Embedding of exogenous B cell epitopes on the surface of UreB structure generates a broadly reactive antibody response against Helicobacter pylori.

Since Helicobacter pylori (H. pylori) resistance to antibiotic regimens has increased, vaccination is becoming an increasingly important alternative therapy to control H. pylori infection. UreB, FlaA, AlpB, SabA, and HpaA proteins of H. pylori were previously proved to be used as candidate vaccine antigens. Here, we developed an engineered antigen based on a recombinant chimeric protein containing a structural scaffold from UreB and B cell epitopes from FlaA, AlpB, SabA, and HpaA. The multi-epitope chimeric antigen, named MECU, could generate a broadly reactive antibody response including antigen-specific antibodies and neutralising antibodies against H. pylori urease and adhesins. Moreover, therapeutic immunisation with MECU could reduce H. pylori colonisation in the stomach and protect the stomach in BALB/c mice. This study not only provides promising immunotherapy to control H. pylori infection but also offers a reference for antigen engineering against other pathogens.

Systematic identification of a panel of strong promoter regions from Listeria monocytogenes for fine-tuning gene expression.

BACKGROUND: Attenuated Listeria monocytogenes (Lm) has been widely used as a vaccine vector in the prevention and treatment of pathogen infection and tumor diseases. In addition, previous studies have proved that the attenuated Lm can protect zebrafish from Vibrio infections, indicating that the attenuated Lm has a good application prospect in the field of aquatic vaccines. However, the limitation mainly lies in the lack of a set of well-characterized natural promoters for the expression of target antigens in attenuated Lm. RESULTS: In our study, candidate strong promoters were identified through RNA-seq analysis, and characterized in Lm through enhanced green fluorescent protein (EGFP). Nine native promoters that showed stronger activities than that of the known strong promoter P36 under two tested temperatures (28 and 37 °C) were selected from the set, and P29 with the highest activity was 24-fold greater than P36. Furthermore, we demonstrated that P29 could initiate EGFP expression in ZF4 cells and zebrafish embryos. CONCLUSIONS: This well-characterized promoter library can be used to fine-tune the expression of different proteins in Lm. The availability of a well-characterized promoter toolbox of Lm is essential for the analysis of yield increase for biotechnology applications.

Identification and evaluation of a panel of strong constitutive promoters in Listeria monocytogenes for improving the expression of foreign antigens.

Attenuated Listeria monocytogenes could be a potential vaccine vector for the immunotherapy of tumors or pathogens. However, the lack of reliable promoters has limited its ability to express foreign antigens. In the present study, 21 promoters were identified from Listeria monocytogenes through RNA-seq analysis under two pH conditions of pH 7.4 and pH 5.5. Based on the constructed fluorescence report system, 7 constitutive promoters exhibited higher strength than Phelp (1.8-fold to 5.4-fold), a previously reported strong promoter. Furthermore, the selected 5 constitutive promoters exhibited higher UreB production activity than Phelp (1.1-fold to 8.3-fold). Notably, a well-characterized constitutive promoter P18 was found with the highest activity of fluorescence intensity and UreB production. In summary, the study provides a panel of strong constitutive promoters for Listeria monocytogenes and offers a theoretical basis for mining constitutive promoters in other organisms. KEY POINTS: • Twenty-one promoters were identified from L. monocytogenes through RNA-seq. • Fluorescent tracer of L. monocytogenes (P18) was performed in vitro and in vivo. • A well-characterized constitutive promoter P18 could improve the expression level of a foreign antigen UreB in L. monocytogenes.

Immunological evaluation of virulence-deficient Listeria monocytogenes strains in C57BL/6 mice.

Attenuated Listeria monocytogenes (L. monocytogenes), which has unique advantages in presenting foreign antigens, was widely used in tumor immunotherapy research. As a live vaccine vector, attenuated L. monocytogenes was required to not only have certain invasiveness but also ensure safety, while the lack of different virulence factors may cause L. monocytogenes to show different safety and invasiveness. To evaluate the potential of virulence-deficient L. monocytogenes strains as a vaccine vector, four mutant strains EGD-eΔactA, EGD-eΔactA/inlB, EGD-eΔhly, and EGD-eΔprfA were used to infect C57BL/6 mice for determining related immune indexes. Compared with EGD-e, mutant strains showed significantly decreased invasion in C57BL/6 mice and caused relatively minor damage to spleen and liver. However, EGD-eΔactA and EGD-eΔactA/inlB were superior to EGD-eΔhly and EGD-eΔprfA in the comprehensive evaluation of inflammatory factor transcription level, immune cell differentiation and antibody level, which proved that they have a stronger adjuvant effect as a vaccine vector.

Tigecycline-resistance mechanisms and biological characteristics of drug-resistant Salmonella Typhimurium strains in vitro.

Increased drug resistance of Gram-negative bacteria to tetracycline caused by the unreasonable overuse of tigecycline has attracted extensive attention to reveal potential mechanisms. Here, we identified a tigecycline-resistant strain called TR16, derived from Salmonella Typhimurium ATCC13311 (AT), and examined its biological characteristics. Compared with AT, the TR16 strain showed significantly higher resistance to amoxicillin but lower resistance to gentamicin. Although the growth curves of TR16 and AT were similar, TR16 showed a significantly increased capacity for biofilm formation and a notably decreased motility compared to AT. Furthermore, transcriptome sequencing and reverse transcription-quantitative PCR (RT-qPCR) were implemented to evaluate the genetic difference between AT and TR16. Whole genome sequencing (WGS) analysis was also conducted to identify single nucleotide polymorphism (SNPs) and screened out two genetic mutations (lptD and rpsJ). The acrB gene of TR16 was knocked out through CRISPR/Cas9 system to further elucidate underlying mechanisms of tigecycline resistance in Salmonella Typhimurium. The up-regulation of acrB in TR16 was verified by RNA-seq and RT-qPCR, and the lack of acrB resulted in a 16-fold reduction in tigecycline resistance in TR16. Collectively, these results implied that AcrB efflux pump plays a key role in the tigecycline resistance of Salmonella, shedding light on the potential of AcrB efflux pump as a novel target for the discovery and development of new antibiotics.

The effects of NDM-5 on Escherichia coli and the screening of interacting proteins.

Carbapenem-resistant Escherichia coli (E. coli) strains are widely distributed and spreading rapidly, creating significant challenges for clinical therapeutics. NDM-5, a novel mutant of New Delhi Metallo-ß-Lactamase-1 (NDM-1), exhibits high hydrolase activity toward carbapenems. Since the genetic backgrounds of clinically isolated carbapenem-resistant E. coli are heterogeneous, it is difficult to accurately evaluate the impact of blaNDM-5 on antibiotic resistance. Herein, E. coli BL21 was transformed with a plasmid harboring blaNDM-5, and the resultant strain was named BL21 (pET-28a-blaNDM-5). Consistent with the findings of previous studies, the introduction of exogenous blaNDM-5 resulted in markedly greater resistance of E. coli to multiple ß-lactam antibiotics. Compared with BL21 (pET-28a), BL21 (pET-28a-blaNDM-5) exhibited reduced motility but a significant increase in biofilm formation capacity. Furthermore, transcriptome sequencing was conducted to compare the transcriptional differences between BL21 (pET-28a) and BL21 (pET-28a-blaNDM-5). A total of 461 differentially expressed genes were identified, including those related to antibiotic resistance, such as genes associated with the active efflux system (yddA, mcbR and emrY), pili (csgC, csgF and fimD), biofilm formation (csgD, csgB and ecpR) and antioxidant processes (nuoG). Finally, the pGS21a plasmid harboring blaNDM-5 was transformed into E. coli Rosetta2, after which the expression of the NDM-5 protein was induced using isopropyl-ß-D-thiogalactoside (IPTG). Using glutathione-S-transferase (GST) pull-down assays, total proteins from E. coli were scanned to screen out 82 proteins that potentially interacted with NDM-5. Our findings provide new insight into the identified proteins to identify potential antibiotic targets and design novel inhibitors of carbapenem-resistant bacteria.

Evaluation of an attenuated Listeria monocytogenes as a vaccine vector to control Helicobacter pylori infection.

The increasing resistance of Helicobacter pylori (H. pylori) to antibiotics has limited the efficacy of antibiotic therapy in the treatment of H. pylori-associated gastric diseases. The vaccine as an alternative method is becoming a safe and effective way to address this problem. In previous studies, live vector vaccines have proved to be effective in controlling H. pylori infection. Attenuated Listeria monocytogenes (L. monocytogenes) is a potential candidate vector applied in clinical trials, which can deliver foreign antigens and induce a broad immune response. To further explore the effectiveness of L. monocytogenes as a vaccine vector against H. pylori, attenuated L. monocytogenes-based vaccine EGDeΔactA/inlB(EGDeAB)-MECU was constructed to secrete a multi-epitope chimeric antigen (MECU) containing multiple B cell epitopes from H. pylori antigens. EGDeAB-MECU could secrete MECU stably. After immunized by gavage and intravenous injection, both EGDeAB and EGDeAB-MECU could significantly decrease gastric H. pylori colonization and induce a high level of specific antibodies against H. pylori. In conclusion, attenuated L. monocytogenes had an immunotherapeutic effect on H. pylori-infected mice, indicating its further development as a promising candidate vaccine vector for the H. pylori vaccine.

A Novel Design of Multi-epitope Vaccine Against Helicobacter pylori by Immunoinformatics Approach.

Helicobacter pylori (H. pylori) is a gram-negative spiral bacterium that caused infections in half of the world's population and had been identified as type I carcinogen by the World Health Organization. Compared with antibiotic treatment which could result in drug resistance, the vaccine therapy is becoming a promising immunotherapy option against H. pylori. Further, the multi-epitope vaccine could provoke a wider immune protection to control H. pylori infection. In this study, the in-silico immunogenicity calculations on 381 protein sequences of H. pylori were performed, and the immunogenicity of selected proteins with top-ranked score were tested. The B cell epitopes and T cell epitopes from three well performed proteins UreB, PLA1, and Omp6 were assembled into six constructs of multi-epitope vaccines with random orders. In order to select the optimal constructs, the stability of the vaccine structure and the exposure of B cell epitopes on the vaccine surface were evaluated based on structure prediction and solvent accessible surface area analysis. Finally Construct S1 was selected and molecular docking showed that it had the potential of binding TLR2, TLR4, and TLR9 to stimulate strong immune response. In particular, this study provides good suggestions for epitope assembly in the construction of multi-epitope vaccines and it may be helpful to control H. pylori infection in the future. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s10989-020-10148-x) contains supplementary material, which is available to authorized users.

A review: Progress in the development of fish Vibrio spp. vaccines.

During the past decades, Vibrio spp. infections have been a serious problem that causes fish blight and very large economic losses, especially in China, Japan, Europe, and North America. Hitra disease caused by Vibrio salmonicida and winter ulcer disease caused by Vibrio viscosus have seriously affected cultured salmon in Norway and Iceland. In Asia, Vibriosis has caused huge losses in cultured grass carp and turbot of Chinese and cultured eels of Japan. Antibiotics and other chemical agents are the most common methods currently to prevent and control diseases but they have led to the emergence of drug-resistant strains and antibiotic residues. The vaccine is superior in high efficiency, safety, and convenience. It can also obtain greater economic benefits, which make it the most suitable method to control fish diseases. The most traditional method is inactivated vaccines with adjuvant, which is administered by intraperitoneal injection. With the development of molecular biology, a combination of specific pathogen components and new adjuvants can provide enhanced immune protection. This review provides the research progress of different types of Vibrio spp. vaccines, including inactivated vaccines, live attenuated vaccines, subunit vaccines, DNA vaccines, and live vector vaccines.