Brain glucose induces tolerance of Cryptococcus neoformans to amphotericin B during meningitis.

Antibiotic tolerance is the ability of a susceptible population to survive high doses of cidal drugs and has been shown to compromise therapeutic outcomes in bacterial infections. In comparison, whether fungicide tolerance can be induced by host-derived factors during fungal diseases remains largely unknown. Here, through a systematic evaluation of metabolite-drug-fungal interactions in the leading fungal meningitis pathogen, Cryptococcus neoformans, we found that brain glucose induces fungal tolerance to amphotericin B (AmB) in mouse brain tissue and patient cerebrospinal fluid via the fungal glucose repression activator Mig1. Mig1-mediated tolerance limits treatment efficacy for cryptococcal meningitis in mice via inhibiting the synthesis of ergosterol, the target of AmB, and promoting the production of inositolphosphorylceramide, which competes with AmB for ergosterol. Furthermore, AmB combined with an inhibitor of fungal-specific inositolphosphorylceramide synthase, aureobasidin A, shows better efficacy against cryptococcal meningitis in mice than do clinically recommended therapies.

TLR2 regulates Moraxella catarrhalis adhesion to and invasion into alveolar epithelial cells and mediates inflammatory responses.

Moraxella catarrhalis is a major cause of chronic obstructive pulmonary disease. Toll-like receptor 2 (TLR2) plays an important role in the inflammatory response in host respiratory epithelial cells. M. catarrhalis induces an inflammatory immune response in respiratory epithelial cells that is mostly dependent on TLR2. However, the mechanisms by which this pathogen adheres to and invades the respiratory epithelium are not well understood. The present study aimed to reveal the role of TLR2 in M. catarrhalis adhesion to and invasion into alveolar epithelial cells, using molecular techniques. Pretreatment with the TLR2 inhibitor TLR2-IN-C29 enhanced M. catarrhalis adhesion to A549 cells but reduced its invasion, whereas the agonist Pam3CSK4 reduced both M. catarrhalis adhesion and invasion into A549 cells. Similarly, M. catarrhalis 73-OR strain adhesion and invasion were significantly reduced in TLR2-/- A549 cells. Moreover, the lung clearance rate of the 73-OR strain was significantly higher in TLR2-/- C57/BL6J mice than in wild-type (WT) mice. Histological analysis showed that inflammatory responses were milder in TLR2-/- C57/BL6J mice than in WT mice, which was confirmed by a decrease in cytokine levels in TLR2-/- C57/BL6J mice. Overall, these results indicate that TLR2 promoted M. catarrhalis adhesion and invasion of A549 cells and lung tissues and mediated inflammatory responses in infected lungs. This study provides important insights into the development of potential therapeutic strategies against M. catarrhalis and TLR2-induced inflammatory responses.

Fungicide-tolerant persister formation during cryptococcal pulmonary infection.

Bacterial persisters, a subpopulation of genetically susceptible cells that are normally dormant and tolerant to bactericides, have been studied extensively because of their clinical importance. In comparison, much less is known about the determinants underlying fungicide-tolerant fungal persister formation in vivo. Here, we report that during mouse lung infection, Cryptococcus neoformans forms persisters that are highly tolerant to amphotericin B (AmB), the standard of care for treating cryptococcosis. By exploring stationary-phase indicator molecules and developing single-cell tracking strategies, we show that in the lung, AmB persisters are enriched in cryptococcal cells that abundantly produce stationary-phase molecules. The antioxidant ergothioneine plays a specific and key role in AmB persistence, which is conserved in phylogenetically distant fungi. Furthermore, the antidepressant sertraline (SRT) shows potent activity specifically against cryptococcal AmB persisters. Our results provide evidence for and the determinant of AmB-tolerant persister formation in pulmonary cryptococcosis, which has potential clinical significance.

Virulence profiling of Cryptococcus gattii isolates in China: insights from a multi-center study.

IMPORTANCE: Our study indicates that the molecular typing of Cryptococcus gattii is unrelated to virulence. The integration of animal experiments and clinical prognosis demonstrated that pathogenicity did not exhibit a direct correlation with in vitro virulence phenotypes or molecular genotypes, emphasizing the intricate nature of virulence. In conclusion, our research holds the potential to provide valuable insights into understanding the microbiological attributes of C. gattii in China.

Effect of probiotic Bacillus cereus DM423 on the flavor formation of fermented sausage.

Insufficient protein and fat hydrolysis capacity of lactic acid bacteria (LAB) limit the flavor formation of fermented sausage. Bacillus is known for its substantial expression of proteases and lipases. However, its application in meat fermentation remains underexplored. In this study, a strain of probiotic Bacillus cereus (B. cereus DM423) was employed as a co-starter to improve the quality of Lactiplantibacillus plantarum (L. plantarum HH-LP56) fermented sausage. The addition of DM423 did not interfere with regular fermentation, but it significantly improved the flavor, as measured by electronic tongue and electronic nose. Further analyses using SDS-PAGE and thin-layer chromatography observed enhanced hydrolysis of protein and fat in sausages in which DM423 was involved in fermentation. GC-IMS identified DM423 mediated upregulation of various flavor compounds, including esters, ketones, furans, and branched-chain fatty acids. In addition, genomic de novo sequencing revealed that DM423 carried an abundance of genes associated with proteolysis, lipolysis, and the production of flavor substances, whereas HH-LP56 lacked these genes. Overall, this study finds that B. cereus DM423 can promote flavor formation in fermented sausages. It may illuminate a promising direction for the development of sausage co-starters from a wider microbial pool.

Polysaccharides from Platycodon grandiflorus attenuates high-fat diet induced obesity in mice through targeting gut microbiota.

The root of Platycodon grandiflorus (PG), abundant in soluble polysaccharides, has a long history in traditional Asian diets and herbal medicine due to its anti-inflammatory activity and anti-obesity effects. Our previous study was the first to establish a link between the beneficial effects of PG and changes in the gut microbiota, and suggested potential roles that the polysaccharide components play. However, more evidence was needed to understand the anti-obesity functions of polysaccharides from PG (PS) and their relationship with the regulation of the gut microbiota. In this study, we first performed an experiment to explore the anti-obesity activities of PS: Male C57BL/6 mice (six-weeks-old) were fed either a standard control diet (CON), or a high-fat diet (HFD) to induce obesity, or a HFD supplemented with PS (HFPS) for 8 weeks. Body weight and food intake were monitored throughout. Lipid metabolism were determined and related gene expression changes in adipose tissues were analyzed by RNA-seq. Amplicon sequencing of the bacterial 16 S rRNA gene was used to explore gut microbiota structure in fecal samples. Then, we performed the second experiment to explore whether the anti-obesity activities of PS were dependent on the regulation of the gut microbiota: Male C57BL/6 mice (six-weeks-old), treated with an antibiotic cocktail to reduce the gut microbial load, were fed either a HFD (A-HFD) or a HFPS (A-HFPS) diet for 8 weeks. Finally, we used in vitro fermentation experiments to verify the effects of PS on the growth and metabolic activities of the gut microbes. We found that PS significantly reduced HFD-induced weight gain and excessive fat accumulation, changed the expression of key genes involved in lipid metabolism, and attenuated HFD-induced changes in the gut microbiota. However, PS did not affect fat accumulation or lipid metabolism in the gut microbiota depleted mice. Overall, our results show that PS has significant effects on the gut microbiota in the mouse model, and the anti-obesity effects of PS are mediated via changes in the gut microbiota composition and metabolic activity.

Bile Acid Derivatives Effectively Prevented High-Fat Diet-Induced Colonic Barrier Dysfunction.

SCOPE: Bile acids (BAs) have recently emerged as important regulators of many physiological and pathological processes. However, the change of colonic BAs induced by high-fat diet (HFD) and their effects on colonic barrier function remain to be further elucidated. METHODS AND RESULTS: C57BL/6 mice are divided into two groups and feed 12 weeks with diets differing for fat content. Higher levels of serum diamine oxidase (DAO) activity, endotoxin (ET), and d-lactate (d-LA) are observed in HFD-fed mice, indicating an increase in intestinal permeability. Real-time quantitative PCR and western blot analyses demonstrate that HFD downregulates tight junction proteins (TJs, including zonula-occludens 1 [ZO-1], Occludin, and Claudin1) and Muc2 expression in the colon. The colonic BA profiles are analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). HFD induces an increase in primary BAs but a decrease in secondary BAs. In human colonic cell line Caco-2, secondary BAs (deoxycholic acid [DCA], lithocholic acid [LCA], their 3-oxo- and iso- derivates) upregulate the expression of TJs and counteract DSS-induced increase in intestinal permeability at physiological concentrations. IsoDCA and isoLCA are the most effective ones. Moreover, supplementation of isoDCA or isoLCA also effectively prevents HFD-induced colonic barrier dysfunction in mice. CONCLUSION: These results demonstrate that secondary BAs (especially isomerized derivatives) may be important protectors for the colonic barrier function.

Correction: Molecular epidemiology and microbiological characteristics of Cryptococcus gattii VGII isolates from China.

[This corrects the article DOI: 10.1371/journal.pntd.0010078.].

Regulatory basis for reproductive flexibility in a meningitis-causing fungal pathogen.

Pathogenic fungi of the genus Cryptococcus can undergo two sexual cycles, involving either bisexual diploidization (after fusion of haploid cells of different mating type) or unisexual diploidization (by autodiploidization of a single cell). Here, we construct a gene-deletion library for 111 transcription factor genes in Cryptococcus deneoformans, and explore the roles of these regulatory networks in the two reproductive modes. We show that transcription factors crucial for bisexual syngamy induce the expression of known mating determinants as well as other conserved genes of unknown function. Deletion of one of these genes, which we term FMP1, leads to defects in bisexual reproduction in C. deneoformans, its sister species Cryptococcus neoformans, and the ascomycete Neurospora crassa. Furthermore, we show that a recently evolved regulatory cascade mediates pre-meiotic unisexual autodiploidization, supporting that this reproductive process is a recent evolutionary innovation. Our findings indicate that genetic circuits with different evolutionary ages govern hallmark events distinguishing unisexual and bisexual reproduction in Cryptococcus.

The effects of high pressure treatment on the structural and digestive properties of myoglobin.

The effects of high pressure treatment (100-400 MPa for 20 min) on the structural and digestive properties of myoglobin were investigated by UV-vis absorption spectroscopy, intrinsic and synchronous fluorescence spectroscopy and molecular dynamics simulation. High pressure treatment induced the exposure of aromatic residues and changed the interaction between heme and globin, which in turn increased the gastrointestinal digestibility of myoglobin. Molecular dynamics simulation indicated that the hydrophobic interaction and hydrogen bonds of the myoglobin-pepsin complex were weakened after high pressure treatment, but the dipolar interaction was strengthened. The findings revealed the mechanisms on high pressure-induced increase in digestibility of myoglobin.

A Velvet Transcription Factor Specifically Activates Mating through a Novel Mating-Responsive Protein in the Human Fungal Pathogen Cryptococcus deneoformans.

Sexual reproduction facilitates infection by the production of both a lineage advantage and infectious sexual spores in the ubiquitous human fungal pathogen Cryptococcus deneoformans. However, the regulatory determinants specific for initiating mating remain poorly understood. Here, we identified a velvet family regulator, Cva1, that strongly promotes sexual reproduction in C. deneoformans. This regulation was determined to be specific, based on a comprehensive phenotypic analysis of cva1Δ under 26 distinct in vitro and in vivo growth conditions. We further revealed that Cva1 plays a critical role in the initiation of early mating events, including sexual cell-cell fusion, but is not important for the late sexual development stages or meiosis. Thus, Cva1 specifically contributes to mating activation. Importantly, a novel mating-responsive protein, Cfs1, serves as the key target of Cva1 during mating, since its absence nearly blocks cell-cell fusion in C. deneoformans and its sister species C. neoformans. Together, our findings provide insight into how C. deneoformans ensures the regulatory specificity of mating. IMPORTANCE The human fungal pathogen C. deneoformans is a model organism for studying fungal sexual reproduction, which is considered to be important to infection. However, the specific regulatory determinants for activation of sexual reproduction remain poorly understood. In this study, by combining transcriptomic and comprehensive phenotypic analysis, we identified a velvet family regulator Cva1 that specifically and critically elicits early mating events, including sexual cell-cell fusion. Significantly, Cva1 induces mating through the novel mating-responsive protein Cfs1, which is essential for cell-cell fusion in C. deneoformans and its sister species C. neoformans. Considering that Cva1 and Cfs1 are highly conserved in species belonging to Cryptococcaeceae, they may play conserved and specific roles in the initiation of sexual reproduction in this important fungal clade, which includes multiple human fungal pathogens.

Molecular epidemiology and microbiological characteristics of Cryptococcus gattii VGII isolates from China.

Cryptococcus gattii (C. gattii) is a fungal pathogen that once caused an outbreak of cryptococcosis on Vancouver Island, and had spread worldwide, while few data were available in China. In this study, seven clinical isolates of C. gattii VGII were collected from 19 hospitals, Multi-locus Sequence Typing (MLST) analysis and whole-genome sequencing (WGS) was performed, combined with published data for phylogenetic analysis. In addition, in vitro antifungal susceptibility testing, phenotypic analysis, and in vivo virulence studies were performed, subsequently, histopathological analysis of lung tissue was performed. C.gattii VGII infected patients were mainly immunocompetent male, and most of them had symptoms of central nervous system (CNS) involvement. MLST results showed that isolates from China exhibited high genetic diversity, and sequence type (ST) 7 was the major ST among the isolates. Some clinical isolates showed a close phylogenetic relationship with strains from Australia and South America. All clinical isolates did not show resistance to antifungal drugs. In addition, there was no correlation between virulence factors (temperature, melanin production, and capsule size) and virulence while in vivo experiments showed significant differences in virulence among strains. Lung fungal burden and damage to lung tissue correlated with virulence, and degree of damage to lung tissue in mice may highlight differences in virulence. Our work seeks to provide useful data for molecular epidemiology, antifungal susceptibility, and virulence differences of C. gattii VGII in China.

The degree of doneness affected molecular changes and protein digestibility of pork.

The degree of doneness has been shown to have a great impact on eating quality of meat, however, it is little known whether it affects protein digestibility of meat. In this study, we explored molecular changes and protein digestibility of pork under different degree of doneness. Pork chops were cooked in a 100°C water bath for about 26 min and a gradient decrease in doneness was obtained from outer to inner layers of samples. Compared with the raw samples, the cooked samples' active and total sulfhydryl contents, surface hydrophobicity, and turbidity increased but its solubility decreased. The inner layers with lower doneness contained higher α-helix, and fluorescence intensities of tryptophan and tyrosine residues than the outer layers with higher doneness. The pepsin and pancreatin digestibility of meat proteins in the inner layers were higher than those of the outer layers. Molecular simulation analysis showed that the most abundant protein in pork, i.e., myosin in the outer layers were more stable with an increased number of hydrogen bonds, making it difficult to be digested. These findings provided a new insight into the heterogeneity of meat nutritional quality due to the existence of doneness gradient.

Effect of Sous-vide cooking on the quality and digestion characteristics of braised pork.

This study aimed to evaluate the effect of Sous-vide (SV) cooking on the quality, flavor and digestion characteristics of braised pork. Traditional (TD) sample had the highest fat content and malondialdehyde (MDA) value, but the lowest protein content and total sulfhydryl (SH) content (P < 0.05). The SH content in SV samples decreased with the heating time, but MDA content increased (P < 0.05). In addition, α-helix content of TD samples was higher but ß-sheet content was lower than SV samples. LC-MS/MS indicated that SV samples at 65 °C for 8 h had potential bioactive and antioxidant peptides. GC-MS mainly identified 395 volatile components and SV samples at 70 °C for 8 h and 75 °C for 8 h had similar flavor compounds to TD samples. Thus, Sous-vide cooking produced better quality and digestion characteristics of braised pork. These findings give a new insight into the associations of processing methods with meat quality.

Application of ultrasound treatment for improving the quality of infant meat puree.

Infant meat puree has an indispensable effect on the oral development and nutritional intake of infants. However, commercially available products have poor texture and relatively low digestibility. In this study, ultrasound (20 kHz and 200 W, 400 W, or 600 W) was applied to the pretreatment of raw meat for preparing infant meat puree for 15 min, 30 min, and 45 min. To assess the impact of ultrasound on infant meat puree, the viscosity, texture, water distribution, particle size and in vitro digestibility were determined. The results showed that, compared with control, viscosity and hardness of meat puree decreased and the texture was better in 400 W and 600 W groups. The content of immobilized water increased in comparison with the control. Ultrasound had no obvious effect on the digestibility of meat puree in gastric phase, but it increased the digestibility in intestinal phase with the highest digestibility (80.85%±3.33) in 600 W, 15 min group. Overall, the ultrasound parameters of 600 W for 15 min can be selected as the best condition to process infant meat puree. The findings provide a new perspective for the improvement of infant meat puree.

Quorum Sensing in Fungal Species.

Quorum sensing (QS) is one of the most studied cell-cell communication mechanisms in fungi. Research in the last 20 years has explored various fungal QS systems that are involved in a wide range of biological processes, especially eukaryote- or fungus-specific behaviors, mirroring the significant contribution of QS regulation to fungal biology and evolution. Based on recent progress, we summarize in this review fungal QS regulation, with an emphasis on its functional role in behaviors unique to fungi or eukaryotes. We suggest that using fungi as genetically amenable eukaryotic model systems to address why and how QS regulation is integrated into eukaryotic reproductive strategies and molecular or cellular processes could be an important direction for QS research.

A cyclin protein governs the infectious and sexual life cycles of Cryptococcus neoformans.

Cell cycle is a fundamental process underlying growth and development in evolutionarily diverse organisms, including fungi. In human fungal pathogens, cell cycle control generally determines their life cycles, either in the environment or during infections. Thus, cell cycle components can potentially serve as important targets for the development of antifungal strategy against fungal infections. Here, in Cryptococcus neoformans, the most common cause of fatal fungal meningitis, we show that a previously uncharacterized B-type cyclin named Cbc1 is essential for both its infectious and sexual cycles. We reveal that Cbc1 coordinates various sexual differentiation and molecular processes, including meiosis. Especially, the absence of Cbc1 abolishes formation of sexual spores in C. neoformans, which are presumed infectious particles. Cbc1 is also required for the major Cryptococcus pathogenic attributes. Virulence assessment using the murine model of cryptococcosis revealed that the cbc1 mutant is avirulent. Together, our results provide an important insight into how C. neoformans employs shared cell cycle regulation to coordinate its infectious and sexual cycles, which are considered crucial for virulence evolution and the production of infectious spores.

Suppression of High-Fat Diet-Induced Obesity by Platycodon Grandiflorus in Mice Is Linked to Changes in the Gut Microbiota.

BACKGROUND: The root of Platycodon grandiflorus (PG) has a long-standing tradition in the Asian diet and herbal medicine, because of its anti-inflammatory and antiobesity effects. Changes in the gut microbiota can have dietary effects on host health, which suggests a relation between the 2. OBJECTIVES: The aim of our study was to investigate the relation between PG-mediated suppression of obesity and the composition and functioning of the gut microbiota. METHODS: Six-week-old male C57BL/6J mice were fed either a control diet (CON, 10% kcal from fat), a high-fat diet (HFD, 60% kcal from fat), or a PG-supplemented HFD for 18 wk. PG was administered by oral gavage at 2 g · kg body weight-1 · d-1. Body weight and food intake were monitored. Lipid metabolism, inflammation, and intestinal barrier function were determined. Amplicon sequencing of the bacterial 16S ribosomal RNA gene was used to explore gut microbiota structure, and nontargeted metabolomics analysis was performed to investigate metabolite concentrations in fecal samples. RESULTS: We found that PG significantly ameliorated HFD-induced inflammation, recovered intestinal barrier integrity (reduced permeability by 39% , P = 0.008), reduced fat accumulation by 26% (P = 0.009), and changed the expression of key genes involved in the development of white adipose tissue (P < 0.05) in HFD-fed mice to similar levels in CON mice. Moreover, PG attenuated HFD-induced changes in the gut microbiota; it especially increased Allobaculum (7.3-fold, P = 0.002) relative to HFD, whereas CON was 15.2-fold of HFD (P = 0.002). These changes by PG were associated with an increase in the production of SCFAs (butyrate and propionate, P < 0.001) and other carbohydrate-related metabolites known to have a major role in disease suppression. CONCLUSIONS: Our study demonstrated that PG beneficially changed the gut microbiota and the gut metabolome in HFD-fed mice, and suggests that the antiobesity effects of PG may be mediated via changes in gut microbiota composition and metabolic activity.

Targeting the gut microbiota with resveratrol: a demonstration of novel evidence for the management of hepatic steatosis.

Resveratrol is a natural polyphenol that has been reported to reduce the risk of obesity and nonalcoholic fatty liver disease (NAFLD). Recent evidence has demonstrated that the gut microbiota plays an important role in the protection against NAFLD and other metabolic diseases. The present study aimed to investigate the relationship between the gut microbiota and the beneficial effects of resveratrol on the amelioration of NAFLD in mice. We observed marked decreases in body weight and liver steatosis and improved insulin resistance in high-fat diet (HFD)-fed mice treated with resveratrol. Furthermore, we found that resveratrol treatment alleviated NAFLD in HFD-fed mice by improving the intestinal microenvironment, including gut barrier function and gut microbiota composition. On the one hand, resveratrol improved gut intestinal barrier integrity through the repair of intestinal mucosal morphology and increased the expression of physical barrier- and physiochemical barrier-related factors in HFD-fed mice. On the other hand, in HFD-fed mice, resveratrol supplementation modulated the gut bacterial composition. The resveratrol-induced gut microbiota was characterized by a decreased abundance of harmful bacteria, including Desulfovibrio, Lachnospiraceae_NK4A316_group and Alistipes, as well as an increased abundance of short-chain fatty acid (SCFA)-producing bacteria, such as Allobaculum, Bacteroides and Blautia. Moreover, transplantation of the HFDR-microbiota into HFD-fed mice sufficiently decreased body weight, liver steatosis and low-grade inflammation and improved hepatic lipid metabolism. Collectively, resveratrol would provide a potentially dietary intervention strategy against NAFLD through modulating the intestinal microenvironment.

Resveratrol reduces obesity in high-fat diet-fed mice via modulating the composition and metabolic function of the gut microbiota.

Resveratrol (RSV) is a natural polyphenol with anti-obesity effects. However, the mechanisms of anti-obesity remain unclear due to its low bioavailability. Recent evidence demonstrates that gut microbiota plays a key role in obesity. This spurred us to investigate whether the anti-obesity effects of RSV are related to modulations in the gut microbiota and metabolic functions. Here, RSV significantly improved metabolic phenotype and intestinal oxidative stress in the high-fat diet (HFD)-fed mice. A multi-omics approach was used to systematically profile the microbial signatures at both the phylogenetic and functional levels using 16S rRNA gene sequencing and metagenome. At the phylogenetic level, RSV treatment significantly modulated the gut microbiota composition in HFD-fed mice, characterized with increased Blautia abundance and decreased Desulfovibrio and Lachnospiraceae_NK4A136_group abundance. At the functional level, RSV significantly decreased the enrichment of pathways linked to host metabolic disease and increased the enrichment of pathways involved in the generation of small metabolites. Besides, the fecal microbiota transplantation experiment showed anti-obesity and microbiota-modulating effects similar to those observed in the oral RSV-feeding experiment. Furthermore, metabolomic analysis and antibiotic treatment verified that 4-hydroxyphenylacetic acid (4-HPA) and 3-hydroxyphenylpropionic acid (3-HPP) were the two gut metabolites of RSV, which contribute to improving lipid metabolism in vitro. Moreover, the content of 4-HPA and 3-HPP exhibited strong correlation with the intestinal oxidative state. We concluded that the RSV-mediated alteration of gut microbiota, related gut metabolites and redox state of the intestinal environment contributed to prevention of metabolic syndrome in HFD-fed mice.