Showing papers by "Jin Zhong published in 2019"

Lactobacillus plantarum PFM 105 Promotes Intestinal Development Through Modulation of Gut Microbiota in Weaning Piglets.

Abstract: Lactobacillus plantarum is a widespread bacterial species and is commonly used as a probiotic. L. plantarum PFM105 was isolated from the rectum of a healthy sow. Here we found that L. plantarum PFM105 showed probiotic effect on weaning piglets in which intestinal inflammation and unbalanced gut microbiota happened frequently. L. plantarum PFM105 was identified to improve the growth of weaning piglet and promote the development of small intestinal villi. Antibiotics are often used in weaning piglet to prevent intestinal infection and promote the growth of animal. We found that weaning piglets feeding with L. plantarum PFM105 showed similar growth promotion but decreased diarrhea incidence compared with those feeding with antibiotics. High-throughput sequencing was used to analyze the gut microbiota in weaning piglets treated with L. plantarum PFM105 or antibiotics. The relative abundance of beneficial microbes Prevotellaceae and Bifidobacteriaceae were increased in colon of weaning piglet feeding L. plantarum PFM105, while antibiotics increased the relative abundance of bacteria associated with pathogenicity, such as Spirochaeta and Campylobacteraceae. L. plantarum PFM 105 increased indicators of intestinal health including serum levels of IgM, IL-10, and TGF-β, and colonic levels of SCFAs. We found strong correlations between the alterations in gut microbiota composition caused by feeding antibiotics and probiotics and the measured growth and health parameters in weaning piglets. The addition of L. plantarum PFM105 could significantly increase the relative abundance of metabolic genes which may important to intestinal microbiota maturation. Altogether, we demonstrated here that L. plantarum PFM 105 could promote intestinal development through modulation of gut microbiota in weaning piglets.

Effects of ferulic acid esterase-producing Lactobacillus fermentum and cellulase additives on the fermentation quality and microbial community of alfalfa silage

Abstract: Background Alfalfa (Medicago sativa) is an important forage material widely used for animal feed production. Ensiling is an effective method for preserving alfalfa, but it has shown some limitations in the production of high-quality alfalfa silage due to its low water soluble carbohydrates (WSC) content and high buffering capacity. Lactic acid bacteria (LAB) and cellulase are often used as silage additives to promote the ensiling process and enhance fermentation quality. Methods Experiments were conducted to investigate the effects of ferulic acid esterase (FAE)-producing Lactobacillus fermentum 17SD-2 (LF) and cellulase (CE) on the fermentation quality and microbial community of alfalfa silage. After 60 days of ensiling, analysis of fermentation quality and bacterial diversity in alfalfa silages were conducted using high-performance liquid chromatography and high-throughput sequencing methods. Results Alfalfa was ensiled with additives (LF, CE, and LF+CE) or without additives for 60 days. All additives increased lactic acid and decreased pH values and ammonia-N contents compared to control. Among all treatments, the combined addition of LF and CE showed lowest pH (4.66) and ammonia-N (NH3-N, 0.57% DM) content, highest contents of lactic acid (LA, 10.51% DM), dry matter (DM, 22.54%) and crude protein (CP, 24.60% DM). Combined addition of LF and CE performed better in reducing neutral detergent fiber (NDF, 29.76% DM) and acid detergent fiber (ADF, 22.86% DM) contents than the addition of LF (33.71, 27.39% DM) or CE (32.07, 25.45% DM) alone. Moreover, the microbial analysis indicated that LF+CE treatments increased the abundance of desirable Lactobacillus and inhibited the growth of detrimental Enterobacter and Clostridia in alfalfa silage. Discussion Combined addition of FAE-producing LF and CE is more effective than treatments of LF or CE alone in improving fermentation quality and nutrition values of alfalfa silage. This is likely due to a synergistic effect of CE and FAE produced by LF on plant cell wall degradation, indicating that these additives promote each other to improve fiber degradation and silage fermentation. In conclusion, combined addition of FAE-producing LF and CE could be a feasible way to improve alfalfa silage quality.

A Nanoparticle-Based Hepatitis C Virus Vaccine With Enhanced Potency.

Abstract: Despite the emergence of new direct-acting antivirals, hepatitis C virus (HCV) chronic infection and its consequent fibrosis and hepatocarcinoma remain a significant burden for public health, thus requiring an effective preventive vaccine. Our group previously showed that a subunit vaccine based on recombinant soluble E2 (sE2) can induce broadly neutralizing antibodies. To improve the immunogenicity of sE2, we designed and produced a fusion protein (sE2-ferritin) comprising sE2 and a ferritin unit in Drosophila S2 cells, which self-assembled into a nanoparticle with sE2 displayed on the surface. The sE2 moiety on the sE2-ferritin nanoparticle not only had nearly natural conformation but also had better affinities than the unfused sE2 to neutralizing antibodies, receptor, and patient serum. Mouse immunization studies showed that sE2-ferritin was more potent than sE2 in inducing anti-HCV broadly neutralizing antibodies. Our results demonstrate that sE2-ferritin is a vaccine candidate superior to previously developed sE2, providing a new possibility for controlling HCV.

ZIKV infection induces robust Th1-like Tfh cell and long-term protective antibody responses in immunocompetent mice.

Abstract: Induction of long-lived antibody responses during infection or vaccination is often essential for subsequent protection, but the relative contributions of T follicular helper (Tfh) cells and T helper 1 (Th1) cells for induction of antigen specific antibody responses to viruses are unclear. Here, we establish an acute Zika virus (ZIKV) infection model in immunocompetent mice, and show that ZIKV infection elicits robust Th1-like Tfh cell and protective antibody responses. While these Th1-like Tfh cells share phenotypic and transcriptomic profiles with both Tfh and Th1 cells, they also have unique surface markers and gene expression characteristics, and are dependent on T-bet for their development. Th1-like Tfh cells, but not Th1 cells, are essential for class switching of ZIKV-specific IgG2c antibodies and maintenance of long-term neutralizing antibody responses. Our study suggests that specific modulation of the Th1-like Tfh cell response during infection or vaccination may augment the induction of antiviral antibody response to ZIKV and other viruses. Here, the authors show that Zika virus (ZIKV) infection induces Th1-like Tfh cells that depend on T-bet for their development and are essential for class switching of ZIKV-specific IgG2c antibodies and maintenance of long-term neutralizing antibody responses.

A trivalent HCV vaccine elicits broad and synergistic polyclonal antibody response in mice and rhesus monkey.

Abstract: Objective Despite the development of highly effective direct-acting antivirals, a prophylactic vaccine is needed for eradicating HCV. A major hurdle of HCV vaccine development is to induce immunity against HCV with high genome diversity. We previously demonstrated that a soluble E2 (sE2) expressed from insect cells induces broadly neutralising antibodies (NAbs) and prevents HCV infection. The objective of this study is to develop a multivalent HCV vaccine to increase the antigenic coverage. Design We designed a trivalent vaccine containing sE2 from genotype 1a, 1b and 3a. Mice and rhesus macaques were immunised with monovalent or trivalent sE2 vaccine, and sera or purified immunoglobulin were assessed for neutralisation against a panel of cell culture-derived virion (HCVcc) of genotype 1–7 in cell culture. Splenocytes from the vaccinated macaques were assessed for HCV-specific T cell response. Results We showed that the trivalent vaccine elicited pangenotypic NAbs in mice, which neutralised HCVcc of all the seven genotypes more potently than the monovalent vaccine. Further analyses demonstrated that each sE2 component of this trivalent vaccine elicited unique spectrum of NAbs which acted synergistically to inhibit HCV infection. Finally, the trivalent vaccine triggered stronger and more uniform multigenotypic neutralising antibody response than the monovalent vaccine in rhesus macaques. Conclusions In summary, we developed a trivalent HCV vaccine that induces broad and synergistic-acting neutralising antibodies in mice and non-human primates.

Ebola virus VP35 has novel NTPase and helicase-like activities

Abstract: Ebola virus (EBOV) is a non-segmented, negative-sense RNA virus (NNSV) in the family Filoviridae, and is recognized as one of the most lethal pathogens in the planet. For RNA viruses, cellular or virus-encoded RNA helicases play pivotal roles in viral life cycles by remodelling viral RNA structures and/or unwinding viral dsRNA produced during replication. However, no helicase or helicase-like activity has ever been found to associate with any NNSV-encoded proteins, and it is unknown whether the replication of NNSVs requires the participation of any viral or cellular helicase. Here, we show that despite of containing no conserved NTPase/helicase motifs, EBOV VP35 possesses the NTPase and helicase-like activities that can hydrolyse all types of NTPs and unwind RNA helices in an NTP-dependent manner, respectively. Moreover, guanidine hydrochloride, an FDA-approved compound and inhibitor of certain viral helicases, inhibited the NTPase and helicase-like activities of VP35 as well as the replication/transcription of an EBOV minigenome replicon in cells, highlighting the importance of VP35 helicase-like activity during EBOV life cycle. Together, our findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster our understanding of EBOV and NNSVs.

Functional expression and characterization of the envelope glycoprotein E1E2 heterodimer of hepatitis C virus

Abstract: Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.

AcrR and Rex Control Mannitol and Sorbitol Utilization through Their Cross-Regulation of Aldehyde-Alcohol Dehydrogenase (AdhE) in Lactobacillus plantarum

Abstract: Lactobacillus plantarum is a versatile bacterium that occupies a wide range of environmental niches. In this study, we found that a bifunctional aldehyde-alcohol dehydrogenase-encoding gene, adhE, was responsible for L. plantarum being able to utilize mannitol and sorbitol through cross-regulation by two DNA-binding regulators. In L. plantarum NF92, adhE was greatly induced, and the growth of an adhE-disrupted (ΔadhE) strain was repressed when sorbitol or mannitol instead of glucose was used as a carbon source. The results of enzyme activity and metabolite assays demonstrated that AdhE could catalyze the synthesis of ethanol in L. plantarum NF92 when sorbitol or mannitol was used as the carbon source. AcrR and Rex were two transcriptional factors screened by an affinity isolation method and verified to regulate the expression of adhE DNase I footprinting assay results showed that they shared a binding site (GTTCATTAATGAAC) in the adhE promoter. Overexpression and knockout of AcrR showed that AcrR was a novel regulator to promote the transcription of adhE The activator AcrR and repressor Rex may cross-regulate adhE when L. plantarum NF92 utilizes sorbitol or mannitol. Thus, a model of the control of adhE by AcrR and Rex during L. plantarum NF92 utilization of mannitol or sorbitol was proposed.IMPORTANCE The function and regulation of AdhE in the important probiotic genus Lactobacillus are rarely reported. Here we demonstrated that AdhE is responsible for sorbitol and mannitol utilization and is cross-regulated by two transcriptional regulators in L. plantarum NF92, which had not been reported previously. This is important for L. plantarum to compete and survive in some harsh environments in which sorbitol or mannitol could be used as carbon source. A novel transcriptional regulator AcrR was identified to be important to promote the expression of adhE, which was unknown before. The cross-regulation of adhE by AcrR and Rex is important to balance the level of NADH in the cell during sorbitol or mannitol utilization.

Transcriptional Regulator AcrR Increases Ethanol Tolerance through Regulation of Fatty Acid Synthesis in Lactobacillus plantarum.

Abstract: Lactobacillus plantarum is a versatile bacterium with significant adaptability to harsh habitats containing excessive ethanol concentrations. It was found that the L. plantarum NF92-TetR/AcrR family regulator, AcrR, significantly enhanced the growth rate of this lactic acid bacterium in the presence of ethanol. Through screening 172 ethanol-resistant related genes by electrophoretic mobility shift and quantitative reverse transcription-PCR (RT-qPCR) assays, six genes were identified to be regulated by AcrR under ethanol stress. Among these was a gene coding for a 3-hydroxyacyl-ACP dehydratase (fabZ1) regulated by AcrR under ethanol stress. AcrR regulated fabZ1 under ethanol stress by binding to its promoter, P fabZ1 DNase I footprinting analysis indicated that there were two specific AcrR binding sites on P fabZ1 RT-PCR results showed fabZ1 could cotranscribe with its downstream 12 genes and conform a fatty acid de novo biosynthesis (fab) gene cluster under the control of P fabZ1 Both RT-qPCR of the fab gene cluster in acrR knockout and overexpression strains and fatty acid methyl ester analysis of the acrR knockout strain showed that AcrR could promote fatty acid synthesis in L. plantarum NF92. Membrane fluorescence anisotropy analysis of acrR knockout and overexpression strains showed that AcrR could increase membrane fluidity under ethanol stress. Thus, AcrR could regulate fatty acid synthesis and membrane fluidity to promote the adaption of L. plantarum NF92 to a high ethanol concentration.IMPORTANCE Ethanol tolerance is essential for L. plantarum strains living in substances with more than 9% ethanol, such as wine and beer. The details regarding how L. plantarum adapts to ethanol are still lacking. This study demonstrates that AcrR regulates the de novo synthesis of fatty acids in L. plantarum adapting to toxic levels of ethanol. We also identified the ability of the TetR/AcrR family regulator to bind to the fatty acid biosynthesis gene promoter, P fabZ1 , in L. plantarum and defined the binding sites. This finding facilitates the induction of the adaptation of L. plantarum strains to ethanol for food fermentation applications.

A molecular phylogeny of Schizothoracinae (Teleostei: Cypriniformes: Cyprinidae) based on 12 protein-coding mitochondrial genes and RAG1 gene analysis

Abstract: The ever-increasing interest in the investigation of origin and speciation of schizothoracine fishes can be dated to 20th century. However, molecular phylogeny of Schizothoracinae and their phylogenetic relationships, as well as the divergence times still remain controversial. In this study, two DNA sets consisting of 12 protein-coding mitochondrial genes from 254 individuals and RAG1 gene from 106 individuals were used to reconstruct the phylogenetic relationships and calculate the divergence times among the subfamily schizothoracinae. Our results indicated that both of the data sets supported a non-monophyletic relationship due to involving of species of Barbinae . However, the phylogenetic relationships based on mtDNA genes were more reliable than that inferred from RAG1 gene. The highly specialized grade formed a monophyletic group, together with Ptychobarbus as a sister group of Diptychus and Gymnodiptychus , which was belonging to specialized grade, indicating that Ptychobarbus may be transition species to involve to highly specialized schizothoracianae. In addition, the primitive grade clustered with Percocypris pingi , a species of Barbinae . Based on mtDNA gene, the speciation time of Schizothoracinae was 66 Ma, and the divergence time of the primitive grade and Percocypris pingi was 64 Ma. The speciation times of the three grades Schizothoracinae were 57 Ma, 51 Ma and 43 Ma, respectively; and the divergence time of specialized and highly specialized grade was 46 Ma. The divergence times of three grades were not consistent with the three stages of uplift of Qinghai-Tibet Plateau, which is older than the times. Keywords: Schizothoracinae, protein-coding mtDNA, RAG1, phylogeny, divergence