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Cryptococcus neoformansposes a threat to human health, but anticryptococcal therapy is hampered by the emergence of drug resistance, whose underlying mechanisms remain poorly understood. Herein, we discovered that Isw1, an imitation switch chromatin remodeling ATPase, functions as a master modulator of genes responsible for multidrug resistance inC. neoformans. Cells with the disruptedISW1gene exhibited profound resistance to multiple antifungal drugs. Mass spectrometry analysis revealed that Isw1 is both acetylated and ubiquitinated, suggesting that an interplay between these two modification events exists to govern Isw1 function. Mutagenesis studies of acetylation and ubiquitination sites revealed that the acetylation status of Isw1K97coordinates with its ubiquitination processes at Isw1K113and Isw1K441through modulating the interaction between Isw1 and Cdc4, an E3 ligase. Additionally, clinical isolates ofC. neoformansoverexpressing the degradation-resistantISW1K97Qallele showed impaired drug-resistant phenotypes. Collectively, our studies revealed a sophisticated acetylation-Isw1-ubiquitination regulation axis that controls multidrug resistance inC. neoformans.

The amino sugar N-acetyl-d-glucosamine (GlcNAc) is the key constituent of cell wall components and plays an important role in pathogenesis in a wide range of fungi. However, catabolism of GlcNAc has not been studied in basidiomycete fungi. In this study, we identified and characterized a gene cluster essential for GlcNAc utilization in Cryptococcus deneoformans, an environmental human fungal pathogen. The C. deneoformans genome contains a GlcNAc transporter (Ngt1), a GlcNAc kinase (Hxk3), a GlcNAc-6-phosphate deacetylase (Dac1), and a glucosamine-6-phosphate deaminase (Nag1). Their expression levels were highly induced in cultures containing GlcNAc as the sole carbon source, and the corresponding mutants showed severe growth defects in the presence of GlcNAc. Functional and biochemical analyses revealed that HXK3 encodes a novel GlcNAc kinase. Site-directed mutations of conserved residues of Hxk3 indicated that ATP binding and GlcNAc binding are essential for GlcNAc kinase activities. Taken together, the results from this study provide crucial insights into basidiomycete GlcNAc catabolism. IMPORTANCE N-Acetylglucosamine (GlcNAc) is recognized as not only the building block of chitin but also an important signaling molecule in fungi. The catabolic pathway of GlcNAc also plays an important role in vital biological processes in fungi. However, the utilization pathway of GlcNAc in the phylum Basidiomycota, which contains more than 41,000 species, remains unknown. Cryptococcus deneoformans is a representative basidiomycetous pathogen that causes life-threatening meningitis. In this study, we characterized a gene cluster essential for GlcNAc utilization in C. deneoformans and identified a novel GlcNAc kinase. The results of this study provide important insights into basidiomycete GlcNAc catabolism and offer a starting point for revealing its role in pathogenesis.

The emergence of drug-resistant fungal pathogens poses great threats to an increasing number of vulnerable populations worldwide, and the need for novel antifungal agents is imperative. In this work, a series of lipo-γ-AA peptides were synthesized and evaluated for their biological activities. One lead, MW

The Prp8 intein is one of the most widespread eukaryotic inteins, present in important pathogenic fungi, including Cryptococcus and Aspergillus species. Because the processed Prp8 carries out essen...

The increasing emergence and spread of drug resistant Candida albicans represent a serious challenge for effective treatment and call for the development of new therapeutic options. To address this need, we synthesized a series of polypyridyl iridium(III) complexes and studied their antimicrobial activities. Herein, one lead iridium(III) complex Ir-3 [(ptpy)

Coronavirus disease 2019 (COVID-19), which emerged in Wuhan, China in December 2019, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has become a major global public h...

Inspired by the fact that chitosan is a representative constituent of the ectocellular structure of Cryptococcus neoformans and a typical biomaterial for improving drug oral absorption, we designed an elegant and efficient C. neoformans-targeted drug delivery system via oral administration. A chitosan-binding peptide screened by phage display was used as the targeting moiety, followed by conjugation to the surface of poly(lactic- co-glycolic acid) nanoparticles as the drug carrier, which was then incubated with free chitosan. The noncovalently bound chitosan adheres to mucus layers and significantly enhances penetration of nanoparticles through the oral absorption barrier into circulation and then re-exposed the targeting ligand for later recognition of the fungal pathogen at the site of infection. After loading itraconazole as a model drug, our drug delivery system remarkably cleared lung infections of C. neoformans and increased survival of model mice. Currently, targeted drug delivery is mainly performed intravenously; however, the system described in our study may provide a universal means to facilitate drug targeting to specific tissues and disease sites by oral administration and may be especially powerful in the fight against increasingly severe fungal infections.

Cryptococcus neoformans is the most common cause of deadly fungal meningitis. This fungus has a complex inositol acquisition and utilization system, and our previous studies have shown the importance of inositol utilization in cryptococcal development and virulence. However, how inositol utilization is regulated in this fungus remains unknown. In this study, we found that inositol, irrespective of the presence of glucose in the media, represses the expression of C. neoformans genes involved in inositol pyrophosphate biosynthesis, including the gene encoding inositol hexakisphosphate kinase Kcs1. Kcs1 was recently reported to regulate inositol metabolism in Saccharomyces cerevisiae and to impact virulence in C. neoformans. To examine the potential role of Kcs1 in inositol regulation in C. neoformans, we generated the kcs1Δ mutant and compared its phenotype with the wild type strain. We found that Kcs1 negatively regulates inositol uptake and catabolism in C. neoformans, but, in contrast to Kcs1 function in S. cerevisiae, does not appear to regulate inositol biosynthesis. Together, these results show that Kcs1 functions to fine-tune inositol acquisition to maintain inositol homeostasis in C. neoformans.

Aims The wblA gene, encoding a homolog of the WhiB family protein, was identified in the sequenced genome of daptomycin producer S.roseosporus. To explore the function of wblA, we construct wblA disruption strains, complemented strains and overexpression strains to test whether it can affect the production of secondary metabolites and morphogenesis. Methods and results We constructed disruption strains by homologous recombination in S.roseosporus. The disruption mutant of wblA could block aerial mycelium sporulation and enhance the production of daptomycin by 51%. In contrast, overexpression of wblA resulted in significantly decreased the yield of daptomycin. In agreement with it, the transcription of key daptomycin regulatory genes atrA, dptR2 and dptR3 and structural gene dptE remarkably increased in the wblA disruption mutant. Conclusions wblA plays a key role in control of daptomycin biosynthesis and is essential for sporulation. The disruption of wblA could accumulate the transcription level of some key genes involving in daptomycin biosynthesis. Significance and Impact of the Study Daptomycin is an important antibiotic with potent activity against a variety of Gram-positive pathogens. This study demonstrates that valuable improvement in the yield of daptomycin can be achieved through modulating the expression of wblA transcription regulator. This article is protected by copyright. All rights reserved.

Deletion of the Lsr2 gene in Streptomyces roseosporus up-regulated silent gene clusters and produced new secondary metabolites. An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS) method was used to analyze metabolites of the mutant and wild-type strains, and recognize previously unreported sulfur-containing compounds based on their molecular formulas and fragmentation ions. The targeted isolation of unidentified compounds afforded six new sulfur-containing compounds, pyrismycins A–F (1–6), together with seven known analogues 7–13. Their cytotoxic effects were evaluated using four clinically relevant human cancer cell lines, gastric carcinoma SGC7901, breast carcinoma MDA-MB-231, lung carcinoma A549 and hepatocellular carcinoma HepG2. Compound 7 exhibited the most potent cytotoxicity with IC50 values of 1.7, 5.8 and 6.3 μM against the SGC7901, HepG2 and MDA-MB-231, respectively.

Most current technologies can hardly simultaneously reduce the accumulation of arsenic (As) and cadmium (Cd) in crops. In this study, root application of selenite [Se (IV)] and selenate [Se (VI)] was used to assess their abilities to reduce the accumulation of As and Cd, and maintain the yields and quality of rice grains. The results show that Se (IV) showed a weaker ability than Se (VI) to maintain the grain contents of many essential elements, but a stronger ability to decrease As and Cd contents in rice grains, and maintain the yields, photosynthesis rate and stomatal conductance, and increase the grain contents of several amino acids (AAs), total Se, selenomethionine (SeMet) and selenocysteine (SeCys). The best outcomes resulted at a relatively high application of 5 mg kg −1 Se (IV), reflecting in the highest total Se, SeCys and SeMet content (14.95, 118.70 and 864.73 μg kg −1 , respectively) in the grains, highest grain yield, and lowest grain As and Cd content (0.36 and 0.07 mg kg −1 , respectively). In addition, the application of 1–5 mg kg −1 Se (IV) seemed to facilitate the formation of SeMet in the grains, but most inorganic Se in the grains were transformed into SeCys and SeMet under Se (VI) treatments. This study provides a new idea to resolve the problems of high accumulation of As and Cd in rice grains and insufficiency of Se intake in China.

Overcoming the reticuloendothelial system (RES) has long been a vital challenge to nanoparticles as drug carriers. Modification of nanoparticles with polyethylene glycol helps them avoid clearance by macrophages but also suppresses their internalization by target cells. To overcome this paradox, we developed an RES-specific blocking system utilizing a "don't-eat-us" strategy. First, a CD47-derived, enzyme-resistant peptide ligand was designed and placed on liposomes (d-self-peptide-labeled liposome, DSL). After mainline administration, DSL was quickly adsorbed onto hepatic phagocyte membranes (including those of Kupffer cells and liver sinusoidal endothelial cells), forming a long-lasting mask that enclosed the cell membranes and thus reducing interactions between phagocytes and subsequently injected nanoparticles. Compared with blank conventional liposomes (CL), DSL blocked the RES at a much lower dose, and the effect was sustained for a much longer time, highly prolonging the elimination half-life of the subsequently injected nanoparticles. This "don't-eat-us" strategy by DSL was further verified on the brain-targeted delivery against a cryptococcal meningitis model, providing dramatically enhanced brain accumulation of the targeted delivery system and superior therapeutic outcome of model drug Amphotericin B compared with CL. Our study demonstrates a strategy that blocks the RES by masking phagocyte surfaces to prolong nanoparticle circulation time without excess modification and illustrates its utility in enhancing nanoparticle delivery.

Cell membrane cloaking is an emerging field in drug delivery in which specific functions of parent cells are conferred to newly formed biomimetic vehicles. A growing variety of delivery systems with diverse surface properties have been utilized for this strategy, but it is unclear whether the affinity of membrane-core pairs could guarantee effective and proper camouflaging. In this study, we propose a concise and effective "molecular affinity" strategy using the intracellular domain of transmembrane receptors as "grippers" during membrane coating. Red blood cell (RBC) membranes and cationic liposomes were adopted for fabrication, and a peptide ligand derived from the cytoplasmic protein P4.2 was prepared to specifically recognize the cytoplasmic domain of band 3, a key transmembrane receptor of erythrocytes. Once anchored onto the liposome surface, the P4.2-derived peptide would interact with the isolated RBC membrane, forming a "hidden peptide button", which ensures the right-side-out orientation. The membrane-coated liposomes exhibited an appropriate size distribution around 100 nm and high stability, with superior circulation durations compared with those of conventional PEGylated liposomes. Importantly, they possessed the ability to target Candida albicans by the interaction between the pathogenic fungus and host erythrocytes and to neutralize hemotoxin secreted by the pathogenic fungi. The curative effect of the model drug was thus substantially improved. In summary, the "molecular affinity" strategy may provide a powerful and universal approach for the construction of cell membrane-coated biomaterials and nanomedicines at both laboratory and industrial scales.

A novel sesquiterpenoid, roseosporol A (1), together with 12 known compounds (2–13) were isolated from the ethyl acetate extract of Lsr2-deletion mutant strain of Streptomyces roseosporus. Their structures were determined by spectroscopic methods, including NMR, HRMS, UV, IR and ECD.

Our goal was to improve treatment outcomes for C. neoformans infection by designing nanocarriers that enhance drug-encapsulating capacity and stability. Thus, a noncovalent complex of methoxy poly(ethylene glycol)-poly(lactide)-poly(&beta, amino ester) (MPEG-PLA-PAE) and amphotericin B (AMB) was developed and characterized. The MPEG-PLA-PAE copolymer was synthesized by a Michael-type addition reaction, the copolymer was then used to prepare the AMB-loaded nanocomplex. AMB was in a highly aggregated state within complex cores. A high encapsulation efficiency (&gt, 90%) and stability of the AMB-loaded nanocomplex were obtained via electrostatic interaction between AMB and PAE blocks. This nanocomplex retained drug activity against C. neoformans in vitro. Compared with micellar AMB, the AMB nanocomplex was more efficient in terms of reducing C. neoformans burden in lungs, liver, and spleen, based on its improved biodistribution. The AMB/MPEG-PLA-PAE complex with enhanced drug-loading capacity and stability can serve as a platform for effective treatment of C. neoformans infection.

Paddy soils in many regions of China have been seriously polluted by multiple heavy metals or metalloids, such as arsenic (As), cadmium (Cd) and lead (Pb). In order to ensure the safety of food and take full advantage of the limited farmland resources of China, exploring an effective technology to repair contaminated soils is urgent and necessary. In this study, three technologies were employed, including variety screening, water management and foliage dressing, to assess their abilities to reduce the accumulation of Cd and As in the grains of different rice varieties, and meanwhile monitor the related yields. The results of variety screening under insufficient field drying condition showed that the As and Cd contents in the grains of only four varieties [Fengliangyouxiang 1 (P6), Zhongzheyou 8 (P7), Guangliangyou 1128 (P10), Y-liangyou 696 (P11)] did not exceed their individual national standard. P6 gained a relatively high grain yield but accumulated less As and Cd in the grains despite of the relatively high As and Cd concentrations in the rhizosphere soil. However, long-playing field drying in water management trial significantly increased Cd but decreased As content in the grains of all tested three varieties including P6, suggesting an important role of water supply in controlling the accumulation of grain As and Cd. Selenium (Se) showed a stronger ability than silicon (Si) to reduce As and Cd accumulation in the grains of Fengliangyou 4 (P2) and Teyou 524 (P13), and keep the yields. The results of this study suggest that combined application of water management and foliage dressing may be an efficient way to control As and Cd accumulation in the grains of paddy rice exposing to As- and Cd-contaminated soils.

Antimony (Sb) is a toxic element for plant; however, its toxic mechanisms, in particular for its toxicity to plant root growth, are unclear. Selenium (Se) can detoxify Sb, but its associated detoxification mechanisms have not been fully clarified. The aim of the present study was to investigate the effects of Sb alone, or accompanying with Se, on the root growth, antioxidative systems and the uptake of Se and Sb in paddy rice. Two nested hydroponic experiments were performed based on a two-factor, five-level central composite design. The results showed that Sb and Se could each reduce the contents of the other in both the shoots and roots. With 0.8 mg L−1 Se in the solution, increasing Sb levels from 1 to 9 mg L−1 significantly reduced the shoot biomass but enhanced the root biomass; however, in the presence of 5 mg L−1 Sb in the solution, the opposite results were observed with increased Se levels from 0.1 to 1.5 mg L−1. In the presence of 0.8 mg L−1 Se, the addition of Sb increased the values of most root growth parameters, such as root length, root area, root forks, and the numbers of fine roots, medium roots and thick roots, suggesting a counteracting effect on root growth already negatively affected by 0.8 mg L−1 Se. However, the increased values of the root growth parameters cannot explain the significantly inhibited Se uptake in both the shoots and roots, suggesting an unknown mechanism in this respect. The addition of Se counteracted the negative effects of Sb on the cell membrane lipids and shoot biomass, but reduced the values of the root growth parameters. Thus, the enzymes SOD, APX and CAT might play important roles in rebalancing the excess ROS resulting from exposure to Se and/or Sb. These results support the hypothesis that Se affects the root growth to inhibit Sb uptake and simultaneously regulate the antioxidant systems to reduce oxidative stress.

Due to potent bactericidal activity and low rate of drug-resistance, daptomycin is recognized as first line antibiotic to treat serious infections caused by drug-resistant Gram-positive pathogens. However, the incidence of daptomycin resistance is increasing due to its widespread application. Alteration of cell wall homeostasis and membrane phospholipid metabolism is involved in daptomycin resistance. The unique mode of action underlying daptomycin resistance in important pathogens, including Staphylococcus aureus and Enterococci, is presented in this paper.环脂肽抗生素达托霉素抗菌活性强，致病菌不容易产生耐药性，已成为治疗革兰氏阳性菌特别是耐药菌感染的一线药物。但由于广泛使用，仍然出现了达托霉素耐药菌。细胞膜磷脂代谢和细胞壁结构动态与致病菌达托霉素耐药密切相关。文中综述了达托霉素作用机制和耐药机制，以期对药物研发和临床用药有所裨益。.

A novel sesquiterpenoid, roseosporol A (

Novel agents are urgently needed to rapidly kill drug-resistant Mycobacterium tuberculosis. Noble metal complexes, particularly polypyridyl iridium complexes serving as therapeutic agents, have attracted considerable interest recently, due to their significant cytotoxic or antimicrobial activities. Here, we reported an polypyridyl iridium dimer complex [Ir(ppy)2Cl]2 (3), with ppy = phenylpyridine, which was found to be active against both exponential growing and non-replicating M. smegmatis, with minimum inhibitory concentration values of 2 μg/mL, and exhibited rapid bactericidal kinetics, killing pathogens within 30–60 min. Moreover, 3 was demonstrated to generate a large amount of reactive oxygen species and to be effective in drug-resistant strains. Taken together, the selectively active iridium(III) dimer complex showed promise for use as a novel drug candidate for the treatment of M. tuberculosis infection.

N(ɛ)-Acetylation of lysine residues represents a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Mycobacterium tuberculosis is the causative agent of tuberculosis, one of the most formidable public health threats. Many aspects of the biology of M. tuberculosis remain elusive, in particular the extent and function of N(ɛ)-lysine acetylation. With a combination of anti-acetyllysine antibody-based immunoaffinity enrichment with high-resolution mass spectrometry, we identified 1128 acetylation sites on 658 acetylated M. tuberculosis proteins. GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis. Six types of acetylated peptide sequence motif were revealed from the acetylome. Twenty lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Salmonella enterica, Bacillus subtilis and Streptomyces roseosporus, with several acetylation sites highly conserved among four or five bacteria, suggesting that acetylated proteins are more conserved. Notably, several proteins including isocitrate lyase involved in the persistence, virulence and antibiotic resistance are acetylated, and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity, indicating major roles of KAc in these proteins engaged cellular processes. Our data firstly provides a global survey of M. tuberculosis acetylation, and implicates extensive regulatory role of acetylation in this pathogen. This may serve as an important basis to address the roles of lysine acetylation in M. tuberculosis metabolism, persistence and virulence.

An antibiotic-affinity strategy was designed to detect pathogenic bacterial whole cells based on the strong affinity of the antibiotic agent to bind to the cell wall of the bacterium. Vancocin, a powerful glycopeptide antibiotic, was used as a molecular recognition agent for Staphylococcus aureus. To improve its specificity, IgG was utilized as the second recognition agent based on the binding between the Fc region of IgG and protein A in the cell wall.

Octahedral transition metal complexes have been shown to have tremendous applications in chemical biology and medicinal chemistry. Meanwhile, structural transition metals can be replaced by inert octahedral silicon in a proof-of-principle study. We here introduce the first example of octahedral silicon complexes, which can very well serve as an efficient antimicrobial agent. The typical silicon arenediolate complex 1 {[(phen)₂Si(OO)](PF₆)₂, with phen = 1,10-phenanthroline, OO = 9,10-phenanthrenediolate} exhibited significant inhibition towards the growth of Cryptococcus neoformans with MIC and MFC values of 4.5 and 11.3 μM, respectively. Moreover, it was fungicidal against both proliferative and quiescent Cryptococcus cells. This work may set the stage for the development of novel antifungal drugs based upon hexacoodinate silicon scaffolds.

Aims: Daptomycin, one of the A21978C factors produced by Streptomyces roseosporus, is an acidic cyclic lipopeptide antibiotic with potent activity against a variety of Gram-positive pathogens. To increase the titre of this extensively used and clinically important antibiotic, we applied a reported-guided rpsL mutation selection system to generate strains producing high levels of A21978C. Methods and Results: In the reporter design, dptE was chosen as the overexpressing target, and neo-encoding neomycin phosphotransferase as the reporter. Using this reporter-guided selection system, 20% of the selected, streptomycin-resistant mutants produced greater amounts of A21978C than the starting strain. The selection system increased the screening efficiency about 10-fold with a frequency of 1·7% A21978C overproducing strains among strr mutants. A21978C production was increased approximately 2·2-fold in the rpsL K43N mutant. Conclusions: The combination of ribosome engineering and reporter-guided mutant selection generated an A21978C overproducing strain that produced about twice as much A21978C as the parental strain. Significance and Impact of the Study: The strategies presented here, which integrated the advantages of both ribosome engineering and reporter-guided mutation selection, could be applied to other bacteria to improve their yield of secondary metabolites.

Recent developments of therapeutic agents based on transition metals have attracted a great deal of attention. Metal drugs have advantages over other small molecule drugs, and it was demonstrated that, in a number of studies, they played an important role in pharmaceutical chemical research and clinical chemotherapy of cancers. It is worthwhile mentioning that octahedral polypyridyl ruthenium(II) complexes have shown remarkable applications in chemical biology and medicinal chemistry over the last decade. However, only very recently has there been comprehensive interest in their antimicrobial properties due to metal-related toxic concerns or neglected potential roles in microbiological systems. Our review will highlight the recent developments in octahedral polypyridyl ruthenium(III) complexes that have exhibited significant antimicrobial activities and will discuss the relationship between the chemical structure and biological process of ruthenium complexes, in both bacterial and fungal cells.

Background Streptomyces, as the main source of antibiotics, has been intensively exploited for discovering new drug candidates to combat the evolving pathogens. Disruption of wblA, an actinobacteria-specific gene controlling major developmental transition, can cause the alteration of phenotype and morphology in many species of Streptomyces. One wblA homologue was found in Streptomyces ansochromogenes 7100 by using the Basic Local Alignment Search Tool. It is interesting to identify whether novel secondary metabolites could be produced by the wblA disruption mutant as evidenced in other Streptomyces. Results The wblA disruption mutant of S. ansochromogenes 7100 (ΔwblA) was constructed by homologous recombination. ΔwblA failed to produce spores and nikkomycin, the major product of S. ansochromogenes 7100 (wild-type strain) during fermentation. Antibacterial activity against Staphylococcus aureus and Bacillus cereus was observed with fermentation broth of ΔwblA but not with that of the wild-type strain. To identify the antibacterial compounds, the two compounds (compound 1 and compound 2) produced by ΔwblA were characterized as 16-membered macrolides by mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical structure of these compounds shows similarity with tylosin, and the bioassays indicated that the two compounds inhibited the growth of a number of gram-positive bacteria. It is intriguing that they displayed much higher activity than tylosin against Streptococcus pneumoniae. Conclusions Two novel tylosin analogues (compound 1 and 2) were generated by ΔwblA. Bioassays showed that compound 1 and 2 displayed much higher activity than tylosin against Streptococcus pneumoniae, implying that these two compounds might be used to widen the application of tylosin. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0359-5) contains supplementary material, which is available to authorized users.

To study the regulation of laeA overexpression on mevastatin production and sporulation in Penicillium citrinum.We cloned the laeA gene from Penicillium citrinum and constructed the vector pGiHTGi-laeA. The plasmid pGiHTGi-laeA was transformed in Penicillium citrinum by agrobacterium tumefaciens-mediated transformation. Positive transformants were detected by cloning the hygromycin gene. The mevastatin production of the wild type and OE:: laeA was compared by HPLC. The conidia number was counted by blood counting chamber. The biosynthetic gene cluster expression quantity of mevastatin in the wild type and OE: :laeA were analyzed by qRT-PCR.We constructed the plasmid pGiHTGi-laeA, and screened the positive transformants that overexpress the laeA in Penicillium citrinum. With the overexpression of laeA, the mevastatin production was increased from (0.69 ± 0.12) mg/g to (4.02 ± 0.50) mg/g dry cell weight. Compared to the wild type strain, the laeA expression quantity in the OE :: laeA strain increased 29%, and the mlcB expression increased 72%, the mlcR expression increased 153%. Moreover, the overexpression of laeA would decrease the conidia number.Overexpression of LaeA enhances mevastatin production and reduces sporulation of Penicillium citrinum, with increases expression of pathway-regulator mlcR, and biosynthetic gene MlcR. These results could guide global regulatory mechanism of mevastatin biosynthesis and the exploitation of high-production strain.

Additional file 1. Figure S1. NMR Spectra of compound 1. (A) Summary of key correlations between protons and carbons in compound 1 based on NMR spectroscopic data. (B) 1H-1H COSY spectrum of compound 1. (C) 1H-13C HSQC spectrum of compound 1. (D) 1H-13C HMBC spectrum of compound 1. Figure S2. NMR Spectra of compound 2. (A) Summary of key correlations between protons and carbons in compound 2 based on NMR spectroscopic data. (B) 1H-1H COSY spectrum of compound 2. (C) 1H-13C HSQC spectrum of compound 2. (D) 1H-13C HMBC spectrum of compound 2. Table S1. Antimicrobial activities of fermentation broth from S. ansochromogenes 7100 and Î wblA by agar diffusion assays.

With the accelerated globalization of the world economies, the role of intellectual property in the the competition is increasingly important. The universities are important base to instill the intellectual property awareness to the young generation. However, current model of intellectual property education cannot meet the needs of undergraduates. In this paper, we take the first patent issued for CRISPR/Cas9 system as a teaching example, and together with personal teaching experience in biomedicine related intellectual property, we propose a new way for intellectual property education which consists of two stages: enlightenment stage and in-depth training stage. In the former stage, we integrate the intellectual property education with the basic major courses. In the latter stage, students are encouraged to devote into intellectual property related career. This model can somehow solve the the current shortage of qualified teachers for biotechnology related intellectual property education and will facilitate the popularization of intellectual property in college students. Since genetics plays a pivotal role in biomedicine, this effort is illustrated by the novel genome editing technology based on the CRISPR/Cas9 system, which is one hotspot of recent studies. The trajectory of CRISPR/Cas9 from basic microbial genetics discovery to major tools for genome editing exeplified the essence of biomedicine related intellectual property education.

Lysine acetylation is emerging as a ubiquitous and conserved posttranslational modification in living cells. While the role of lysine acetylation in regulating primary metabolism is well-established, its function in secondary metabolism remains largely elusive. To gain insight into the nature, extent and biological function of lysine acetylation in Streptomyces reseosporus , a producer of daptomycin, dubiously dubbed as the trump card antibiotic, we used immunoaffinity-based acetyllysine peptide enrichment integrated with high resolution mass spectrometry to comprehensively characterize lysine acetylated proteins in this microbe. We identified a total of 667 proteins with 1143 unique sites, representing the largest acetylproteome reported to date in bacteria. Acetylated proteins belong to various functional classes such as metabolism and gene expression according to the gene ontology. We demonstrated for the first time that proteins involved in the biosynthesis of diverse secondary metabolites are acetylated, such as a nonribosomal peptide synthetase, enzymes essential for hydroxamate siderophore and phosphinic acid natural products biosynthesis, implying an important role of acetylation in these processes. Taken together, this proteomic analysis revealed a surprising breadth of cellular processes affected by lysine acetylation and also furnishes some fresh intervention nodes for the rational improvement of the antibiotic producer. Biological significance Despite considerable efforts have been devoted to elucidating the mechanism underlying secondary metabolism in Streptomyces , which are prolific producers of secondary metabolites with diverse biological activities, such as bacteriocides and antitumors, the full map of regulation and corresponding network is still far from perfect. Protein lysine acetylation is an evolutionarily conserved protein post-translational modification, abundantly existing in proteins with diverse biological context. We took advantage of integrated high throughput PTM proteomics followed by intensive bioinformatic analysis to profile lysine acetylome of Streptomyces roseosporus . In total, 1134 unique Kac sites in 667 lysine acetylated substrates were identified, representing the largest aceylomics in prokaryotes to date. Significantly, a nonribosomal peptide synthetase, an enzyme essential for hydroxamate siderophore and phosphinic acid natural products biosynthesis, was found to be acetylated. Given the conservation of these enzymes in biosynthesis of diverse secondary metabolites, lysine acetylation likely plays an important role in regulating secondary metabolism in Streptomyces .

Daptomycin-resistance in environmental actinomycetes was studied to provide warning systems for emerging clinical resistance.In total 49 soil and 10 endophytic acitnomycetes were used in this study. The daptomycin resistant strains were identified by measuring daptomycin resistance profile. Subsequently, daptomycin inactivating assay was preformed to distinguish resistance from other nondestructive mechanisms. Then, the strains of interest were determined by morphology and 16S rRNA gene sequence analysis. Finally, PCR analysis was used to detect the daptomycin acylase gene (dpa) encoding daptomycin acylase in those strains.All strains tested in this study were resistant towards daptomycin. Of them 24 soil acitnomycetes and 4 endophytic actinomycetes had the ability to inactivate daptomycin, while the remaining strains used other measures to confer resistance. Sequence analysis demonstrated that strains inactivating daptomycin were Streptomyces, Micromonospora and Norcadia. PCR analysis shows that 17.9% strains contained the dpa.There is very high percentage of resistance in environmental actinomycetes and inactivating daptomycin is one of the main resistant mechanisms.

Chong Li, Xiaolin Zhang, Xinliang Huang, Xiaoying Wang, Guojian Liao, Zhangbao ChenCollege of Pharmaceutical Sciences, Southwest University, Chongqing, People’s Republic of ChinaAbstract: The purpose of this study was to investigate flexible nanoliposomes for mediating topical delivery of daptomycin, and to document permeation rates and bacteriostatic activity towards skin infections. Response surface methodology was used to optimize the daptomycin-loaded flexible nanoliposomes (DAP-FL), and the amount of drug loaded into the particles was evaluated as the investigation index. The optimal lipid ratio was lecithin to sodium cholate 17:1 (w/w) and the lipid to drug ratio was 14:1 (w/w). The hydration temperature was set at 37°C and the duration of treatment with ultrasound was 20 minutes. The DAP-FL obtained had a small mean particle size (55.4 nm) with a narrow size distribution (polydispersity index 0.15). The mean entrapment efficiency was 87.85% ± 2.15% and the mean percent drug loading was 5.61% ± 0.14%. Using skin mounted between the donor and receptor compartments of a modified Franz diffusion cell, the percentage and quantity of cumulative daptomycin permeation from DAP-FL within 12 hours were measured at 96.28% ± 0.70% and (132.23 ± 17.73) µg/cm2 *5 = 661.15 ± 88.65 µg/cm2, directly, showing rapid and efficient antibacterial activity against Staphylococcus aureus. Following local administration of DAP-FL, daptomycin was detected in multilayer tissues within the skin and underlying structures in the dorsal skin of the mouse. Effective therapeutic concentrations were maintained for several hours, and significantly inhibited bacterial growth and injury-induced biofilms. These results demonstrate that the DAP-FL can enhance the ability of daptomycin to permeate the skin efficiently, where it has a powerful antibacterial action and activity against biofilms. This novel formulation of daptomycin has potential as a new approach in the clinical application of daptomycin.Keywords: daptomycin, liposomes, response surface methodology, biofilm