Cryptic Gene Research Articles

Pepticinnamins N, O, and P, Nonribosomal Peptides from the Soil-Derived Streptomyces mirabilis P8-A2.

Cinnamoyl moiety containing nonribosomal peptides represented by pepticinnamin E are a growing family of natural products isolated from different Streptomyces species and possess diverse bioactivities. The soil bacterium Streptomyces mirabilis P8-A2 harbors a cryptic pepticinnamin biosynthetic gene cluster, producing azodyrecins as major products. Inactivation of the azodyrecin biosynthetic gene cluster by CRISPR-BEST base editing led to the activation and production of pepticinnamin E (1) and its analogues, pepticinnamins N, O, and P (2-4), the structures of which were determined by detailed NMR spectroscopy, HRMS data, and Marfey's reactions. These new compounds did not show a growth inhibitory effect against the LNCaP and C4-2B prostate cancer lines, respectively.

Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus.

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.IMPORTANCEThere is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20-60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

The application of targeted RNA sequencing for the analysis of fusion genes, gene mutations, IKZF1 intragenic deletion, and CRLF2 overexpression in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is characterized by highly genetic heterogeneity, owing to recurrent fusion genes, gene mutations, intragenic deletion, and gene overexpression, which poses significant challenges in clinical detection. RNA sequencing (RNA-seq) is a powerful tool for detecting multiple genetic abnormalities, especially cryptic gene rearrangements, in a single test. Sixty samples (B-ALL, n = 49; T-ALL, n = 9; mixed phenotype acute leukemia (MPAL), n = 2) and 20 controls were analyzed by targeted RNA-seq panel of 507 genes developed by our lab. Of these, 16 patients were simultaneously analyzed for gene mutations at the DNA level using a next-generation sequencing panel of 51 genes. Fusion genes, CRLF2 expression, and IKZF1 intragenic deletion were also detected by reverse transcription-polymerase chain reaction (RT-PCR). Karyotype analysis was performed using the R-banding and G-banding technique on bone marrow cells after 24 hours of culture. Partial fusion genes were analyzed using fluorescence in situ hybridization (FISH). Compared with the results of Karyotype analysis, FISH, and RT-PCR, the detection rate of fusion genes by targeted RNA-seq increased from 48.3% to 58.3%, and six unexpected fusion genes were discovered, along with one rare isoform of IKZF1 intragenic deletion (IK10). The DNA sequencing analysis of 16 ALL patients revealed that 96.2% (25/26) of gene mutations identified at the DNA level were also detectable at the RNA level, except for one mutation with a low variant allele fraction. The detection of CRLF2 overexpression exhibited complete concordance between RT-PCR and RNA-seq. The utilization of RNA-seq enables the identification of clinically significant genetic abnormalities that may go undetected through conventional detection methods. Its robust analytical performance might bring great application value for clinical diagnosis, prognosis, and therapy in ALL.

Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Evolution-Based Discovery of Polyketide Acylated Valine from a Cytochalasin-Like Gene Cluster in Simplicillium lamelliciola HDN13430.

Utilizing a gene evolution-oriented approach for gene cluster mining, a cryptic cytochalasin-like gene cluster (sla) in Antarctic-derived Simplicillium lamelliciola HDN13430 was identified. Compared with the canonical cytochalasin biosynthetic gene clusters (BGCs), the sla gene cluster lacks the key α,β-hydrolase gene. Heterologous expression of the sla gene cluster led to the discovery of a new compound, slamysin (1), characterized by an N-acylated amino acid structure and demonstrating weak anti-Bacillus cereus activity. These findings underscore the potential of genetic evolution in uncovering novel compounds and indicating specific adaptive evolution within specialized habitats.

Molecular Identification of Chlamydia trachomatis among Infertile Females

Chlamydia trachomatis is one among the sexually transmitted diseases causing genital tract infection frequently associated with complications of infertility. The aim of our study is to detect the presence of C. trachomatis infection (CTI) in female infertility by nested Polymerase Chain Reaction (PCR) in a tertiary care center. A cross-sectional study was done with 230 infertile women attending the OPD of Reproductive Medicine and Surgery. CTI was detected among the study participants by screening for momp and cryptic plasmid gene using nested PCR. Based on the history and clinical presentation, the enrolled patients categorized as primary and secondary infertility. The results of the nested PCR for the primary and secondary infertile women were tabulated and compared for the statistical significance using Epi info version 7 and Chi-square test. A p-value of < 0.05 considered significant. In the study, participants 1 (20%) was primary and 4 (80%) belonged to secondary infertility. Of the 230 infertile women screened 2.2% of them had PCR positive for either momp or cryptic plasmid gene. CTI was seen more (80%) in secondary infertile than in primary infertile women. CTI was seen more in the age group 26-30 years (60%) followed by 21-25 years and 31-35 years (20%). The results of our study showed CTI is associated with infertility and recurrent spontaneous abortion. It’s imperative to screen for CTI by molecular method in young females which necessitates early therapy and prevention of long term complications like infertility.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from Marine Cyanobacterium Moorena producens JHB Discovered via Coculture with Candida albicans.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies on this strain led to the discovery of several novel compounds such as hectochlorins and jamaicamides. However, bioinformatic analyses of its genome indicate the presence of numerous cryptic biosynthetic gene clusters that have yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was cocultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that coculture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria.

P450-Modified Multicyclic Cyclophane-Containing Ribosomally Synthesized and Post-Translationally Modified Peptides.

Cyclic peptides with cyclophane linkers are an attractive compound type owing to the fine-tuned rigid three-dimensional structures and unusual biophysical features. Cytochrome P450 enzymes are capable of catalyzing not only the C-C and C-O oxidative coupling reactions found in vancomycin and other nonribosomal peptides (NRPs), but they also exhibit novel catalytic activities to generate cyclic ribosomally synthesized and post-translationally modified peptides (RiPPs) through cyclophane linkage. To discover more P450-modified multicyclic RiPPs, we set out to find cryptic and unknown P450-modified RiPP biosynthetic gene clusters (BGCs) through genome mining. Synergized bioinformatic analysis reveals that P450-modified RiPP BGCs are broadly distributed in bacteria and can be classified into 11 classes. Focusing on two classes of P450-modified RiPP BGCs where precursor peptides contain multiple conserved aromatic amino acid residues, we characterized 11 novel P450-modified multicyclic RiPPs with different cyclophane linkers through heterologous expression. Further mutation of the key ring-forming residues and combinatorial biosynthesis study revealed the order of bond formation and the specificity of P450s. This study reveals the functional diversity of P450 enzymes involved in the cyclophane-containing RiPPs and indicates that P450 enzymes are promising tools for rapidly obtaining structurally diverse cyclic peptide derivatives.

Analysis of the cryptic biosynthetic gene cluster encoding the RiPP curacozole reveals a phenylalanine-specific peptide hydroxylase

Curacozole belongs to an unusual cyanobactin-like sub-family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The molecule is distinguished by its small, macrocyclic structure, a poly-azole sequence that includes a...

Synthetic-bioinformatic natural product-inspired peptides.

Covering: 2016 to 2024Natural products, particularly cyclic peptides, are a promising source of bioactive compounds. Nonribosomal peptide synthetases (NRPSs) play a key role in biosynthesizing these compounds, which include antibiotic and anticancer agents, immunosuppressants, and others. Traditional methods of discovering natural products have limitations including cryptic biosynthetic gene clusters (BGCs), low titers, and currently unculturable organisms. This has prompted the exploration of alternative approaches. Synthetic-bioinformatic natural products (syn-BNPs) are one such alternative that utilizes bioinformatics techniques to predict nonribosomal peptides (NRPs) followed by chemical synthesis of the predicted peptides. This approach has shown promise, resulting in the discovery of a variety of bioactive compounds including peptides with antibacterial, antifungal, anticancer, and proteasome-stimulating activities. Despite the success of this approach, challenges remain especially in the accurate prediction of fatty acid incorporation, tailoring enzyme modifications, and peptide release mechanisms. Further work in these areas will enable the discovery of many bioactive peptides that are currently inaccessible.

Untargeted Metabolomics to Investigate the Influence of Epigenetic Modifiers on the Metabolism of Fusarium verticillioides.

Toxigenic fungi are capable of producing toxic metabolites, called mycotoxins. But the presence of silent and lowly expressed genes represents the main challenge for the discovery of novel mycotoxins, especially their lesser-known forms, commonly referred to as "emerging mycotoxins." Epigenetic modifiers (EMs) are compounds that are able to alter the production of metabolites through the induction of silent biosynthetic pathways leading to an enhanced chemical diversity. The aim of this study was to assess the effects of different chemical modulators on the metabolic profiles of the well-known toxigenic fungal species, Fusarium verticillioides. Four EMs, 5-azacytidine, sodium butyrate, nicotinamide (NIC), and sodium valproate (SV), were used. Following their addition to Fusarium verticillioides cultures, the metabolic profiles were analyzed by using UHPLC-HRMS/MS under targeted and untargeted metabolomics approaches. Metabolites were putatively annotated through the use of MS-DIAL and MS-FINDER. Our results show that the treatment with SV induced the most important alteration of the secondary metabolic profile of F. verticillioides, by promoting the expression of cryptic genes. Among the 50 most discriminating metabolites across five culture conditions, 12 were fusarins or fusarin analogs. In contrast, SB and NIC had little impact on these metabolites. The study highlights SV's ability to alter gene expression by inhibiting DNA deacetylation in fungal strains. This research could have significant implications for agriculture and food industry, especially in regions facing major mycotoxin challenges.

Panel-based RNA fusion sequencing improves diagnostics of pediatric acute myeloid leukemia

New methods like panel-based RNA fusion sequencing (RNA-FS) promise improved diagnostics in various malignancies. We here analyzed the impact of RNA-FS on the initial diagnostics of 241 cases with pediatric acute myeloid leukemia (AML). We show that, compared to classical cytogenetics (CCG), RNA-FS reliably detected risk-relevant fusion genes in pediatric AML. In addition, RNA-FS strongly improved the detection of cryptic fusion genes like NUP98::NSD1, KMT2A::MLLT10 and CBFA2T3::GLIS2 and thereby resulted in an improved risk stratification in 25 patients (10.4%). Validation of additionally detected non-risk-relevant high confidence fusion calls identified PIM3::BRD1, C22orf34::BRD1, PSPC1::ZMYM2 and ARHGAP26::NR3C1 as common genetic variants and MYB::GATA1 as recurrent aberration, which we here describe in AML subtypes M0 and M7 for the first time. However, it failed to detect rare cytogenetically confirmed fusion events like MNX1::ETV6 and other chromosome 12p-abnormalities. As add-on benefit, the proportion of patients for whom measurable residual disease (MRD) monitoring became possible was increased by RNA-FS from 44.4 to 75.5% as the information on the fusion transcripts’ sequence allowed the design of new MRD assays.

Assessment of Optical Genome Mapping for Front-Line Diagnostic Evaluation of Acute Leukemia: A Canadian Single-Center Evaluation of Added Yield in 69 Informative Cases

Optical genome mapping (OGM) is a novel method currently being evaluated internationally as a diagnostic assay for genomic profiling of malignancies. OGM uses imaging and reference alignment of fluorescently labeled ultra-long DNA molecules to detect structural and copy number variants genome-wide at significantly higher resolution than is attainable with current standard-of-care (SOC) cytogenetic analysis. OGM is nearing deployment as a first-line diagnostic test for patients with acute leukemia at Vancouver General Hospital, the largest tertiary care hospital in British Columbia. Here, we describe our experience in the evaluation of OGM as a diagnostic assay relative to SOC karyotyping and fluorescence in situ hybridization (FISH) in newly diagnosed and relapsed acute leukemia. A selected cohort of 69 bone marrow specimens produced adequate quality OGM data for analysis: 52 acute myeloid leukemia (AML), 12 B-lymphoblastic leukemia (B-ALL), 4 mixed phenotype acute leukemia (MPAL; 2 B/myeloid, 1 B/T, and 1 T/myeloid), and 1 blast phase (T) myeloid/lymphoid neoplasm with tyrosine kinase gene fusion (M/L-N-Eo). Variants were filtered using population thresholds and with pan-cancer (669 genes) and disease targeted (myeloid: 185; B-lymphoid: 130) gene lists to facilitate detection of variants of interest. OGM results were compared to SOC to identify additional diagnostic yield and changes to risk stratification or diagnosis resulting from OGM findings. In these 69 acute leukemias, filtering to remove common polymorphisms using a manufacturer-provided control dataset yielded a median of 53 variants (interquartile range (IQR) 39-86); using a pan-cancer list yielded a median of 6 (IQR 2-16); and disease-specific lists a median of 4 (IQR 1-10.5). In 11% of cases, diagnosis or risk stratification was altered by OGM (13% of AML; 8% of B-ALL), including a complex karyotype being resolved as normal, downgrading cytogenetic risk; identification of several cryptic KMT2A rearrangements, changing diagnostic classification; and identification of cryptic MECOM::RUNX1 (class-modifying), NUP98::NSD1 (risk-upgrading), and NUP214::ABL1 (potentially targetable and class- and risk-modifying) fusions. In 26% of cases, driver rearrangements were either newly identified by OGM or novel partner genes were resolved (23% of AML; 33% of B-ALL; 40% of others), including recurrent rearrangements of the IGH locus with MIR125B1/ BLID in B-ALL; KMT2A partial tandem duplication; novel MECOM::PAN3, JAKMIP2::PDGFRB, and NUP214::FRMD4B fusions; and a cryptic deletion juxtaposing FLT3 and PAN3 known to result in FLT3 overexpression. Additional findings, including copy number changes and clarifications of complex events, were seen in 54% of cases (44% of AML; 83% of B-ALL; 80% of others), mostly accounted for by small deletions or disruptive rearrangements in key disease genes,likely resulting in loss of function, including but not limited to RUNX1, GATA2 and ETV6 in AML; CDKN2A, PAX5, and RB1 in B-ALL; and TP53 in MPAL. Several cases harboured small MLLT4 copy number abnormalities that have been reported to be linked to cancer predisposition, and one potentially germline FANCA deletion was observed. Details of comparison between SOC methods (karyotyping and pertinent FISH where applicable) are detailed in the Figure and Table (bold text: newly resolved or clarified potential driver or resolution of normal karyotype; underline: change in diagnosis or risk stratification resulting from OGM findings). Notably, OGM failed to identify one hypodiploid B-ALL clone masked by endoreduplication, and further evaluation of this technology's performance in this context and in prospective practice is required. This study demonstrates the significant potential added diagnostic yield of OGM relative to SOC as a clinical diagnostic and research assay. High-resolution genome-wide evaluation not reliant on cell culture substantially expands the scope of detectable variants, for many of which determining the biologic and clinical implications will require larger scale prospective study. Importantly, application of OGM in the clinical setting is likely to result in clinically meaningful disease reclassification and restratification of risk, and in some cases may permit patients access to previously inaccessible targeted therapies (as in the instance of cytogenetically cryptic tyrosine kinase gene fusions).

Detection of Cryptic Gene Fusion and Chimeric RNA Variants in Relapsed/Refractory Acute Myeloid Leukemia Patients Diagnosed with KMT2A/Afdn Chromosomal Translocation

Chromosomal rearrangements, such as translocations, play a significant role in creating chimeric genes and oncofusion proteins initiating leukemogenesis in 52% of acute myeloid leukemia (AML) patients, and are associated with an adverse prognosis. Currently, there are limited targeted therapies available for AML patients with chromosome rearrangements primarily due to a lack of accurate molecular and genetic characterization of the mechanism of leukemic progression along the treatment course. In our study, we investigated the pathological landscape of treatment resistance and relapse in AML patients with KMT2A-rearranged gene fusions (previously known as mixed lineage leukemia, MLL gene, 11q23), which is one of the most common gene rearrangements in AML with a dismal prognosis. Our approach included chromosome analysis, fluorescence in situ hybridization (FISH), transcriptomic analyses (RNA-seq), polymerase chain reaction (PCR), DNA-sequencing and quantitative PCR (qPCR) were applied to monitor RNA- and genome-wide changes among newly diagnosed, refractory and relapsed AML patients. Bone marrow samples of seven AML patients with different chromosome were obtained and two samples were found to possess the chromosomal translocation t(6;11)(q27;q23) which leads to KMT2A/AFDN gene fusion. In these patients, RNA-seq identified a cryptic CCDC32 (15q15.1) /CBX3 (7p15.2) gene fusion in both newly diagnosed and relapsed samples, which is previously unreported in KMT2A/AFDN-rearranged AML patients. Quantitative gene expression measurement by qPCR demonstrated that CCDC32/CBX3 gene fusion significantly affected the expression of wild-type genes of CCDC32 (essential for embryonic development) and CBX3 (an oncogene for solid tumors) during the relapse, suggesting the potential oncogenic role for the CCDC32/CBX3 chimera in AML development. Our result is consistent with a previous study showing multiple breakpoints in AML patients with KMT2A/AFDN translocations. KMT2A rearrangements are known to have an unfavorable prognosis in AML and the specific t(6;11)(q27;23) translocation in AML patients leads to a median overall survival of only 12 months. Our novel findings also suggest that one reason for the adverse outcomes of KMT2A chromosomal rearrangements is their complex ability to generate chimeric variants and more aggressive subclones to elude the treatment. Our study implies that standard cytogenetic analyses and emerging NGS are not sufficient to fully diagnose a patient with chromosome rearrangement. Particularly, short-read RNA-seq presents problems because it cannot detect gene fusions with low expression level, like the KMT2A/AFDN gene fusion that was undetected in patient 7 by our short-read RNA-seq. Instead, long-read RNA-seq, combined with cytogenetics and molecular profiling through PCR/Sanger DNA-sequencing, should be utilized to identify complex fusion transcripts and different chimeric variants of multiple gene fusions.

Cytochrome P450 Mediated Cyclization in Eunicellane Derived Diterpenoid Biosynthesis**

AbstractTerpene cyclization, one of the most complex chemical reactions in nature, is generally catalyzed by two classes of terpene cyclases (TCs). Cytochrome P450s that act as unexpected TC‐like enzymes are known but are very rare. In this study, we genome‐mined a cryptic bacterial terpenoid gene cluster, named ari, from the thermophilic actinomycete strain Amycolatopsis arida. By employing a heterologous production system, we isolated and characterized three highly oxidized eunicellane derived diterpenoids, aridacins A−C (1–3), that possess a 6/7/5‐fused tricyclic scaffold. In vivo and in vitro experiments systematically established a noncanonical two‐step biosynthetic pathway for diterpene skeleton formation. First, a class I TC (AriE) cyclizes geranylgeranyl diphosphate (GGPP) into a 6/10‐fused bicyclic cis‐eunicellane skeleton. Next, a cytochrome P450 (AriF) catalyzes cyclization of the eunicellane skeleton into the 6/7/5‐fused tricyclic scaffold through C2−C6 bond formation. Based on the results of quantum chemical computations, hydrogen abstraction followed by electron transfer coupled to barrierless carbocation ring closure is shown to be a viable mechanism for AriF‐mediated cyclization. The biosynthetic logic of skeleton construction in the aridacins is unprecedented, expanding the catalytic capacity and diversity of P450s and setting the stage to investigate the inherent principles of carbocation generation by P450s in the biosynthesis of terpenoids.

Cytochrome P450 Mediated Cyclization in Eunicellane Derived Diterpenoid Biosynthesis.

Terpene cyclization, one of the most complex chemical reactions in nature, is generally catalyzed by two classes of terpene cyclases (TCs). Cytochrome P450s that act as unexpected TC-like enzymes are known but are very rare. In this study, we genome-mined a cryptic bacterial terpenoid gene cluster, named ari, from the thermophilic actinomycete strain Amycolatopsis arida. By employing a heterologous production system, we isolated and characterized three highly oxidized eunicellane derived diterpenoids, aridacins A-C (1-3), that possess a 6/7/5-fused tricyclic scaffold. In vivo and in vitro experiments systematically established a noncanonical two-step biosynthetic pathway for diterpene skeleton formation. First, a class I TC (AriE) cyclizes geranylgeranyl diphosphate (GGPP) into a 6/10-fused bicyclic cis-eunicellane skeleton. Next, a cytochrome P450 (AriF) catalyzes cyclization of the eunicellane skeleton into the 6/7/5-fused tricyclic scaffold through C2-C6 bond formation. Based on the results of quantum chemical computations, hydrogen abstraction followed by electron transfer coupled to barrierless carbocation ring closure is shown to be a viable mechanism for AriF-mediated cyclization. The biosynthetic logic of skeleton construction in the aridacins is unprecedented, expanding the catalytic capacity and diversity of P450s and setting the stage to investigate the inherent principles of carbocation generation by P450s in the biosynthesis of terpenoids.

Nobachelins, new siderophores from Nocardiopsisbaichengensis protecting Caenorhabditiselegans from Pseudomonasaeruginosa infection

The biosynthetic potential of actinobacteria to produce novel natural products is still regarded as immense. In this paper, we correlated a cryptic biosynthetic gene cluster to chemical molecules by genome mining and chemical analyses, leading to the discovery of a new group of catecholate-hydroxamate siderophores, nobachelins, from Nocardiopsisbaichengensis DSM 44845. Nobachelin biosynthesis genes are conserved in several bacteria from the family Nocardiopsidaceae. Structurally, nobachelins feature fatty-acylated hydroxy-ornithine and a rare chlorinated catecholate group. Intriguingly, nobachelins rescued Caenorhabditiselegans from Pseudomonasaeruginosa-mediated killing.

Acidonemycins A-C, Glycosylated Angucyclines with Antivirulence Activity Produced by the Acidic Culture of Streptomyces indonesiensis.

The genome of Streptomyces indonesiensis is highly enriched with cryptic biosynthetic gene clusters (BGCs). The majority of these cryptic BGCs are transcriptionally silent in normal laboratory culture conditions as determined by transcriptome analysis. When cultured in acidic pH (pH 5.4), this strain has been shown to produce a set of new metabolites that were not observed in cultures of neutral pH (pH 7.4). The organic extract of the acidic culture displayed an antivirulence activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report the structures of new glycosylated aromatic polyketides, named acidonemycins A-C (1-3), belonging to the family of angucyclines. Type II polyketide synthase BGC responsible for the production of 1-3 was identified by a transcriptome comparison between acidic (pH 5.4) and neutral (pH 7.4) cultures and further confirmed by heterologous expression in Streptomyces albus J1074. Of the three new compounds, acidonemycins A and B (1 and 2) displayed antivirulence activity against MRSA. The simultaneous identification of both antivirulent compounds and their BGC provides a starting point for the future effort of combinatorial biosynthesis.

Structural homology screens reveal host-derived poxvirus protein families impacting inflammasome activity

Viruses acquire host genes via horizontal transfer and can express them to manipulate host biology during infections. Some homologs retain sequence identity, but evolutionary divergence can obscure host origins. We use structural modeling to compare vaccinia virus proteins with metazoan proteomes. We identify vaccinia A47L as a homolog of gasdermins, the executioners of pyroptosis. An X-ray crystal structure of A47 confirms this homology, and cell-based assays reveal that A47 interferes with caspase function. We also identify vaccinia C1L as the product of a cryptic gene fusion event coupling a Bcl-2-related fold with a pyrin domain. C1 associates with components of the inflammasome, a cytosolic innate immune sensor involved in pyroptosis, yet paradoxically enhances inflammasome activity, suggesting differential modulation during infections. Our findings demonstrate the increasing power of structural homology screens to reveal proteins with unique combinations of domains that viruses capture from host genes and combine in unique ways.

A Well-Curated Cost-Effective Next-Generation Sequencing Panel Identifies a Diverse Landscape of Pathogenic and Novel Germline Variants in a Bone Marrow Failure Cohort in a Resource-Constraint Setting

Current study is a 4 year experience in diagnosis and screening of inherited and immune bone marrow failure cases using a targeted sequencing panel. A total of 171 cases underwent targeted NGS and were categorized as suspected inherited bone marrow failure syndrome (IBMFS) group (106; 62%) and immune/idiopathic aplastic anemia (IAA) group (65; 38%) based on clinical and laboratory criteria. 110 (64%) were pediatric (0-12 years) and 61 (36%) adolescent and adults (13-47 years). In suspected IBMFS group, 47 (44%) and in IAA group, 8 (12%) revealed a likely germline pathogenic variation. Whole exome sequencing performed in 15/59 suspected IBMFS group cases negative on targeted panel, revealed a clinically important variation in 3 (20%) cases. A total of 11 novel variants were identified. The targeted panel helped establish a diagnosis in 44% (27/61) of unclassified bone marrow failure syndrome cases and also led to amendment of clinical diagnosis in 5 (4.7%) cases. Overall, diagnostic yield of this well curated small panel was comparable to Western studies with larger gene panels. Moreover, this was achievable at a much lower cost, making it suitable for resource constraint settings. In addition, high frequency (>10%) of cryptic pathogenic IBMFS gene variations in IAA cohort suggests routine incorporation of targeted NGS screening in these cases.