: Conjugate vaccines are known to be one of the most effective and safest types of vaccines against bacterial pathogens. Previously, vaccine biosynthesis has been performed by using N-linked glycosylation systems. However, the structural specificity of these systems for sugar substrates has hindered their application. Here, we report a novel protein glycosylation system (O-linked glycosylation viaNeisseria meningitidis) that can transfer virtually any glycan to produce a conjugate vaccine. We successfully established this system inShigellaspp., avoiding the construction of an expression vector for polysaccharide synthesis. We further found that different protein substrates can be glycosylated using this system and that the O-linked glycosylation system can also effectively function in other Gram-negative bacteria, including some strains whose polysaccharide structure was not suitable for conjugation using the N-linked glycosylation system. The results from a series of animal experiments show that the conjugate vaccine produced by this O-linked glycosylation system offered a potentially protective antibody response. Furthermore, we elucidated and optimized the recognition motif, named MOOR, for theO-glycosyltransferase PglL. Finally, we demonstrated that the fusion of other peptides recognized by major histocompatibility complex class II around MOOR had no adverse effects on substrate glycosylation, suggesting that this optimized system will be useful for future vaccine development. Our results expand the glycoengineering toolbox and provide a simpler and more robust strategy for producing bioconjugate vaccines against a variety of pathogens.IMPORTANCERecently, the rapid development of synthetic biology has allowed bioconjugate vaccines with N-linked protein glycosylation to become a reality. However, the difficulty of reestablishing the exogenous polysaccharide synthetic pathway inEscherichia colihinders their application. Here, we show that an O-linked protein glycosylation system fromNeisseria meningitidis, which has a lower structure specificity for sugar substrates, could be engineered directly in attenuated pathogens to produce effective conjugate vaccines. To facilitate the further design of next-generation bioconjugate vaccines, we optimized a novel short motif consisting of 8 amino acids that is sufficient for glycosylation. Our results expand the application potential of O-linked protein glycosylation and demonstrate a simpler and more robust strategy for producing bioconjugate vaccines against different pathogens. In the future, bacterial antigenic polysaccharides could be attached to major histocompatibility complex binding peptides to improve immunological memory or attached to protein subunit vaccine candidates to provide double immune stimulation.

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.

Recent years have seen enormous advances in nanovaccines for both prophylactic and therapeutic applications, but most of these technologies employ chemical or hybrid semi‐biosynthetic production methods. Thus, production of nanovaccines has to date failed to exploit biology‐only processes like complex sequential post‐translational biochemical modifications and scalability, limiting the realization of the initial promise for offering major performance advantages and improved therapeutic outcomes over conventional vaccines. A Nano‐B5 platform for in vivo production of fully protein‐based, self‐assembling, stable nanovaccines bearing diverse antigens including peptides and polysaccharides is presented here. Combined with the self‐assembly capacities of pentamer domains from the bacterial AB₅ toxin and unnatural trimer peptides, diverse nanovaccine structures can be produced in common Escherichia coli strains and in attenuated pathogenic strains. Notably, the chassis of these nanovaccines functions as an immunostimulant. After showing excellent lymph node targeting and immunoresponse elicitation and safety performance in both mouse and monkey models, the strong prophylactic effects of these nanovaccines against infection, as well as their efficient therapeutic effects against tumors are further demonstrated. Thus, the Nano‐B5 platform can efficiently combine diverse modular components and antigen cargos to efficiently generate a potentially very large diversity of nanovaccine structures using many bacterial species.

Shigella species possess a type III secretion system (T3SS), which is required for human infection and that delivers effector proteins into target host cells. Here, we show that the effector, IpaH4.5 dampens the pro‐inflammatory cytokine response. In both the Sereny test and a murine lung infection model, the Shigella ΔipaH4.5 mutant strain caused more severe inflammatory responses and significantly induced higher pro‐inflammatory cytokine levels (MIP‐2 and TNF‐α) in the lung homogenates of wild type‐infected mice. Moreover, there was a threefold decrease in bacterial colonization of the mutant compared with the WT and ΔipaH4.5/ipaH4.5‐rescued strains. Yeast two‐hybrid screening showed that IpaH4.5 specifically interacts with the p65 subunit of NF‐κB. Ten truncated versions of IpaH4.5 and p65 spanning different regions were constructed and expressed to further map the IpaH binding sites with p65. The results revealed thatthe p65 region spanning amino acids 1–190 of p65 interacted with the IpaH4.5/1–293 N‐terminal region. In vitro, IpaH4.5 displayed ubiquitin ligase activity towards ubiquitin and p65. Furthermore, the transcriptional activity of NF‐κB was shown to be inhibited by IpaH4.5 utilizing a dual‐luciferase reporter gene detection system containing NF‐κB promoter response elements. Thus, we conclude that the IpaH4.5 protein is an E3 ubiquitin ligase capable of directly regulating the host inflammatory response by inhibiting the NF‐κB signalling pathway.