Physical exercise plays a role in rebalancing the bile acids of enterohepatic axis in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is considered as one of the most common diseases of lipid metabolism disorders, which is closely related to bile acids disorders and gut microbiota disorders. Bile acids are synthesized from cholesterol in the liver, and processed by gut microbiota in intestinal tract, and participate in metabolic regulation through the enterohepatic circulation. Bile acids not only promote the consumption and absorption of intestinal fat but also play an important role in biological metabolic signaling network, affecting fat metabolism and glucose metabolism. Studies have demonstrated that exercise plays an important role in regulating the composition and function of bile acid pool in enterohepatic axis, which maintains the homeostasis of the enterohepatic circulation and the health of the host gut microbiota. Exercise has been recommended by several health guidelines as the first-line intervention for patients with NAFLD. Can exercise alter bile acids through the microbiota in the enterohepatic axis? If so, regulating bile acids through exercise may be a promising treatment strategy for NAFLD. However, the specific mechanisms underlying this potential connection are largely unknown. Therefore, in this review, we tried to review the relationship among NAFLD, physical exercise, bile acids, and gut microbiota through the existing data and literature, highlighting the role of physical exercise in rebalancing bile acid and microbial dysbiosis.

Current Progress and Challenges in the Study of Adjuvants for Oral Vaccines.

Over the past 20 years, a variety of potential adjuvants have been studied to enhance the effect of oral vaccines in the intestinal mucosal immune system; however, no licensed adjuvant for clinical application in oral vaccines is available. In this review, we systematically updated the research progress of oral vaccine adjuvants over the past 2 decades, including biogenic adjuvants, non-biogenic adjuvants, and their multi-type composite adjuvant materials, and introduced their immune mechanisms of adjuvanticity, aiming at providing theoretical basis for developing feasible and effective adjuvants for oral vaccines. Based on these insights, we briefly discussed the challenges in the development of oral vaccine adjuvants and prospects for their future development.

Deletion of the cheZ gene results in the loss of swimming ability and the decrease of adhesion ability to Caco-2 cells in Escherichia coli Nissle 1917.

The aim of this study was to elucidate the biological functions of the motility regulatory protein CheZ in the probiotic strain Escherichia coli Nissle 1917. A cheZ gene deletion strain Nissle 1917ΔcheZ was constructed using the CRISPR/Cas9 two-plasmid system, and the corresponding complemented strain Nissle 1917ΔcheZ/pBR322-cheZ was established. Combined studies of growth kinetics testing, motility assays, swarming motility assays, and bacterial adherence assays were performed to study the motility regulatory protein CheZ-mediated functions in the prototype Nissle 1917 strain, its isogenic cheZ mutant, and the corresponding complemented strain. The growth rate of the cheZ mutant strain was lower than that of the wild-type strain in the exponential growth phase. The motility of the cheZ mutant strain was significantly lower than that of the wild-type strain. And the adhesion ability of ΔcheZ mutant to the Caco-2 cells was significantly lower than that of the wild-type strain and complemented strain. In conclusion, the results presented in our study suggested that the deletion of the cheZ gene in E. coli Nissle 1917 led to a significant reduction of its swimming ability and a subsequent marked decrease of adhesion to the Caco-2 cells.

VP39 of Spodoptera litura multicapsid nucleopolyhedrovirus cannot efficiently rescue the nucleocapsid assembly of vp39-null Autographa californica multiple nucleopolyhedrovirus.

BACKGROUND: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vp39 is conserved in all sequenced baculovirus genomes. In previous studies, VP39 has been identified as the major capsid structure protein of baculoviruses and found to be essential for nucleocapsid assembly. The nucleocapsid composition and structure of Group I and II NPVs of the Alphabaculovirus genus are very similar. It is not clear whether the major capsid structure protein VP39 of Group I NPVs is functionally identical to or substitutable with the Group II NPV VP39. In this study, the function of Group II Spodoptera litura MNPV (SpltMNPV) VP39 in Group I AcMNPV was characterized. METHODS: Sequence alignment of AcMNPV VP39 and SpltMNPV VP39 was performed using Clustal X and edited with GeneDoc. To determine whether VP39 of Group I NPVs can be functionally substituted by Group II NPV VP39, a vp39-null AcMNPV (vAcvp39KO) and a vp39-pseudotyped AcMNPV (vAcSpltvp39:FLAG), in which the Group I AcMNPV vp39 coding sequence was replaced with that of SpltMNPV from Group II NPVs, were constructed via homologous recombination in Escherichia coli. Using an anti-FLAG monoclonal antibody, immunoblot analysis was performed to examine SpltMNPV VP39 expression. Fluorescence and light microscopy were used to monitor viral replication and infection. Viral growth curve analysis was performed using a fifty percent tissue culture infective dose (TCID50) endpoint dilution assay. Viral morphogenesis was detected using an electron microscope. RESULTS: Sequence alignment indicated that the N-termini of AcMNPV VP39 and SpltMNPV VP39 are relatively conserved, whereas the C-terminus of SpltMNPV VP39 lacks the domain of amino acid residues 306-334 homologous to AcMNPV VP39. Immunoblot analysis showed that SpltMNPV VP39 was expressed in vAcSpltvp39:FLAG. Fluorescence and light microscopy showed that vAcSpltvp39:FLAG did not spread by infection. Viral growth curve analysis confirmed a defect in infectious budded virion production. Electron microscopy revealed that although masses of abnormally elongated empty capsid structures existed inside the nuclei of Sf9 cells transfected with vAcSpltvp39:FLAG, no nucleocapsids were observed. CONCLUSION: Altogether, our results demonstrated that VP39 from SpltMNPV cannot efficiently substitute AcMNPV VP39 during nucleocapsid assembly in AcMNPV.

Coexistence of Fosfomycin Resistance Determinant fosA and fosA3 in Enterobacter cloacae Isolated from Pets with Urinary Tract Infection in Taiwan.

To analyze the characteristics of fosA and fosA3 in Enterobacter cloacae isolated from aspirated and catheterized urine culture specimens of companion pets in Taiwan. A total of 19 E. cloacae isolates from pets with urinary tract infection were screened for the presence of fosA, fosA3, and fosC2 and for the genetic context of them by PCR amplification and sequencing. The transferability, resistance phenotypes, plasmid replicon typing properties and genetic environments of fosA- and/or fosA3-positive strains were characterized. Five E. cloacae isolates were positive for fosA and three coharbored fosA and fosA3. No fosC determinant was detected. Transconjugants of fosA3 were successfully acquired, while the acquisition of fosA transconjugants was failed. The minimum inhibitory concentrations (MICs) of the three fosA3-positive isolates and their transconjugants were ≥256 mg/L, whereas the MICs of the five fosA-positive isolates ranged from 64 mg/L to 256 mg/L. Three plasmid replicons (InCFrepB, InCL/M, and InCHI2) were identified in fosA- and fosA3-positive E. cloacae isolates. Different genetic contexts lay in the downstream region of fosA and fosA3, respectively. Eight distinct patterns based on the similarity value of more than 80% were typed for all the 8 fosA-positive isolates. In conclusion, the fosA concomitant with fosA3 were found in E. cloacae isolates. The fosA3 not only exhibits stronger activity of inactivating fosfomycin than fosA but also possesses stronger potential to spread than fosA. Different genetic backgrounds exist in these fosA- and fosA3-positive isolates, and different mobile elements may confer the dissemination of fosA and fosA3.

Transcriptomic Analysis of Staphylococcus aureus Under the Stress Condition Caused by Litsea cubeba L. Essential Oil via RNA Sequencing.

Litsea cubeba L. essential oil (LCEO) is a natural essential oil with considerable antimicrobial activity, and it can gradually replace some chemical additives in the food industry. However, the genetic evidences of stress response of bacteria under sub-lethal treatment with LCEO is limited. To this end, transcriptomic analysis of Staphylococcus aureus 29213 under a low concentration of LCEO was performed. Bacterial RNA samples were extracted from 1/4 MIC (0.07 µL/mL) of LCEO-treated and non-treated S. aureus 29213. The transcriptional results were obtained by RNA sequencing (RNA-Seq). After treated with LCEO of S. aureus 29213, 300, and 242 genes were significantly up and down-regulated. Up-regulated genes were mainly related to cell membrane (wall) stress stimulon including genes related to two-component regulatory system (VraS), histidine metabolism (hisABCD etc.) and L-lysine biosynthesis (thrA, lysC, asd etc.). Significant differences were also founded between LCEO-treated and non-treated groups in peptidoglycan biosynthesis related pathways. Down-regulated genes were related to nitrogen metabolism (NarGHIJ etc.), carotenoid biosynthesis (all) and pyruvate metabolism (phdA, pflB, pdhC etc.) of S. aureus 29213 in an LCEO-existing environment compared to the control. At the same time, we confirmed that LCEO can significantly affect the staphyloxanthin level of S. aureus 29213 for the first time, which is closely related to the redox state of S. aureus 29213. These evidences expanded the knowledge of stress response of S. aureus 29213 strain under sub-lethal concentration of LCEO.

Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

Targeting ideal oral vaccine vectors based on probiotics: a systematical view.

Probiotics have great potential to be engineered into oral vaccine delivery systems, which can facilitate elicitation of mucosal immunity without latent risks of pathogenicity. Combined with the progressive understanding of probiotics and the mucosal immune system as well as the advanced biotechniques of genetic engineering, the development of promising oral vaccine vectors based on probiotics is available while complicated and demanding. Therefore, a systematical view on the design of practical probiotic vectors is necessary, which will help to logically analyze and resolve the problems that might be neglected during our exploration. Here, we attempt to systematically summarize several fundamental issues vital to the effectiveness of the vector of probiotics, including the stability of the engineered vectors, the optimization of antigen expression, the improvement of colonization, and the enhancement of immunoreactivity. We also compared the existent strategies and some developing ones, attempting to figure out an optimal strategy that might deserve to be referred in the future development of oral vaccine vectors based on probiotics.

Techniques for chromosomal integration and expression optimization in Escherichia coli.

Due to the inherent expression stability and low metabolic burden to the host cell, the expression of heterologous proteins in the bacterial chromosome in a precise and efficient manner is highly desirable for metabolic engineering and live bacterial applications. However, obtaining suitable chromosome expression levels is particularly challenging. In this minireview, we briefly present the technologies available for the integration of heterologous genes into Escherichia coli chromosomes and strategies to optimize the expression levels of heterologous proteins.

Genetic engineering of probiotic Escherichia coli Nissle 1917 for clinical application.

Escherichia coli strain Nissle 1917 (EcN) has been used as a probiotic. Genetic engineering has enhanced the utility of EcN in several vaccine and pharmaceutical preparations. We discuss in this mini review the genetics and physical properties of EcN. We also discuss the numerous genetic engineering strategies employed for EcN-based vaccine development, including recombinant plasmid transfer, genetic engineering of cryptic plasmids or the EcN chromosome, EcN bacterial ghosts and its outer membrane vesicles. We also provide a current update on the progress and the challenges regarding the use of EcN in vaccine development.

Impact of acquisition of 16S rRNA methylase RmtB on the fitness of Escherichia coli.

The aim of this study was to elucidate the biological phenotypes of 16S rRNA methylase RmtB in Escherichia coli and the impact of RmtB acquisition on the fitness of the target bacterium. An rmtB in-frame deletion mutant in E. coli was constructed using a suicide vector (pDMS197)-based double crossover allelic exchange, and its corresponding complemented strain was established. Combined studies of microdilution susceptibility testing, conjugation experiments, growth kinetics assays, competitive experiments, biofilm formation tests and motility assays were performed to study the rmtB-mediated fitness among the prototype E. coli strain, its isogenic mutant and the corresponding complemented strain. The minimum inhibitory concentrations (MICs) of 4,6-disubstituted 2-deoxystreptamines for the rmtB wild-type strain, its isogenic mutant and the complemented strain were ≥1024, ≤2 and ≥1024mg/L, respectively. Both the growth rates and the competitive abilities of the wild-type and complemented strains were relatively inferior to the ΔrmtB mutant. There was no significant difference in biofilm formation and motility among the three strains. In conclusion, the data presented here suggest that acquisition of the 16S rRNA methylase gene rmtB in E. coli can exact a fitness cost on the bacteria, subsequently reducing the growth rate slightly and decreasing the competitive capacity of the bacterium, whereas it does not affect biofilm formation or motility.

The flagellin hypervariable region is a potential flagella display domain in probiotic Escherichia coli strain Nissle 1917.

The most studied probiotic, Escherichia coli strain Nissle 1917 (EcN) possesses flagella of serotype H1. To explore the potential to use EcN flagellin in flagella display applications, we investigated the effect of deleting amino acids in the hypervariable region of flagellin on EcNc (EcN cured of its two cryptic plasmids pMUT1 and pMUT2). Two EcNc flagellin isogenic mutants with deletions of amino acid residual from 277 to 286 and from 287 to 296 in the hypervariable domain were constructed. Both mutants were flagellated, adherent to IPEC-J2 cells, and colonized BALB/c mice. These hypervariable regions may have future utility in the display of heterologous epitopes.