Activation of secondary metabolite gene clusters in Chaetomium olivaceum via the deletion of a histone deacetylase.

Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: • Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. • Three polyketides and one asterriquinone were isolated from HDAC deleted strain. • Two different PKSs were reported in C. olivaceum SD-80A.

Podocyte Pathogenic Bone Morphogenetic Protein-2 Pathway and Immune Cell Behaviors in Primary Membranous Nephropathy.

Primary membranous nephropathy (PMN) is one of the leading causes of end-stage renal disease, and the most frequent cause of massive proteinuria in nondiabetic adults, resulting in fatal complications. However, the underlying pathomechanisms of PMN remain largely unclear. Here, single-cell RNA sequencing is employed to analyze kidney biopsies from eleven PMN patients and seven healthy subjects. Profiling 44 060 cells from patients allowed us to characterize the cellular composition and cell-type-specific gene expression in the PMN kidney. The complement-induced BMP2/pSMAD1/COL4 pathway is identified as the pathogenic pathway in podocytes, bridging two key events, i.e., complement system activation and glomerular basement membrane thickening in PMN. Augmented infiltration and activation of myeloid leukocytes and B lymphocytes are found, profiling delicate crosstalk of immune cells in PMN kidneys. Overall, these results provide valuable insights into the roles of podocytes and immune cells in PMN, and comprehensive resources toward the complete understanding of PMN pathophysiology.

Development of Antibacterial Peptides with Membrane Disruption and Folate Pathway Inhibitory Activities against Methicillin-Resistant Staphylococcus aureus.

Antimicrobial peptides (AMPs) offer an opportunity to overcome multidrug resistance. Here, novel peptides were designed based on AMP fragments derived from sea cucumber hemolytic lectin to enhance anti-methicillin-resistant Staphylococcus aureus (MRSA) activity with less side effects. Two designed peptides, CGS19 (LARVARRVIRFIRRAW-NH2) and CGS20 (RRRLARRLIFFIRRAW-NH2), exhibited strong antibacterial activities against clinically isolated MRSA with MICs of 3-6 µM, but no obvious cytotoxicity was observed. Consistently, CGS19 and CGS20 exerted rapid bactericidal activity and effectively induced 5.9 and 5.8 log reduction of MRSA counts in mouse subeschar, respectively. Further, CGS19 and CGS20 kill bacteria not only through disturbing membrane integrity but also by binding formate-tetrahydrofolate ligase, a key enzyme in the folate metabolism pathway, thereby inhibiting the folate pathway of MRSA. CGS19 and CGS20 are promising lead candidates for drug development against MRSA infection. The dual mechanisms on the identical peptide sequence or scaffold might be an underappreciated manner of treating life-threatening pathogens.

Synthesis and anticancer evaluation of acetylated-lysine conjugated gemcitabine prodrugs.

Gemcitabine is an antimetabolite drug approved for the treatment of various cancers. However, its use is limited due to several issues such as stability, toxicity and drug resistance. Herein, we present the design and synthesis of a series of gemcitabine prodrugs with modifications on the 4-N-amino group by employing an acetylated l- or d-lysine moiety masked by different substitutions. Prodrugs 1-3 and 6-8 showed up to 2.4 times greater anticancer activity than gemcitabine in A549 lung cells, while they exhibited potent activity against BxPC-3 pancreatic cells with IC50 values in the range of 7-40 nM. Moreover, prodrugs 2-3 and 7-8 were found to be less potent against CTSL low expression Caco-2 cells and at least 69-fold less toxic towards human normal HEK-293T cells compared to gemcitabine, leading to improved selectivity and safety profiles. Further stability studies showed that representative prodrugs 2 and 7 exhibited enhanced metabolic stability in human plasma, human liver microsomes and cytidine deaminase. Prodrug 1 can be cleaved by tumor cell-enriched CTSL to release parent drug gemcitabine. Overall, these results demonstrated that acetylated lysine conjugated gemcitabine prodrugs could serve as promising leads for further evaluation as new anticancer drugs.

Investigation of anti-aging and anti-infection properties of Jingfang Granules using the Caenorhabditis elegans model.

Jingfang Granule (JFG), a traditional Chinese medicine, is frequently employed in clinical settings for the treatment of infectious diseases. Nevertheless, the anti-aging and anti-infection effects of JFG remain uncertain. In the present study, these effects were evaluated using the Caenorhabditis elegans (C. elegans) N2 as a model organism. The results demonstrated that JFG significantly increased the median lifespan of C. elegans by 31.2% at a dosage of 10 mg/mL, without any discernible adverse effects, such as alterations in the pharyngeal pumping rate or nematode motility. Moreover, JFG notably increased oviposition by 11.3%. Subsequent investigations revealed that JFG enhanced oxidative stress resistance in C. elegans by reducing reactive oxygen species levels and significantly improved survival rates in nematodes infected with Pseudomonas aeruginosa ATCC 9027. These findings suggest that JFG delays reproductive senescence in C. elegans and protects them from oxidative stress, thereby extending their lifespan. Additionally, JFG improves the survival of P. aeruginosa-infected nematodes. Consequently, JFG has potential as a candidate for the development of anti-aging and anti-infection functional medicines.

"Toolbox" construction of an extremophilic nitrile hydratase from Streptomyces thermoautotrophicus for the promising industrial production of various amides.

Nitrile hydratase (NHase; EC 4.2.1.84) is widely used to synthesize the corresponding amides from nitriles, which is the most successful green biocatalyst. However, the limited acceptability of substrates and instability under harsh reaction conditions have hindered its widespread industrial application. Here, a gene encoding an extremophilic NHase from Streptomyces thermoautotrophicus (S.t NHase) was successfully overexpressed in Escherichia coli. The enzyme exhibited excellent thermostability, retaining >50 % of residual activity after heat treatment at 65 °C for 252 min. To further improve the catalytic performance of S.t NHase, semi-rational engineering of its substrate access tunnel was performed. A mutant ßL48D showed a specific activity of 566.18 ± 18.86 U/mg towards 3-cyanopyridine, which was 7.7 times higher than its parent enzyme (73.80 ± 5.76 U/mg). Molecular dynamics simulation showed that the introduction of aspartic acid into ßLeu48 resulted in a larger and more frequent opening of the substrate access tunnel entrance. On this basis, a "toolbox" containing various mutants on the substrate access tunnel was further established, whose catalytic activity towards various nitrile substrates was extensively improved, showing great potential for efficient synthesis of multiple high-value amides.

Investigation of chetomin as a lead compound and its biosynthetic pathway.

Chaetomium fungi produce a diversity of bioactive compounds. Chaetomium cochliodes SD-280 possesses 91 secondary metabolite gene clusters and exhibits strong antibacterial activity. One of the active compounds responsible for that activity, chetomin, has a minimum inhibitory concentration (MIC) for anti-methicillin-resistant Staphylococcus aureus (MRSA) of 0.05 µg/mL (vancomycin: 0.625 µg/mL). This study demonstrated that the addition of glutathione (GSH) can enhance chetomin yield dramatically, increasing its production 15.43-fold. Following genome sequencing, cluster prediction, and transcriptome and proteome analyses of the fungus were carried out. Furthermore, a relatively complete chetomin biosynthetic gene cluster was proposed, and the coding sequences were acquired. In the cluster of GSH-treated cells, proteome analysis revealed two up-regulated proteins that are critical enzymes for chetomin biosynthesis. One of these enzymes, a nonribosomal peptide synthetase (NRPS), was heterologously expressed in Aspergillus nidulans, and one of its metabolites was determined to be an intermediate in the chetomin biosynthetic pathway. We present here, to our knowledge, the first experimental evidence that chetomin exhibits strong bioactivity against MRSA. Our work also provides extensive insights into the biosynthetic pathway of chetomin, in particular identifying two key enzymes (glutathione S-transferase (CheG) and NRPS (CheP)) that substantially up-regulate chetomin. These mechanistic insights into chetomin biosynthesis will provide the foundation for further investigation into the anti-pathogenic properties and applications of chetomin. KEY POINTS: • Chetomin exhibits strong anti-MRSA activity with MIC of 0.05 µg/mL. • Addition of glutathione improved the yield of chetomin by 15.43-fold. • CheG and CheP involved in the chetomin biosynthesis were revealed for the first time.

Transcriptome analysis method based on differential distribution evaluation.

Identifying differential genes over conditions provides insights into the mechanisms of biological processes and disease progression. Here we present an approach, the Kullback-Leibler divergence-based differential distribution (klDD), which provides a flexible framework for quantifying changes in higher-order statistical information of genes including mean and variance/covariation. The method can well detect subtle differences in gene expression distributions in contrast to mean or variance shifts of the existing methods. In addition to effectively identifying informational genes in terms of differential distribution, klDD can be directly applied to cancer subtyping, single-cell clustering and disease early-warning detection, which were all validated by various benchmark datasets.

Mollicellins S-U, three new depsidones from Chaetomium brasiliense SD-596 with anti-MRSA activities.

Fungi are important resources for drug development, as they have a diversity of genes, that can produce novel secondary metabolites with effective bioactivities. Here, five depsidone-based analogs were isolated from the rice media of Chaetomium brasiliense SD-596. Their structures were elucidated using NMR and mass spectrometry analysis. Five compounds, including three new depsidone analogs, mollicellin S (1), mollicellin T (2), and mollicellin U (3), and two known compounds, mollicellin D (4) and mollicellin H (5), exhibited significant inhibition against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), with MIC values ranging from 6.25 to 12.5 µg ml-1. Herein, we identified the predicted plausible biosynthetic cluster of the compounds and discussed the structure-activity relationship. Finally, we found that the introduction of aldehyde and methoxyl groups provide marked improvement for the inhibition against MRSA.

Potassium resistance of halotolerant and alkaliphilic Halomonas sp. Y2 by a Na+-induced K+ extrusion mechanism.

In most halophiles, K+ generally acts as a major osmotic solute for osmotic adjustment and pH homeostasis. However, strains also need to extrude excessive intracellular K+ to avoid its toxicity. In the halotolerant and alkaliphilic Halomonas sp. Y2, an Na+-induced K+ extrusion process was observed when the cells were confronted with high extracellular K+ pressure and supplementation by millimolar Na+ ions. Among three mechanosensitive channels (KefA) and two K+/H+ antiporters founded in the genome of the strain, ke1 displayed around 3-5-fold upregulation to ion stress at pH 8.0, while much higher upregulation of Ha-mrp was observed at pH 10.0. Compared to the growth of wild-type Halomonas sp. Y2, deletion of these genes from the strain resulted in different growth phenotypes in response to the osmotic pressure of potassium. In combination with the transcriptional response of these genes, we proposed that the KefA channel of Ke1 is the main contributor to the K+-extrusion process under weak alkalinity, while the Mrp system plays critical roles in alleviating K+ contents at high pH. The combination of these strategies allows Halomonas sp. Y2 to grow over a range of extracellular pH and ion concentrations, and thus protect cells under high osmotic stress conditions.

Genome sequence of Halomonas hydrothermalis Y2, an efficient ectoine-producer isolated from pulp mill wastewater.

Halophilic microorganisms have great potentials towards biotechnological applications. Halomonas hydrothermalis Y2 is a halotolerant and alkaliphilic strain that isolated from the Na+-rich pulp mill wastewater. The strain is dominant in the bacterial community of pulp mill wastewater and exhibits metabolic diversity in utilizing various substrates. Here we present the genome sequence of this strain, which comprises a circular chromosome 3,933,432 bp in size and a GC content of 60.2%. Diverse genes that encoding proteins for compatible solutes synthesis and transport were identified from the genome. With a complete pathway for ectoine synthesis, the strain could produce ectoine from monosodium glutamate and further partially secreted into the medium. In addition, around 20% ectoine was increased by deleting the ectoine hydroxylase (EctD). The genome sequence we report here will provide genetic information regarding adaptive mechanisms of strain Y2 to its harsh habitat, as well as facilitate exploration of metabolic strategies for diverse compatible solutes, e.g., ectoine production.

Critical Functions of Region 1-67 and Helix XIII in Retaining the Active Structure of NhaD Antiporter in Halomonas sp. Y2.

NhaD-type antiporters are mainly distributed in various Proteobacteria, especially in marine microorganisms and human pathogens. This distribution as well as the pathogenic properties of these strains suggest that these antiporters contribute to the regulation of high osmoregulation and are potential drug targets. Two NhaD homologs, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. Y2 exhibits similar, high in vitro activity, but remarkably different in vivo functions. To search for critical domains or residues involved in these differences of physiological functions, various chimeras composed of NhaD1 and NhaD2 segments were generated. Two regions at residues 1-67 and 464-492 were found to be responsible for the robust in vivo function of NhaD2, and region 464-492 is also crucial to the pH response of the antiporter. In particular, the completely abolished activity of KNabc/N463r, highly recovered activity while very weakly recovered ion resistance of the KNabc/N463r-C7 chimera, suggested that transmembrane helix (TM) XIII is crucial for the robust ion resistance of NhaD2. Using site-directed mutagenesis, seven hydrophobic residues in TM XIII were identified as key residues for the ion translocation of NhaD2. Compared with the fluorescence resonance energy transfer (FRET) profile in the wild-type NhaD2, the reduced FRET efficiency of N463r chimeras provided solid evidence for conformational changes in the N463r fusion protein and consequently verified the structural functions of TM XIII in the pH activation and physiological functions of NhaD2.

Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2.

Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na+ and Li+ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na+ and Li+ expulsion, as well as complementary growth following exposure to 300 mM Na+ and 100 mM Li+ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile.IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu38, in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.

TP53INP2-related basal autophagy is involved in the growth and malignant progression in human liposarcoma cells.

BACKGROUND: Understanding the function of autophagy may allow us to develop a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes in the treatment of cancers. Here, we studied the contribution of basal autophagy in human liposarcoma. METHODS: The levels of basal autophagy were analyzed by measuring autophagy-related protein expression and autophagosome formation. TP53INP2 expression was determined by real-time PCR, western blot and tissue microarray. Genetic inhibition or overexpression of TP53INP2 was performed to examine its effects on autophagic activity and cell growth. RESULTS: Compared with human liposarcoma cell line SW872, low level of basal autophagy were present in SW872-S cells with high malignancy. Moreover, a decrease of TP53INP2 expression was found accompanying liposarcoma malignant progression in cell lines and primary tissues. TP53INP2 expression was required for autophagic activity in liposarcoma cells. Autophagy inhibition with chloroquine suppressed the growth of liposarcoma cells. TP53INP2-related basal autophagy rendered liposarcoma cells to be more resistant to bortezomib-induced inhibition of cell growth. CONCLUSIONS: The results reveal the association of TP53INP2-related basal autophagy with cell growth and malignant progression of human liposarcoma, which helps re-evaluate targeting autophagy for cancer therapy, and suggest that TP53INP2 expression might be used as a prognostic marker to predict human liposarcoma malignancies.

Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production.

Alkaline Response of a Halotolerant Alkaliphilic Halomonas Strain and Functional Diversity of Its Na+(K+)/H+ Antiporters.

Halomonas sp. Y2 is a halotolerant alkaliphilic strain from Na+-rich pulp mill wastewater with high alkalinity (pH >11.0). Transcriptome analysis of this isolate revealed this strain may use various transport systems for pH homeostasis. In particular, the genes encoding four putative Na+/H+ antiporters were differentially expressed upon acidic or alkaline conditions. Further evidence, from heterologous expression and mutant studies, suggested that Halomonas sp. Y2 employs its Na+/H+ antiporters in a labor division way to deal with saline and alkaline environments. Ha-NhaD2 displayed robust Na+(Li+) resistance and high transport activities in Escherichia coli; a ΔHa-nhaD2 mutant exhibited growth inhibition at high Na+(Li+) concentrations at pH values of 6.2, 8.0, and 10.0, suggesting its physiological role in osmotic homeostasis. In contrast, Ha-NhaD1 showed much weaker activities in ion exporting and pH homeostasis. Ha-Mrp displayed a combination of properties similar to those of Mrp transporters from some Bacillus alkaliphiles and neutrophiles. This conferred obvious Na+(Li+, K+) resistance in E. coli-deficient strains, as those ion transport spectra of some neutrophil Mrp antiporters. Conversely, similar to the Bacillus alkaliphiles, Ha-Mrp showed central roles in the pH homeostasis of Halomonas sp. Y2. An Ha-mrp-disrupted mutant was seriously inhibited by high concentrations of Na+(Li+, K+) but only under alkaline conditions. Ha-NhaP was determined to be a K+/H+ antiporter and shown to confer strong K+ resistance both at acidic and alkaline stresses.

Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic Halomonas sp. Y2.

Na(+)/H(+) antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na(+)/H(+) antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na(+) and Li(+) translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0-8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na(+) and Li(+), while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues.