Genus-wide analysis of Trichoderma antagonism toward Pythium and Globisporangium plant pathogens and the contribution of cellulases to the antagonism.

Parasitism is an important lifestyle in the Trichoderma genus but has not been studied in a genus-wide way toward Pythium and Globisporangium hosts. Our approach screened a genus-wide set of 30 Trichoderma species in dual culture assays with two soil-borne Pythium and three Globisporangium plant-parasitic species and used exo-proteomic analyses, with the aim to correlate Trichoderma antagonism with potential strategies for attacking Pythium and Globisporangium. The Trichoderma spp. showed a wide range of antagonism from strong to weak, but the same Trichoderma strain showed similar levels toward all the Pythium and Globisporangium species. The Trichoderma enzymes from strong (Trichoderma asperellum, Trichoderma atroviride, and Trichoderma virens), moderate (Trichoderma cf. guizhouense and Trichoderma reesei), and weak (Trichoderma parepimyces) antagonists were induced by the autoclaved mycelia of one of the screened Pythium species, Pythium myriotylum. The variable proportions of putative cellulases, proteases, and redox enzymes suggested diverse as well as shared strategies amongst the antagonists. There was a partial positive correlation between antagonism from microscopy and the cellulase activity induced by autoclaved P. myriotylum mycelia in different Trichoderma species. The deletion of the cellulase transcriptional activator XYR1 in T. reesei led to lower antagonism toward Pythium and Globisporangium. The antagonism of Pythium and Globisporangium appears to be a generic property of Trichoderma as most of the Trichoderma species were at least moderately antagonistic. While a role for cellulases in the antagonism was uncovered, cellulases did not appear to make a major contribution to T. reesei antagonism, and other factors are also likely contributing.IMPORTANCETrichoderma is an important genus widely distributed in nature with broad ecological impacts and applications in the biocontrol of plant diseases. The Pythium and Globisporangium genera of fungus-like water molds include many important soil-borne plant pathogens that cause various diseases. Most of the Trichoderma species showed at least a moderate ability to compete with or antagonize the Pythium and Globisporangium hosts, and microscopy showed examples of parasitism (a slow type of killing) and predation (a fast type of killing). Hydrolytic enzymes such as cellulases and proteases produced by Trichoderma likely contribute to the antagonism. A mutant deficient in cellulase activity had reduced antagonism. Interestingly, Pythium and Globisporangium species contain cellulose in their cell walls (unlike true fungi such as Trichoderma), and the cellulolytic ability of Trichoderma appears beneficial for antagonism of water molds.

The recombinant swinepox virus expressing sseB could provide piglets with strong protection against Salmonella typhimurium challenge.

Salmonella spp. poses a great threat to the livestock, food safety and public health. A recombinant swinepox virus expressing a protective antigen sseB was constructed by homologous recombination to develop a vaccine against Salmonella infection. The rSPV-sseB was verified using PCR, Western blot and indirect immunofluorescence assay. The immune responses and protective efficacy of rSPV-sseB were assessed in piglets. Forty piglets were immunized with rSPV-sseB, inactive Salmonella vaccine, wild-type SPV (wtSPV), or PBS. The results showed that the level of the sseB-specific antibody of the rSPV-sseB-vaccinated piglets was significantly higher at all time points post-vaccination than those of the inactivated Salmonella vaccine (P < 0.05), wtSPV (P < 0.001) or mock treated piglets (P < 0.001). The IL-4 and IFN-γ in the rSPV-sseB group were significantly higher than the other three groups at all post-infection time points. rSPV-sseB provided piglets with strong protection against the challenge of S. typhimurium with lethal dose. These results suggest the possibility of using recombinant swinepox virus rSPV-sseB as a promising vaccine to prevent Salmonella infection.

Detailed Characterization of Antipathogenic Properties of Human Milk N-Glycome, against Staphylococcus aureus, Indicating Its Targeting on Cell Surface Proteins.

Human milk N-glycome was previously identified to have strong antipathogenic activities. This study is aimed to characterize the detailed antibacterial properties and the potential function mechanism of human milk N-glycome against Staphylococcus aureus. A serials of traditional antibacterial assays showed that human milk N-glycome possessed both bacteriostatic and bactericidal activities, which was further confirmed by the cell structure disruption including the change of transmembrane potential and leakage of intracellular contents. The results of the bacterial surface zeta potential and hydrophobicity, bacterial binding assay, gel shift assay, and fluorescence spectra and the different synergistic effects of human milk N-glycome combined with different antibiotics indicated that the bacterial surface proteins could be the targets of human milk N-glycome. Moreover, human milk N-glycome also showed antibiofilm activity. In conclusion, human milk N-glycome exhibited good potential for acting as an antibacterial substance against S. aureus and the antibacterial mechanism was a cell surface targeting action.

The mechanism of probiotic action of human milk N-glycome towards B. infantis ATCC 15697 and identification of the principal functional components.

Human milk N-glycome promotes the growth of Bifidobacterium longum subsp. infantis ATCC 15697. However, the action mode of, and the major functional components for, the bifidogenic function of human milk N-glycome remain unclear. In this study, we demonstrated that milk N-glycome was transferred in an intact form from culture into the bacterial cell and then decomposed intracellularly, evidenced by the following facts: (1) No UHPLC peak shift of N-glycome recovered from culture was observed. (2) No milk N-glycan specific monosugar was detected in culture supernatant. (3) High intracellular exoglycosidase activities were detected. (4) Fluorescently labeled N-glycans were found to be located intracellularly using Laser Scanning Confocal Microscopy (LSCM). Regarding the principal components identification, a novel sequential deglycosylation-based strategy was established. Degalactosylation, defucosylation-desialylation, and defucosylation-desialylation-degalactosylation treatments of human milk N-glycome showed that galactose-containing glycans were the principal components for the probiotic function of human milk N-glycome towards B. infantis ATCC 15697.

Protein Glycosylation and Gut Microbiota Utilization Can Limit the In Vitro and In Vivo Metabolic Cellular Incorporation of Neu5Gc.

SCOPE: Red meat intake is reported to be correlated with chronic diseases. A potential causal factor is N-glycolylneuraminic acid (Neu5Gc) which metabolically incorporates into diverse glycoconjugates in humans. This study aims to investigate the impact of exposure to Neu5Gc-rich red meat on healthy cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase (Cmah) knock-out mice and the underlying mechanisms. METHODS AND RESULTS: CMAH-/- mice are fed Neu5Gc-rich diet for short-term (4 months) and long-term (10 months). Health status and levels of inflammatory cytokines are assessed. Caco-2 cells are used to investigate the intestinal absorption of Neu5Gc-containing glycoprotein, and in vitro fermentation is used to investigate the Neu5Gc utilization by gut microbiota. Neu5Gc-rich diets show neither measurable abnormality in physio-biochemical and inflammatory indexes nor observable alterations of liver tissue in mice. Glycosylation of lactoferrin limits its intestinal epithelial absorption, and the absorption of Neu5Gc attach onto glycoprotein is thus limited. Neu5Gc is also simultaneously utilized by microorganisms under simulated gut conditions. CONCLUSION: The results indicate that the long-term intake of Neu5Gc-rich red meat has no adverse effect on the health of CMAH-/- mice, which may be related to the limited absorption of Neu5Gc that is regulated by protein glycosylation, and the metabolism of Neu5Gc by gut microorganisms.

Effects of Dietary Protein from Different Sources on Biotransformation, Antioxidation, and Inflammation in the Rat Liver.

In this work, the effects of different sources of meat protein on liver metabolic enzymes were investigated. Rats were fed for 90 days with semisynthetic diets in which casein was fully replaced by isolated soybean, fish, chicken, pork, or beef proteins. Then, liver proteomics was performed using iTRAQ and LC-ESI-MS/MS. The results indicated that intake of meat protein diets significantly reduced the protein levels of CYP450s, GSTs, UGTs, and SULTs compared to those of the casein and soybean protein diet groups. The total antioxidant capacity and lipid peroxidation values did not differ between four meat protein diet groups and the casein diet group. However, GSH activity in the fish, chicken, and beef protein groups was significantly higher than those of the casein and soybean protein groups. The beef protein diet significantly upregulated the expression of immune-related proteins. The Keap1-Nrf2-ARE signaling pathway was suggested to involve the diet-mediated regulation of biotransformation, inflammation, and redox status.

Beef, Chicken, and Soy Proteins in Diets Induce Different Gut Microbiota and Metabolites in Rats.

Previous studies have paid much attention to the associations between high intake of meat and host health. Our previous study showed that the intake of meat proteins can maintain a more balanced composition of gut bacteria as compared to soy protein diet. However, the associations between dietary protein source, gut bacteria, and host health were still unclear. In this study, we collected colonic contents from the growing rats fed with casein, beef, chicken or soy proteins for 90 days, and analyzed the compositions of gut microbiota and metabolites. Compared to the casein group (control), the chicken protein group showed the highest relative abundance of Lactobacillus and the highest levels of organic acids, including lactate, which can in turn promote the growth of Lactobacillus. The soy protein group had the highest relative abundance of Ruminococcus but the lowest relative abundance of Lactobacillus. Long-term intake of soy protein led to the up-regulation of transcription factor CD14 receptor and lipopolysaccharide-binding protein (LBP) in liver, an indicator for elevated bacterial endotoxins. In addition, the intake of soy protein also increased the levels of glutathione S-transferases in liver, which implicates elevated defense and stress responses. These results confirmed that meat protein intake may maintain a more balanced composition of gut bacteria and reduce the antigen load and inflammatory response from gut bacteria to the host.

Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria.

Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from rats fed with proteins from red meat (beef and pork), white meat (chicken and fish) and other sources (casein and soy). The results showed significant differences in profiles of gut bacteria between the six diet groups. Rats fed with meat proteins had a similar overall structure of caecal bacterial communities separated from those fed non-meat proteins. The beneficial genus Lactobacillus was higher in the white meat than in the red meat or non-meat protein groups. Also, rats fed with meat proteins and casein had significantly lower levels of lipopolysaccharide-binding proteins, suggesting that the intake of meat proteins may maintain a more balanced composition of gut bacteria, thereby reducing the antigen load and inflammatory response in the host.