Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.

PacBio high-throughput multi-locus sequencing reveals high genetic diversity in mushroom-forming fungi.

Multi-locus sequence data are widely used in fungal systematic and taxonomic studies to delimit species and infer evolutionary relationships. We developed and assessed the efficacy of a multi-locus pooled sequencing method using PacBio long-read high-throughput sequencing. Samples included fresh and dried voucher specimens, cultures and archival DNA extracts of Agaricomycetes with an emphasis on the order Cantharellales. Of the 283 specimens sequenced, 93.6% successfully amplified at one or more loci with a mean of 3.3 loci amplified. Our method recovered multiple sequence variants representing alleles of rDNA loci and single copy protein-coding genes rpb1, rpb2 and tef1. Within-sample genetic variation differed by locus and taxonomic group, with the greatest genetic divergence observed among sequence variants of rpb2 and tef1 from corticioid Cantharellales. Our method is a cost-effective approach for generating accurate multi-locus sequence data coupled with recovery of alleles from polymorphic samples and multi-organism specimens. These results have important implications for understanding intra-individual genomic variation among genetic loci commonly used in species delimitation of fungi.

Genomic determination of breeding systems and trans-specific evolution of HD MAT genes in suilloid fungi.

Studying the signatures of evolution can help to understand genetic processes. Here, we demonstrate how the existence of balancing selection can be used to identify the breeding systems of fungi from genomic data. The breeding systems of fungi are controlled by self-incompatibility loci that determine mating types between potential mating partners, resulting in strong balancing selection at the loci. Within the fungal phylum Basidiomycota, two such self-incompatibility loci, namely HD MAT locus and P/R MAT locus, control mating types of gametes. Loss of function at one or both MAT loci results in different breeding systems and relaxes the MAT locus from balancing selection. By investigating the signatures of balancing selection at MAT loci, one can infer a species' breeding system without culture-based studies. Nevertheless, the extreme sequence divergence among MAT alleles imposes challenges for retrieving full variants from both alleles when using the conventional read-mapping method. Therefore, we employed a combination of read-mapping and local de novo assembly to construct haplotypes of HD MAT alleles from genomes in suilloid fungi (genera Suillus and Rhizopogon). Genealogy and pairwise divergence of HD MAT alleles showed that the origins of mating types predate the split between these two closely related genera. High sequence divergence, trans-specific polymorphism, and the deeply diverging genealogy confirm the long-term functionality and multiallelic status of HD MAT locus in suilloid fungi. This work highlights a genomics approach to studying breeding systems regardless of the culturability of organisms based on the interplay between evolution and genetics.

Fungal heavy metal adaptation through single nucleotide polymorphisms and copy-number variation.

Human-altered environments can shape the evolution of organisms. Fungi are no exception, although little is known about how they withstand anthropogenic pollution. Here, we document adaptation in the mycorrhizal fungus Suillus luteus driven by soil heavy metal contamination. Genome scans across individuals from recently polluted and nearby unpolluted soils in Belgium revealed low divergence across isolates and no evidence of population structure based on soil type. However, we detected single nucleotide polymorphism divergence and gene copy-number variation, with different genetic combinations potentially conferring the ability to persist in contaminated soils. Variants were shared across the population but found to be under selection in isolates exposed to pollution and located across the genome, including in genes involved in metal exclusion, storage, immobilization and reactive oxygen species detoxification. Together, our results point to S. luteus undergoing the initial steps of adaptive divergence and contribute to understanding the processes underlying local adaptation under strong environmental selection.

Rhinovirus respiratory tract infection in hospitalized adult patients is associated with TH2 response irrespective of asthma.

OBJECTIVES: We assessed the immunological response of hospitalized adult patients with rhinovirus infection, including critically-ill patients. METHODS: The differential white blood cell (WBC) count and the levels of 29 plasma cytokines/chemokines were compared between 50 adult hospitalized patients with rhinovirus infection and 100 age-matched controls with influenza virus infection. RESULTS: The demographics and comorbidities were similar between rhinovirus and influenza patients, but severe disease was more common for the rhinovirus cohort. Rhinovirus patients had significantly higher WBC counts than influenza patients, especially for eosinophil (P = 3.1 × 10-8). The level of the TH2 cytokine IL-5 was significantly higher among rhinovirus patients, while the levels of 9 other cytokines/chemokines were significantly lower among rhinovirus patients. The levels of CXCL-10 (IP-10), CCL-2 (MCP-1), IFN-α2, IFN-γ, IL-10, and IL-15 remained significantly lower among rhinovirus patients after correction for multiple comparisons. Notably, CXCL-10 had the highest area under the receiver operating characteristic curve (AUC) in differentiating rhinovirus from influenza patients (AUC, 0.918). In the patient subgroup without asthma, the difference in the WBC count and cytokine/chemokine levels between rhinovirus and influenza patients remained statistically significant. CONCLUSIONS: Rhinovirus infection was characterized by a prominent TH2 response, even in patients without asthma. CXCL-10 (IP-10) is a potential biomarker in differentiating rhinovirus from influenza infection.

Correlations between ACE single nucleotide polymorphisms and prognosis of patients with septic shock.

The aim of the present study is to investigate association between septic shock (SS) and angiotensin I-converting enzyme (ACE) single nucleotide polymorphisms (SNPs). From October 2009 to December 2016, 238 SS patients and 242 healthy individuals were selected for our study. ACE activity was detected, ACE rs4291 and rs4646994 polymorphisms were detected using PCR-restriction fragment length polymorphism (PCR-RFLP). The Kaplan-Meier survival curve was employed to evaluate the association between ACE SNPs and patients' survival and univariate and multivariate analyses to estimate risk factors for SS. ACE activity in the case group was increased in comparison with the control group. Allele and genotype frequencies of rs4291 and rs4646994 were different between the case and control groups. The TT genotype frequency of the rs4291 polymorphisms and the DD genotype of the rs4646994 polymorphisms of the case group were higher than those in the control group. The AT and TT genotypes indicated a significant elevation of ACE activity than the AA genotype, while a significant decline was found in the DI and II genotypes in comparison with the DI genotype. Patients with TT or DD genotypes had increased fatality rate within 7 and 30 days when compared with those with non-TT or non-DD genotypes. Lower sepsis-related organ failure assessment (SOFA) scores, rs4291, serum ACE and rs4646994 were all considered as risky factors for SS patients. The study demonstrates that TT genotype of rs4291 or DD genotype of rs4646994 may be indicative of a higher risk of SS and a poorer prognosis in SS patients.

Talaromyces marneffei Mp1p Is a Virulence Factor that Binds and Sequesters a Key Proinflammatory Lipid to Dampen Host Innate Immune Response.

Talaromyces (Penicillium) marneffei is one of the leading causes of systemic mycosis in immunosuppressed or AIDS patients in Southeast Asia. How this intracellular pathogen evades the host immune defense remains unclear. We provide evidence that T. marneffei depletes levels of a key proinflammatory lipid mediator arachidonic acid (AA) to evade the host innate immune defense. Mechanistically, an abundant secretory mannoprotein Mp1p, shown previously to be a virulence factor, does so by binding AA with high affinity via a long hydrophobic central cavity found in the LBD2 domain. This sequesters a critical proinflammatory signaling lipid, and we see evidence that AA, AA's downstream metabolites, and the cytokines interleukin-6 and tumor necrosis factor α are downregulated in T. marneffei-infected J774 macrophages. Given that Mp1p-LBD2 homologs are identified in other fungal pathogens, we expect that this novel class of fatty-acid-binding proteins sequestering key proinflammatory lipid mediators represents a general virulence mechanism of pathogenic fungi.

Lipid metabolites as potential diagnostic and prognostic biomarkers for acute community acquired pneumonia.

Early diagnosis of acute community-acquired pneumonia (CAP) is important in patient triage and treatment decisions. To identify biomarkers that distinguish patients with CAP from non-CAP controls, we conducted an untargeted global metabolome analysis for plasma samples from 142 patients with CAP (CAP cases) and 97 without CAP (non-CAP controls). Thirteen lipid metabolites could discriminate between CAP cases and non-CAP controls with area-under-the-receiver-operating-characteristic curve of >0.8 (P ≤ 10(-9)). The levels of glycosphingolipids, sphingomyelins, lysophosphatidylcholines and L-palmitoylcarnitine were higher, while the levels of lysophosphatidylethanolamines were lower in the CAP cases than those in non-CAP controls. All 13 metabolites could distinguish CAP cases from the non-infection, extrapulmonary infection and non-CAP respiratory tract infection subgroups. The levels of trihexosylceramide (d18:1/16:0) were higher, while the levels of lysophosphatidylethanolamines were lower, in the fatal than those of non-fatal CAP cases. Our findings suggest that lipid metabolites are potential diagnostic and prognostic biomarkers for CAP.

Lipid mediators of inflammation as novel plasma biomarkers to identify patients with bacteremia.

OBJECTIVES: Rapid diagnostic tests for bacteremia are important for early treatment to improve clinical outcome. We sought to identify plasma biomarkers that can identify patients with bacteremia using an untargeted global metabolomic analysis. METHODS: Plasma metabolomic profiles were analyzed for 145 adult patients with (cases) and without (controls) bacteremia using ultra-high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). All metabolites were compared between cases and controls using a 2-tier filtering approach, and each metabolite underwent receiver operating characteristic (ROC) curve analysis. Individual metabolites that distinguish between cases and controls were characterized. Subgroup analysis was performed to identify metabolites with prognostic significance. RESULTS: After 2-tier filtering, 128 molecular features were identified to be potential biomarkers that could distinguish cases from controls. Five metabolites had an area under the ROC curve (AUC) of >0.8 in ROC curve analysis, including a sphingolipid, an acylcarnitine, a fatty acid ester, and 2 glycerophosphocholines. These metabolites could distinguish cases from controls in the unsupervised hierarchical clustering analysis. Subgroup analysis of bacteremic patients showed that the level of trans-2,3,4-trimethoxycinnamate was lower in fatal than non-fatal cases. CONCLUSIONS: Plasma lipid mediators of inflammation can distinguish bacteremia cases from non-bacteremia controls. These biomarkers may be used as targets for rapid test in clinical practice.

Two rare ophiocordycipitaceous fungi newly recorded in Taiwan.

BACKGROUND: Ophiocordycipitaceae is a highly diverse fungal family parasitizing a wide range of arthropods and hypogeous fungi. We collected two ophiocordycipitaceous species previously unknown in Taiwan: one emerged from hypogeous fruiting bodies of an Elaphomyces fungus and the other was associated with dragonflies. RESULTS: Based on gross morphology, microscopic features, ITS sequences, and hosts, the two ophiocordycipitaceous fungi were identified as Tolypocladium japonicum and Ophiocordyceps odonatae. We isolated axenic cultures of these two fungi, and their anamorphs were obtained. The simplicillium-like anamorph of T. japonicum is described herein for the first time. The anamorph of O. odonatae produce conidia holoblastically in sympodial sequence and is assignable to Hymenostilbe. A dichotomous key to the species of Ophiocordycipitaceae reported in Taiwan is provided. CONCLUSION: A thorough literature study indicates that the two fungi reported herein have rarely been collected. Our identifications of T. japonicum and O. odonatae agree well with descriptions in the literature and are highly supported by DNA sequence analysis.

Backbone and side-chain ¹H, ¹³C and ¹5N assignments of the PPIase domain of macrophage infectivity potentiator (Mip) protein from Coxiella burnetii.

Coxiella burnetii is an obligate intracellular gram-negative bacterium uniquely evolved to thrive in the inhospitable phagolysosome of macrophage. C. burnetii causes Q fever in humans and animals, which is emerging as a global public health concern. It is highly infectious and designated as a category B biowarfare agent because of its ubiquitous nature, abundant natural reservoirs, high resistance to environmental conditions, ease of transmission and low infectious dose. The lack of knowledge and awareness of C. burnetii leads to under-reporting and under-diagnosing of Q fever cases. Therefore, further understanding of the interactions between the infected host and the bacteria is necessary. C. burnetii macrophage infectivity potentiator (cb-Mip) is a secreted protein of 230 amino acids involving in intracellular survival of the pathogen. cb-Mip belongs to the family of FK506 binding protein, which possesses peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a PPIase, Mip protein homolog has been identified as virulence factor of many intracellular pathogenic microorganisms. In the present study, we report the near complete resonance assignments of the PPIase domain-containing region of Mip protein of C. burnetii. Secondary structure prediction based on chemical shift index analysis indicates that the protein adopts a predominately beta-strand structure, which is consistent with the crystal structure of homologous Mip protein in Legionella pneumophila.