The function and application of edible fungal polysaccharides.

Edible fungi, commonly known as mushrooms, are precious medicinal and edible homologous gifts from nature to us. Edible fungal polysaccharides (EFPs) are a variety of bioactive macromolecular which isolated from fruiting bodies, mycelia or fermentation broths of edible or medicinal fungus. Increasing researches have confirmed that EFPs possess multiple biological activities both in vitro and in vivo settings, including antioxidant, antiviral, anti-inflammatory, immunomodulatory, anti-tumor, hypoglycemic, hypolipidemic, and regulating intestinal flora activities. As a result, they have emerged as a prominent focus in the healthcare, pharmaceutical, and cosmetic industries. Fungal EFPs have safe, non-toxic, biodegradable, and biocompatible properties with low immunogenicity, bioadhesion ability, and antibacterial activities, presenting diverse potential applications in the food industries, cosmetic, biomedical, packaging, and new materials. Moreover, varying raw materials, extraction, purification, chemical modification methods, and culture conditions can result in variances in the structure and biological activities of EFPs. The purpose of this review is to provide comprehensively and systematically organized information on the structure, modification, biological activities, and potential applications of EFPs to support their therapeutic effects and health functions. This review provides new insights and a theoretical basis for prospective investigations and advancements in EFPs in fields such as medicine, food, and new materials.

A novel mutation of walK confers vancomycin-intermediate resistance in methicillin-susceptible Staphylococcus aureus.

With the treatment failure by vancomycin and poor clinical outcomes, the emergence and spread of vancomycin intermediate-resistant Staphylococcus aureus (VISA) has raised more concerns in recent years. While most VISA strains are isolated from methicillin-resistant S. aureus (MRSA), the mechanism underlying the generation of VISA from methicillin-susceptible S. aureus (MSSA) is still largely unknown. Here, we identified a total of 10 mutations in 9 genes through comparative genome analysis from laboratory-derived VISA strain. We verified the role of a novel mutation of WalK (I237T) and our results further indicated that the introduction of WalK (I237T) by allelic replacement can confer vancomycin resistance in MSSA with common VISA characteristics, including thickened cell walls, reduced autolysis, and attenuated virulence. Consistent with these phenotypes, real-time quantitative reverse transcription-PCR revealed the altered expression of several genes associated with cell wall metabolism and virulence control. In addition, electrophoretic mobility shift assay indicated that WalR can directly bind to the promoter regions of oatA, sle1, and mgt, fluorescence-based promoter activity and ß-galactosidase assays revealed WalK (I237T) can alter promoter activities of oatA, mgt, and sle1, thus regulating genes expression. These findings broaden our understanding of the regulatory network by WalKR system and decipher the molecular mechanisms of developmental VISA resistance in MSSA with point mutations.

Construction of a Gene Knockdown System Based on Catalytically Inactive ("Dead") Cas9 (dCas9) in Staphylococcus aureus.

There has been an absence of an efficient method of gene knockdown in the important human pathogen Staphylococcus aureus like RNA interference in eukaryotes. The previously developed antisense RNA technology is mainly applied for forward genetic screening but is rather limited in specific gene knockdown because of the lack of rational antisense RNA design strategies. Here we report an efficient and specific system for gene knockdown in S. aureus based on the type II clustered regularly interspaced short palindromic repeat (CRISPR) system from Streptococcus pyogenes We can achieve gene silencing with the coexpression of dCas9, an RNA-guided DNA binding protein, and a small guide RNA complementary to the target gene. With this system, we have successfully silenced diverse sets of genes varying in size and expression level in different S. aureus strains. This system exhibited high-efficiency knockdown of both essential and nonessential genes, and its effect is inducible and reversible. In addition, the system can repress the expression of multiple genes simultaneously and silence an entire operon or part of it. This RNA-guided DNA targeting system thus provides a simple, rapid, and affordable method for selective gene knockdown in S. aureusIMPORTANCEStaphylococcus aureus is an important human and animal pathogen that can cause a diversity of infectious diseases. Molecular genetic study of S. aureus has provided an avenue for the understanding of its virulence, pathogenesis, and drug resistance, leading to the discovery of new therapies for the treatment of staphylococcal infections. However, methodologies developed for genetic manipulation of S. aureus usually involve either low efficiency or laborious procedures. Here we report an RNA-guided system for gene knockdown in S. aureus and show its high efficiency and simplicity for selective gene silencing in different strains of S. aureus This simple, rapid, and affordable system may serve as a promising tool for functional gene study in S. aureus, especially for the study of essential genes, thus facilitating the understanding of this pathogen and its interaction with its hosts.

SigmaB regulates ccrAB expression and SCCmec excision in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide pathogen that is resistant to practically the entire class of ß-lactam antibiotics due to the presence of the mecA gene. The mecA gene is located on a large mobile genetic element referred to as staphylococcal cassette chromosome mec (SCCmec), and the excision and integration of SCCmec are mediated by the Ccr recombinase encoded by ccrAB or ccrC, which are also located on SCCmec. Previous studies have shown that the ccrAB genes are only expressed in a minority of cells and that their expression levels can be affected by certain environmental stimuli, but the molecular mechanisms controlling these phenotypes remain elusive. Here, we found that overexpression of SigB can dramatically enhance ccrA transcription and SCCmec excision in MRSA strain N315, revealing an important role for this alternative sigma factor in the lateral transfer of SCCmec. Further primer extension-blot analysis and 5'RACE (Rapid Amplification of cDNA Ends) indicated that an unrecognized SigB-dependent promoter region, which exists in certain SCCmec type II and IV strains, is responsible for the enhancement, and the ccrAB genes are in fact transcribed in a two-promoter pattern with a low activity of the SigB-dependent promoter under normal growth conditions.

Effects of copper on induction of thiol-compounds and antioxidant enzymes by the fruiting body of Oudemansiella radicata.

Oudemansiella radicata has been found to have ability to tolerate and accumulate heavy metals. In this study, to know about the metal tolerance and detoxification strategy of O. radicata, the tolerance responses in both cap and stipe of the fruiting body, including the copper content, the changes of thiol compounds production and antioxidant enzymes activities, caused by various copper stress (150-600 mg kg(-1)) during 2-6 days were investigated. Results showed that Cu content in the fruiting bodies increased with the increasing Cu concentrations and growing time, which was higher in cap than that in stipe. For thiols contents, the maximum level was in the sample at 300 mg kg(-1) Cu after 2 d both in cap and stipe, in accordance with superoxide dismutase (SOD) and glutathione reductase (GR) activities. Guaicol peroxidase (POD) activities reached maximum at 150 mg kg(-1) Cu after 4 d and 6 d, respectively in cap and stipe, while the maximum of catalase (CAT) activities was recorded at 300 and 600 mg kg(-1) Cu after 4 d in the cap and stipe, respectively. As a whole, low concentration of Cu stimulated the production of thiols and activated the antioxidant enzymes activities in the fruiting body of O. radicata after 2/4 d, while high-level Cu decreased the thiols production and enzymes activities after 4/6 d. Furthermore, the cap was more sensitive than the stipe to Cu exposure. Different indicators showed different responses to copper accumulation and the different fruiting part (cap and stipe) of O. radicata had ability to response the oxidative stress caused by Cu. Considering the metal accumulation and its own detoxification with short growing time, mushroom might have the potential to be used as bio-accumulator to deal with Cu exposure in the Cu-contaminated farmland soil.

Transcription tuned by S-nitrosylation underlies a mechanism for Staphylococcus aureus to circumvent vancomycin killing.

Treatment of Staphylococcus aureus infections is a constant challenge due to emerging resistance to vancomycin, a last-resort drug. S-nitrosylation, the covalent attachment of a nitric oxide (NO) group to a cysteine thiol, mediates redox-based signaling for eukaryotic cellular functions. However, its role in bacteria is largely unknown. Here, proteomic analysis revealed that S-nitrosylation is a prominent growth feature of vancomycin-intermediate S. aureus. Deletion of NO synthase (NOS) or removal of S-nitrosylation from the redox-sensitive regulator MgrA or WalR resulted in thinner cell walls and increased vancomycin susceptibility, which was due to attenuated promoter binding and released repression of genes involved in cell wall metabolism. These genes failed to respond to H2O2-induced oxidation, suggesting distinct transcriptional responses to alternative modifications of the cysteine residue. Furthermore, treatment with a NOS inhibitor significantly decreased vancomycin resistance in S. aureus. This study reveals that transcriptional regulation via S-nitrosylation underlies a mechanism for NO-mediated bacterial antibiotic resistance.

Localization of receptor site on insect sodium channel for depressant ß-toxin BmK IT2.

BACKGROUND: BmK IT2 is regarded as a receptor site-4 modulator of sodium channels with depressant insect toxicity. It also displays anti-nociceptive and anti-convulsant activities in rat models. In this study, the potency and efficacy of BmK IT2 were for the first time assessed and compared among four sodium channel isoforms expressed in Xenopus oocytes. Combined with molecular approach, the receptor site of BmK IT2 was further localized. PRINCIPAL FINDINGS: 2 µM BmK IT2 strongly shifted the activation of DmNa(v)1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNa(v)1.2, rNa(v)1.3 and mNa(v)1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu(896), Leu(899), Gly(904) in extracellular loop Domain II S3-S4 of DmNa(v)1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNa(v)1 could be conferred by Domain III. Analysis of subsequent DmNa(v)1 mutants highlighted the residues in Domain III pore loop, esp. Ile(1529) was critical for recognition and binding of BmK IT2. CONCLUSIONS/SIGNIFICANCE: In this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNa(v)1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Na(v)1.2, Na(v)1.3 and Na(v)1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.

Phenotypes and peripheral mechanisms underlying inflammatory pain-related behaviors induced by BmK I, a modulator of sodium channels.

The integrated mechanisms of dynamic signaling of sodium channels involved in clinical pain are still not yet clear. In this study, a new rat inflammatory pain model was developed by using the unilateral intraplantar injection of BmK I, a receptor site 3-specific modulator of sodium channels from the venom of scorpion Buthus martensi Karsch (BmK). It was found that BmK I could induce several kinds of inflammatory pain-related behaviors including spontaneous pain companied with unique episodic paroxysms, primary thermal hypersensitivity, and mirror-image mechanical hypersensitivity with different time course of development, which could be suppressed by morphine, indomethacin, or bupivacaine to a different extent. The dramatic attenuation by pretreatment with resiniferatoxin (RTX), an ultrapotent analog of capsaicin, on BmK I-induced pain-related behaviors, paw edema, and spinal L4-L5 c-Fos expression demonstrated that capsaicin-sensitive primary afferent neurons played important roles in pain induced by BmK I. Furthermore, the electrophysiological recordings showed that BmK I persistently increased whole-cell and tetrodotoxin-resistant (TTX-R) peak sodium currents and significantly delayed the inactivation phase of whole-cell sodium currents but could not enhance capsaicin-evoked inward currents, in acute isolated small dorsal root ganglion neurons of rat. The results strongly suggested that the dynamic modulation of BmK I on sodium channels located in peripheral primary afferent neurons, especially in capsaicin-sensitive neurons, mediated pain sensation. Thus, BmK I may be a valuable pharmacological tool to understand the sodium channel-involved pain mechanisms.