Background Of Host Research Articles

Transcriptome analysis of Bipolaris sorokiniana - Hordeum vulgare provides insights into mechanisms of host-pathogen interaction.

Spot blotch disease incited by Bipolaris sorokiniana severely affects the cultivation of barley. The resistance to B. sorokiniana is quantitative in nature and its interaction with the host is highly complex which necessitates in-depth molecular analysis. Thus, the study aimed to conduct the transcriptome analysis to decipher the mechanisms and pathways involved in interactions between barley and B. sorokiniana in both the resistant (EC0328964) and susceptible (EC0578292) genotypes using the RNA Seq approach. In the resistant genotype, 6,283 genes of Hordeum vulgare were differentially expressed out of which 5,567 genes were upregulated and 716 genes were downregulated. 1,158 genes of Hordeum vulgare were differentially expressed in the susceptible genotype, out of which 654 genes were upregulated and 504 genes were downregulated. Several defense-related genes like resistant gene analogs (RGAs), disease resistance protein RPM1, pathogenesis-related protein PRB1-2-like, pathogenesis-related protein 1, thaumatin-like protein PWIR2 and defensin Tm-AMP-D1.2 were highly expressed exclusively in resistant genotype only. The pathways involved in the metabolism and biosynthesis of secondary metabolites were the most prominently represented pathways in both the resistant and susceptible genotypes. However, pathways involved in MAPK signaling, plant-pathogen interaction, and plant hormone signal transduction were highly enriched in resistant genotype. Further, a higher number of pathogenicity genes of B. sorokiniana was found in response to the susceptible genotype. The pathways encoding for metabolism, biosynthesis of secondary metabolites, ABC transporters, and ubiquitin-mediated proteolysis were highly expressed in susceptible genotype in response to the pathogen. 14 and 11 genes of B. sorokiniana were identified as candidate effectors from susceptible and resistant host backgrounds, respectively. This investigation will offer valuable insights in unraveling the complex mechanisms involved in barley- B. sorokiniana interaction.

Wolbachia and Asaia Distribution among Different Mosquito Vectors Is Affected by Tissue Localization and Host Species.

Microbial communities play an important role in the fitness of mosquito hosts. However, the factors shaping microbial communities in wild populations, with regard to interactions among microbial species, are still largely unknown. Previous research has demonstrated that two of the most studied mosquito symbionts, the bacteria Wolbachia and Asaia, seem to compete or not compete, depending on the genetic background of the reference mosquito host. The large diversity of Wolbachia-Asaia strain combinations that infect natural populations of mosquitoes may offer a relevant opportunity to select suitable phenotypes for the suppression of pathogen transmission and for the manipulation of host reproduction. We surveyed Wolbachia and Asaia in 44 mosquito populations belonging to 11 different species of the genera Anopheles, Aedes, and Culex using qualitative PCR. Through quantitative PCR, the amounts of both bacteria were assessed in different mosquito organs, and through metagenomics, we determined the microbiota compositions in some selected mosquito populations. We show that variation in microbial community structure is likely associated with the species/strain of mosquito, its geographical position, and tissue localization. Together, our results shed light on the interactions among different bacterial species in the microbial communities of mosquito vectors, and this can aid the development and/or improvement of methods for symbiotic control of insect vectors.

Strengthening Bordetella pertussis genomic surveillance by direct sequencing of residual positive specimens.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.

An integrated method for targeted Oxford Nanopore sequencing and automated bioinformatics for the simultaneous detection of bacteria, fungi, and ARG.

The use of metagenomics for pathogen identification in clinical practice has been limited. Here we describe a workflow to encourage the clinical utility and potential of NGS for the screening of bacteria, fungi, and antimicrobial resistance genes (ARGs). The method includes target enrichment, long-read sequencing, and automated bioinformatics. Evaluation of several tools and databases was undertaken across standard organisms (n=12), clinical isolates (n=114), and blood samples from patients with suspected bloodstream infections (n=33). The strategy used could offset the presence of host background DNA, error rates of long-read sequencing, and provide accurate and reproducible detection of pathogens. Eleven targets could be successfully tested in a single assay. Organisms could be confidently identified considering≥60% of best hits of a BLAST-based threshold of e-value 0.001 and a percent identity of>80%. For ARGs, reads with percent identity of>90% and>60% overlap of the complete gene could be confidently annotated. A kappa of 0.83 was observed compared to standard diagnostic methods. Thus, a workflow for the direct-from-sample, on-site sequencing combined with automated genomics was demonstrated to be reproducible. NGS-based technologies overcome several limitations of current day diagnostics. Highly sensitive and comprehensive methods of pathogen screening are the need of the hour. We developed a framework for reliable, on-site, screening of pathogens.

Somatic mouse models of gastric cancer reveal genotype-specific features of metastatic disease.

Metastatic gastric carcinoma is a highly lethal cancer that responds poorly to conventional and molecularly targeted therapies. Despite its clinical relevance, the mechanisms underlying the behavior and therapeutic response of this disease are poorly understood owing, in part, to a paucity of tractable models. Here we developed methods to somatically introduce different oncogenic lesions directly into the murine gastric epithelium. Genotypic configurations observed in patients produced metastatic gastric cancers that recapitulated the histological, molecular and clinical features of all nonviral molecular subtypes of the human disease. Applying this platform to both wild-type and immunodeficient mice revealed previously unappreciated links between the genotype, organotropism and immune surveillance of metastatic cells, which produced distinct patterns of metastasis that were mirrored in patients. Our results establish a highly portable platform for generating autochthonous cancer models with flexible genotypes and host backgrounds, which can unravel mechanisms of gastric tumorigenesis or test new therapeutic concepts.

Antibody Isolation in C. neoformans.

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

Parallel evolution and enhanced virulence upon in vivo passage of an RNA virus in Drosophila melanogaster.

Virus evolution is strongly affected by antagonistic co-evolution of virus and host. Host immunity positively selects for viruses that evade the immune response, which in turn may drive counter-adaptations in host immune genes. We investigated how host immune pressure shapes virus populations, using the fruit fly Drosophila melanogaster and its natural pathogen Drosophila C virus (DCV), as a model. We performed an experimental evolution study in which DCV was serially passaged for ten generations in three fly genotypes differing in their antiviral RNAi response: wild-type flies and flies in which the endonuclease gene Dicer-2 was either overexpressed or inactivated. All evolved virus populations replicated more efficiently in vivo and were more virulent than the parental stock. The number of polymorphisms increased in all three host genotypes with passage number, which was most pronounced in Dicer-2 knockout flies. Mutational analysis showed strong parallel evolution, as mutations accumulated in a specific region of the VP3 capsid protein in every lineage in a host genotype-independent manner. The parental tyrosine at position ninety-five of VP3 was substituted with either one of five different amino acids in fourteen out of fifteen lineages. However, no consistent amino acid changes were observed in the viral RNAi suppressor gene 1A, nor elsewhere in the genome in any of the host backgrounds. Our study indicates that the RNAi response restricts the sequence space that can be explored by viral populations. Moreover, our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.

Heterologous expression of a fully active Azotobacter vinelandii nitrogenase Fe protein in Escherichia coli.

The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the "clean" genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

Whole genome sequencing reveals insights into hepatitis E virus genome diversity, and virus compartmentalization in chronic hepatitis E.

Hepatitis E virus (HEV) is a leading cause of acute hepatitis and can cause chronic infections in immunocompromised patients. Although HEV infections can be treated with ribavirin, antiviral efficacy is hampered by resistance mutations, normally detected by virus sequencing. High-throughput sequencing (HTS) allows for cost-effective complete viral genome sequencing. This enables the discovery and delineation of new subtypes, and revised the recognition of quasispecies and putative resistance mutations. However, HTS is challenged by factors including low viral load, sample degradation, high host background, and high viral diversity. We apply complete genome sequencing strategies for HEV, including a targeted enrichment approach. These approaches were used to investigate sequence diversity in HEV RNA-positive animal and human samples and intra-host diversity in a chronically infected patient. Here, we describe the identification of potential novel subtypes in a blood donation (genotype 3) and in an ancient livestock sample (genotype 7). In a chronically infected patient, we successfully investigated intra-host virus diversity, including the presence of ribavirin resistance mutations. Furthermore, we found convincing evidence for HEV compartmentalization, including the central nervous system, in this patient. Targeted enrichment of viral sequences enables the generation of complete genome sequences from a variety of difficult sample materials. Moreover, it enables the generation of greater sequence coverage allowing more advanced analyses. This is key for a better understanding of virus diversity. Investigation of existing ribavirin resistance, in the context of minorities or compartmentalization, may be critical in treatment strategies of HEV patients.

Host Genetic Background Effect on Vertical Seed Transmission of Epichloë Endophyte Strains in Tall Fescue.

Tall fescue (Lolium arundinaceum (Schreb.) Darbysh.) is a cool-season perennial grass widely grown for forage and turf. Tall fescue lives in association with a fungal endophyte that helps the grass overcome abiotic and biotic stressors. The endophyte is asexual and transmits vertically from the tall fescue plant to the next generation through the seed. Producers of endophyte-infected tall fescue must have endophyte infection in at least 70% of their seed. Therefore, endophyte seed transmission is vital in breeding and seed production. Transfer of endophytes from their native host to different backgrounds of elite tall fescue cultivars can lead to a low seed transmission of the endophyte to the seed. This study screened 23 previously uncharacterized endophyte strains for transmissibility when artificially inoculated into continental and Mediterranean-type host tall fescue. We found no correlation between the rate of successful inoculation and the seed transmission rate of the endophyte in the new host. Nor did the seed transmission rate of the endophyte strains in their native host correlate with the seed transmission rate of the endophyte in the new host. Five strains exhibited seed transmission above 70% in both Mediterranean and Continental host backgrounds and will be characterized further for potential use in cultivar development.

Understanding Autologous Spliceostatin Transcriptional Regulation to Derive Parts for Heterologous Expression in a Burkholderia Bacterial Host.

Burkholderia β-Proteobacteria are emerging sources of natural products. We are interested in developing Burkholderia sp. FERM BP-3421 into a synthetic biology chassis to facilitate natural product discovery. FERM BP-3421 produces autologous spliceostatins on gram per liter scale. We reasoned that transcription factors and promoters that regulate spliceostatin biosynthesis would provide valuable parts for heterologous expression. Herein we demonstrate that fr9A encodes a pathway-specific transcriptional activator of spliceostatin biosynthesis. In-frame deletion of fr9A abolished spliceostatin production, which was restored by genetic complementation. Using transcriptomics and green fluorescent protein (GFP) reporter assays, we identified four fr9 promoters, three of which are activated by LuxR-type regulator Fr9A. We then constructed an Fr9A-regulated promoter system that was compared to benchmarks and effectively applied for GFP and capistruin lasso peptide expression in an optimized host background. Our findings enrich the genetic toolbox for optimizing heterologous expression and promoting the discovery and development of natural products from Burkholderia bacteria.

Clinical evaluation of cell-free and cellular metagenomic next-generation sequencing of infected body fluids

IntroductionPrevious studies have evaluated metagenomic next-generation sequencing (mNGS) of cell-free DNA (cfDNA) for pathogen detection in blood and body fluid samples. However, no study has assessed the diagnostic efficacy of mNGS using cellular DNA. ObjectivesThis is the first study to systematically evaluate the efficacy of cfDNA and cellular DNA mNGS for pathogen detection. MethodsA panel of seven microorganisms was used to compare cfDNA and cellular DNA mNGS assays concerning limits of detection (LoD), linearity, robustness to interference, and precision. In total, 248 specimens were collected between December 2020 and December 2021. The medical records of all the patients were reviewed. These specimens were analysed using cfDNA and cellular DNA mNGS assays, and the mNGS results were confirmed using viral qPCR, 16S rRNA, and internal transcribed spacer (ITS) amplicon next-generation sequencing. ResultsThe LoD of cfDNA and cellular DNA mNGS was 9.3 to 149 genome equivalents (GE)/mL and 27 to 466 colony-forming units (CFU)/mL, respectively. The intra- and inter-assay reproducibility of cfDNA and cellular DNA mNGS was 100%. Clinical evaluation revealed that cfDNA mNGS was good at detecting the virus in blood samples (receiver operating characteristic (ROC) area under the curve (AUC), 0.9814). In contrast, the performance of cellular DNA mNGS was better than that of cfDNA mNGS in high host background samples. Overall, the diagnostic efficacy of cfDNA combined with cellular DNA mNGS (ROC AUC, 0.8583) was higher than that of cfDNA (ROC AUC, 0.8041) or cellular DNA alone (ROC AUC, 0.7545). ConclusionOverall, cfDNA mNGS is good for detecting viruses, and cellular DNA mNGS is suitable for high host background samples. The diagnostic efficacy was higher when cfDNA and cellular DNA mNGS were combined.

Dynamic changes in thymic sub-populations during acute and long-term infections with virulent and virulence-attenuated Salmonella Typhimurium strains in C57BL/6 and autoimmune-prone lpr mice

SalmonellaTyphimurium infection in mice results in drastic loss of immature CD4− CD8− double negative (DN) and CD4+ CD8+ double positive (DP) thymic subsets compared to mature single positive (SP) subsets. We investigated changes in thymocyte sub-populations post infection with a wild type (WT) virulent strain and ΔrpoS, a virulence-attenuated strain, of Salmonella Typhimurium in C57BL/6 (B6) and Fas-deficient autoimmune-prone lpr mice. The WT strain caused acute thymic atrophy with greater loss of thymocytes in lpr mice compared to B6 mice. Infection with ΔrpoS caused progressive thymic atrophy in B6 and lpr mice. Analysis of thymocyte subsets revealed that immature thymocytes including the DN, immature single positive (ISP), and DP thymocytes underwent extensive loss. SP thymocytes were more resistant to loss in WT-infected B6 mice, whereas WT-infected lpr and ΔrpoS-infected mice exhibited depletion of SP thymocytes. Overall, thymocyte sub-populations exhibited differential susceptibilities depending on bacterial virulence and the host background.

Development and Application of Whole-Cell Biosensors for the Detection of Gallic Acid.

Gallic acid is a prevalent secondary plant metabolite distinguished as one of the most effective free-radical scavengers among phenolic acids. This compound is also known for its cytotoxic, anti-inflammatory, and antimicrobial activities. Bulk quantities of gallic acid are conventionally produced by acid hydrolysis of tannins, a costly and environmentally hazardous process. With the aim to develop more sustainable approaches, microbial bioproduction strategies have been attempted recently. To advance synthetic biology and metabolic engineering of microorganisms for gallic acid production, we characterize here a transcription factor-based inducible system PpGalR/PPP_RS13150 that responds to the extracellular gallic acid in a dose-dependent manner in Pseudomonas putida KT2440. Surprisingly, this compound does not mediate induction when PpGalR/PPP_RS13150 is used in non-native host background. We show that the activation of the inducible system requires gallate dioxygenase activity encoded by galA gene. The 4-oxalomesaconic acid, an intermediate of gallic acid-metabolism, is identified as the effector molecule that interacts with the transcription factor GalR mediating activation of gene expression. Introduction of galA gene along galR enables development of biosensors suitable for detection and monitoring of gallic acid extracellularly using non-native hosts such as E. coli and C. necator. Moreover, the P. putida-based biosensor's applicability is demonstrated by detecting and measuring gallic acid in extracts of Camellia sinensis leaves. This study reports the strategy, which can be applied for developing gallic acid biosensors using bacterial species outside Pseudomonas genus.

548. Target Enriched NGS Reveals Wide Breadth of Viruses Causing Acute Undifferentiated Fever in Thailand

Abstract Background Despite extensive laboratory testing, infectious agents were not detected in approximately 50% of patients hospitalized for acute undifferentiated febrile illnesses (AUFI) at Siriraj Hospital, Bangkok, Thailand. Unbiased Metagenomic Next Generation Sequencing (mNGS) enables detection of any microbe present in patient samples. Target enrichment for viruses represents a highly sensitive and cost-effective approach for overcoming host background in clinical specimens. Plasma collected from 800 patients with undiagnosed AUFI between 2013-2020 were analyzed by mNGS coupled to target enrichment to identify known and novel viruses. Methods Plasma was pre-treated with benzonase before extraction on an Abbott m2000 instrument. mNGS libraries were prepared from double-stranded cDNA with Illumina Nextera XT reagents on an epMotion then combined in pools of 24 and hybridized to Comprehensive Viral Research Panel (CVRP; Twist Biosciences) probes covering &amp;gt;15,000 strains of vertebrate viruses. Captured viral sequences were amplified, quantified, and sequenced together on a MiSeq. Reads were taxonomically classified by the SURPI pipeline and aligned in CLC Bio Genomics Workbench software. Results CVRP method optimization enabled sequencing of 24-48 libraries per MiSeq run, for which &amp;gt;50% genome coverage was obtained with model viruses spiked into clinical specimens at 1000 cp/ml. This approach revealed an array of &amp;gt;24 different viruses found in 26% of samples. Dengue was the most prevalent at 7.4%, with all four genotypes detected. Other common causes of AUFI such as Chikungunya, HIV-1, HAV, HBV, HCV, and CMV were present in 1.5-2.5% of cases. Less prevalent (&amp;lt; 1%) infections included HEV, HSV-2, EBV, HHV-6, Enterovirus B&amp;D and Parvovirus B19. We also encountered sporadic cases of Measles, Cardiovirus, West Nile, Rotavirus, Picobirnavirus, Polyomavirus 4&amp;5, Kubovirus, and Rabies lyssavirus. Conclusion Target capture successfully demonstrated that viruses were important etiologies for unresolved cases of AUFI in Thailand. This data has led to a better understanding of the epidemiology of this clinical syndrome and has implications for proper management of AUFI, including lower rates of unnecessary testing and antimicrobial use. Disclosures Julie Yamaguchi, BS, Abbott Labs: Employee Michael G. Berg, PhD, Abbott Labs: Employee Pakpoom Phoompoung, MD, Abbott Labs: Grant/Research Support|Abbott Labs: Employee Jenna Malinauskas, MS, Abbott Labs: Employee Gavin Cloherty, PhD, Abbott: Employee|Abbott: Stocks/Bonds|Abbott Labs: Employee Yupin Suputtamongkol, MD, Abbott Labs: Grant/Research Support.

Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction.

The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.

A Personal Perspective on My Scientific Career

A Personal Perspective on My Scientific Career

The two-component regulatory system CenK-CenR regulates expression of a previously uncharacterized protein required for salinity and oxidative stress tolerance in Sinorhizobium meliloti.

Genes of unknown function constitute a considerable fraction of most bacterial genomes. In a Tn5-based search for stress response genes in the nitrogen-fixing facultative endosymbiont Sinorhizobium (Ensifer) meliloti, we identified a previously uncharacterized gene required for growth on solid media with increased NaCl concentrations. The encoded protein carries a predicted thioredoxin fold and deletion of the gene also results in increased sensitivity to hydrogen peroxide and cumene hydroperoxide. We have designated the gene srlA (stress resistance locus A) based on these phenotypes. A deletion mutant yields phenotypic revertants on high salt medium and genome sequencing revealed that all revertants carry a mutation in genes homologous to either cenK or cenR. srlA promoter activity is abolished in these revertant host backgrounds and in a strain carrying a deletion in cenK. We also observed that the srlA promoter is autoregulated, displaying low activity in a wildtype (wt) host background and high activity in the srl deletion mutant background. The srlA promoter includes a conserved inverted repeat directly upstream of the predicted −35 subsequence. A mutational analysis demonstrated that the site is required for the high promoter activity in the srlA deletion background. Electromobility shift assays using purified wildtype CenR response regulator and a D55E phosphomimetic derivative suggest this protein acts as a likely Class II activator by binding promoter DNA. These results document the first identified CenK–CenR regulon member in S. meliloti and demonstrate this two-component regulatory system and gene srlA influences cellular growth and persistence under certain stress-inducing conditions.

S1.3c Diversity and hybridization in Malassezia furfur

S1.3 Malassezia: genetics, genomics, and biology, September 21, 2022, 11:00 AM - 12:30 PM The Basidiomycetous yeast Malassezia is the most abundant fungal genus on healthy human skin but may also cause various skin disorders such as seborrheic dermatitis, dandruff, and pityriasis versicolor. In recent years, Malassezia has increasingly been implicated in health and disease beyond the skin: as an underestimated cause of Malassezia bloodstream infections (BSIs) in immunocompromised patients and neonates, associated with Crohn's disease, promoting pancreatic oncogenesis, and exacerbating cystic fibrosis. Malassezia furfur is the number one Malassezia BSI cause and is also implicated in many skin disorders. With these new discoveries of Malassezia’s impact on human health, the need for a better understanding of its evolution and pathobiology also became more pressing. Hybridization has been suggested as a biological mechanism of adaptation to new hosts, and may lead to increased pathogenicity. Many examples of major hybrid yeast pathogens exist, such as Candida albicans, C. orthopsilosis, C. metapsilosis, and multiple examples in the Cryptococcus gattii/Cryptococcus neoformans species complex. Here the multiple hybridization events of the Malassezia furfur species complex will be discussed. Two distinct hybridization events occurred between the same parental lineages, and these parental strains were originally also hybrids. The identification of a pseudobipolar mating system and the analysis of the mating-type loci provide evidence that sexual liaisons of mating compatible cells from these parental lineages led to a diploid/aneuploid state in the hybrid lineages. Sequence similarity percentages suggest that both parental lineages in fact are two different species. The genetic diversity of ca 300 strains belonging to this species complex is evaluated in relationship to host background and phenotype.

Identification and validation of a T-cell-related MIR600HG/hsa-mir-21-5p competing endogenous RNA network in tuberculosis activation based on integrated bioinformatics approaches.

Background: T cells play critical roles in the progression of tuberculosis (TB); however, knowledge regarding these molecular mechanisms remains inadequate. This study constructed a critical ceRNA network was constructed to identify the potentially important role of TB activation via T-cell regulation. Methods: We performed integrated bioinformatics analysis in a randomly selected training set from the GSE37250 dataset. After estimating the abundance of 18 types of T cells using ImmuCellAI, critical T-cell subsets were determined by their diagnostic accuracy in distinguishing active from latent TB. We then identified the critical genes associated with T-cell subsets in TB activation through co-expression analysis and PPI network prediction. Then, the ceRNA network was constructed based on RNA complementarity detection on the DIANA-LncBase and mirDIP platform. The gene biomarkers included in the ceRNA network were lncRNA, miRNA, and targeting mRNA. We then applied an elastic net regression model to develop a diagnostic classifier to assess the significance of the gene biomarkers in clinical applications. Internal and external validations were performed to assess the repeatability and generalizability. Results: We identified CD4+ T, Tr1, nTreg, iTreg, and Tfh as T cells critical for TB activation. A ceRNA network mediated by the MIR600HG/hsa-mir-21-5p axis was constructed, in which the significant gene cluster regulated the critical T subsets in TB activation. MIR600HG, hsa-mir-21-5p, and five targeting mRNAs (BCL11B, ETS1, EPHA4, KLF12, and KMT2A) were identified as gene biomarkers. The elastic net diagnostic classifier accurately distinguished active TB from latent. The validation analysis confirmed that our findings had high generalizability in different host background cases. Conclusion: The findings of this study provided novel insight into the underlying mechanisms of TB activation and identifying prospective biomarkers for clinical applications.