 11,404 genes. This genome will contribute to our understanding of X. flabelliformis secondary metabolism and the organism's ability to live as a decomposer as well as an endosymbiont.

Droplet probe: coupling chromatography to the in situ evaluation of the chemistry of nature.

Covering: up to 2019The chemistry of nature can be beautiful, inspiring, beneficial and poisonous, depending on perspective. Since the isolation of the first secondary metabolites roughly two centuries ago, much of the chemical research on natural products has been both reductionist and static. Typically, compounds were isolated and characterized from the extract of an entire organism from a single time point. While there could be subtexts to that approach, the general premise has been to determine the chemistry with very little in the way of tools to differentiate spatial and/or temporal changes in secondary metabolite profiles. However, the past decade has seen exponential advances in our ability to observe, measure, and visualize the chemistry of nature in situ. Many of those techniques have been reviewed in this journal, and most are tapping into the power of mass spectrometry to analyze a plethora of sample types. In nearly all of the other techniques used to study chemistry in situ, the element of chromatography has been eliminated, instead using various ionization sources to coax ions of the secondary metabolites directly into the mass spectrometer as a mixture. Much of that science has been driven by the great advances in ambient ionization techniques used with a suite of mass spectrometry platforms, including the alphabet soup from DESI to LAESI to MALDI. This review discusses the one in situ analysis technique that incorporates chromatography, being the droplet-liquid microjunction-surface sampling probe, which is more easily termed "droplet probe". In addition to comparing and contrasting the droplet probe with other techniques, we provide perspective on why scientists, particularly those steeped in natural products chemistry training, may want to include chromatography in in situ analyses. Moreover, we provide justification for droplet sampling, especially for samples with delicate and/or non-uniform topographies. Furthermore, while the droplet probe has been used the most in the analysis of fungal cultures, we digest a variety of other applications, ranging from cyanobacteria, to plant parts, and even delicate documents, such as herbarium specimens.

Mapping the Fungal Battlefield: Using in situ Chemistry and Deletion Mutants to Monitor Interspecific Chemical Interactions Between Fungi.

Fungi grow in competitive environments, and to cope, they have evolved strategies, such as the ability to produce a wide range of secondary metabolites. This begs two related questions. First, how do secondary metabolites influence fungal ecology and interspecific interactions? Second, can these interspecific interactions provide a way to "see" how fungi respond, chemically, within a competitive environment? To evaluate these, and to gain insight into the secondary metabolic arsenal fungi possess, we co-cultured Aspergillus fischeri, a genetically tractable fungus that produces a suite of mycotoxins, with Xylaria cubensis, a fungus that produces the fungistatic compound and FDA-approved drug, griseofulvin. To monitor and characterize fungal chemistry in situ, we used the droplet-liquid microjunction-surface sampling probe (droplet probe). The droplet probe makes a microextraction at defined locations on the surface of the co-culture, followed by analysis of the secondary metabolite profile via liquid chromatography-mass spectrometry. Using this, we mapped and compared the spatial profiles of secondary metabolites from both fungi in monoculture versus co-culture. X. cubensis predominantly biosynthesized griseofulvin and dechlorogriseofulvin in monoculture. In contrast, under co-culture conditions a deadlock was formed between the two fungi, and X. cubensis biosynthesized the same two secondary metabolites, along with dechloro-5'-hydroxygriseofulvin and 5'-hydroxygriseofulvin, all of which have fungistatic properties, as well as mycotoxins like cytochalasin D and cytochalasin C. In contrast, in co-culture, A. fischeri increased the production of the mycotoxins fumitremorgin B and verruculogen, but otherwise remained unchanged relative to its monoculture. To evaluate that secondary metabolites play an important role in defense and territory establishment, we co-cultured A. fischeri lacking the master regulator of secondary metabolism laeA with X. cubensis. We found that the reduced secondary metabolite biosynthesis of the ΔlaeA strain of A. fischeri eliminated the organism's ability to compete in co-culture and led to its displacement by X. cubensis. These results demonstrate the potential of in situ chemical analysis and deletion mutant approaches for shedding light on the ecological roles of secondary metabolites and how they influence fungal ecological strategies; co-culturing may also stimulate the biosynthesis of secondary metabolites that are not produced in monoculture in the laboratory.

Characterizing the Pathogenic, Genomic, and Chemical Traits of Aspergillus fischeri, a Close 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 A. fischeri causes less human disease than A. fumigatus is unclear. A comparison of A. fischeri and A. fumigatus for pathogenic, genomic, and secondary metabolic traits revealed multiple differences in pathogenesis-related phenotypes. We observed that A. fischeri NRRL 181 is less virulent than A. fumigatus strain CEA10 in multiple animal models of disease, grows slower in low-oxygen environments, and is more sensitive to oxidative stress. Strikingly, the observed differences for some traits are of the same order of magnitude as those previously reported between A. fumigatus strains. In contrast, similar to what has previously been reported, 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. Additionally, 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 nonpathogenic 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.IMPORTANCEAspergillus 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 pathogenesis-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 Remarkably, the observed differences between A. fischeri and A. fumigatus strains examined here closely resembled those previously described for two commonly studied A. fumigatus strains, AF293 and CEA10. A. fischeri and A. fumigatus 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 chemicals that induce the production of reactive oxygen species. 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 report here are the first step toward understanding the evolutionary origin of a major fungal pathogen.