Efficient screening of adsorbed receptors for Salmonella phage LP31 and identification of receptor-binding protein.

The adsorption process is the first step in the lifecycle of phages and plays a decisive role in the entire infection process. Identifying the adsorption mechanism of phages not only makes phage therapy more precise and efficient but also enables the exploration of other potential applications and modifications of phages. Phage LP31 can lyse multiple Salmonella serotypes, efficiently clearing biofilms formed by Salmonella enterica serovar Enteritidis (S. Enteritidis) and significantly reducing the concentration of S. Enteritidis in chicken feces. Therefore, LP31 has great potential for many practical applications. In this study, we established an efficient screening method for phage infection-related genes and identified a total of 10 genes related to the adsorption process of phage LP31. After the construction of strain C50041ΔrfaL 58-358, it was found that the knockout strain had a rough phenotype as an O-antigen-deficient strain. Adsorption rate and transmission electron microscopy experiments showed that the receptor for phage LP31 was the O9 antigen of S. Enteritidis. Homology comparison and adsorption experiments confirmed that the tail fiber protein Lp35 of phage LP31 participated in the adsorption process as a receptor-binding protein. IMPORTANCE A full understanding of the interaction between phages and their receptors can help with the development of phage-related products. Phages like LP31 with the tail fiber protein Lp35, or a closely related protein, have been reported to effectively recognize and infect multiple Salmonella serotypes. However, the role of these proteins in phage infection has not been previously described. In this study, we established an efficient screening method to detect phage adsorption to host receptors. We found that phage LP31 can utilize its tail fiber protein Lp35 to adsorb to the O9 antigen of S. Enteritidis, initiating the infection process. This study provides a great model system for further studies of how a phage-encoded receptor-binding protein (RBP) interacts with its host's RBP binding target, and this new model offers opportunities for further theoretical and experimental studies to understand the infection mechanism of phages.

Application and challenge of bacteriophage in the food protection.

In recent years, foodborne diseases caused by pathogens have been increasing. Therefore, it is essential to control the growth and transmission of pathogens. Bacteriophages (phages) have the potential to play an important role in the biological prevention, control, and treatment of these foodborne diseases due to their favorable advantages. Phages not only effectively inhibit pathogenic bacteria and prolong the shelf life of food, but also possess the advantages of specificity and an absence of chemical residues. Currently, there are many cases of phage applications in agriculture, animal disease prevention and control, food safety, and the treatment of drug-resistant disease. In this review, we summarize the recent research progress on phages against foodborne pathogenic bacteria, including Escherichia coli, Salmonella, Campylobacter, Listeria monocytogenes, Shigella, Vibrio parahaemolyticus, and Staphylococcus aureus. We also discuss the main issues and their corresponding solutions in the application of phages in the food industry. In recent years, although researchers have discovered more phages with potential applications in the food industry, most researchers use these phages based on their host spectrum, and the application environment is mostly in the laboratory. Therefore, the practical application of these phages in different aspects of the food industry may be unsatisfactory and even have some negative effects. Thus, we suggest that before using these phages, it is necessary to identify their specific receptors. Using their specific receptors as the selection basis for their application and combining phages with other phages or phages with traditional antibacterial agents may further improve their safety and application efficiency. Collectively, this review provides a theoretical reference for the basic research and application of phages in the food industry.

A phage for the controlling of Salmonella in poultry and reducing biofilms.

As a natural alternative to traditional antimicrobials, phages are being recognised as highly effective control agents for Salmonella and other foodborne bacteria. Due to the high diversity of Salmonella serotypes and the emergence of phage-resistant strains, attempting to isolate more widespread, strictly lytic Salmonella phages is highly warranted. In this study, a lytic phage, LP31, was isolated from poultry faecal samples. Transmission electron microscopy revealed that the phage had a polyhedral head and a retraction-free tail, indicative of the Siphoviridae family. Adsorption rate experiments showed that LP31 required the participation of lipopolysaccharides, but not flagella, during phage adsorption. Host profile identification showed that LP31 could lyse most Salmonella Enteritidis (S. Enteritidis) (96.15%, N = 104) and Salmonella Pullorum (S. Pullorum) (96.67%, N = 60). Initial applications found that LP31 reduced the concentration of static S. Enteritidis on metal surfaces (0.951 log10 cfu/ml) and in the faeces of chicks (2.14 log10 cfu/g). Notably, LP31 could almost completely remove biofilms formed by S. Enteritidis and S. Pullorum in 1 h. These findings suggest that LP31 has a good prevention and control effect against biofilms and planktonic antibiotic-resistant Salmonella, and is therefore a potentially promising biocontrol agent for controlling the spread of Salmonella in the poultry and food processing industries.

A nanofiber hydrogel derived entirely from ocean biomass for wound healing.

Crustaceans and fish scales in the marine food industry are basically thrown away as waste. This not only wastes resources but also causes environmental pollution. While reducing pollution and waste, biological activity and storage of materials are urgent issues to be solved. In this study, by first preparing dry fibers and then making hydrogels, we prepared a fish scale/sodium alginate/chitosan nanofiber hydrogel (FS-P) by cross-linking the nanofibers in situ. From fish and other organisms, fish gelatin (FG), collagen and CaCO3 were extracted. Fish scale (FS)/sodium alginate/chitosan nanofibers were cross-linked with copper sulfide nanoparticles prepared by a one-step green method to obtain FS-P nanofiber hydrogels under mild conditions without catalyst and additional procedures. These fiber hydrogels not only have good tissue adhesion and tensile properties, but also have the antibacterial effect of natural antibacterial and CuS photothermal synergism, which can achieve 51.32% and 49.96% of the antibacterial effect against Staphylococcus aureus and Escherichia coli respectively, avoiding the generation of superbacteria. The nanofiber hydrogels have 87.56% voidage and 52.68% degradability after 14 days. The combined strategy of using marine bio-based fibers to prepare gels promoted angiogenesis and tissue repair.

Salmonella Typhimurium ST34 Isolate Was More Resistant than the ST19 Isolate in China, 2007 - 2019.

To disclose the antimicrobial susceptibility and wide adaptability of commonly occurring genotypes of Salmonella enterica serovar Typhimurium, the antimicrobial resistance and multilocus sequence typing (MLST) profiles of 196 Salmonella Typhimurium isolates (136 from food-producing animals, 19 from environments, 15 from markets, and 26 from humans) in China between 2007 and 2019 were analyzed. Tests of susceptibility to 19 antimicrobial agents using the broth microdilution method showed that 84.7% of the isolates were resistant to at least one antimicrobial. Antimicrobial susceptibility analysis demonstrated that 66.8% of the isolates were multidrug-resistant (MDR) strains, with resistance to three or more antimicrobials. The highest antidrug resistance was to ampicillin, amoxicillin/clavulanic acid, and tetracycline. Three MLST types were detected, and sequence type (ST) 19 was the most common ST. However, ST34 was associated with a higher MDR rate and more complex MDR patterns, than ST19 and ST99, although the exact mechanism has not been reported. Our study highlights the variation of drug resistance and STs from different sources and the association between STs and drug resistance, providing useful information for epidemiological research and developing a public health strategy.

The rfbN gene of Salmonella Typhimurium mediates phage adsorption by modulating biosynthesis of lipopolysaccharide.

The study of the interaction mechanism between bacteriophage and host is helpful in promoting development of bacteriophage applications. The mechanism of the interaction with the phage was studied by constructing the rfbN gene deletion and complemented with strains of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium, S. Typhimurium) D6. The rfbN gene deletion strain could not be lysed by phage S55 and led to a disorder of lipopolysaccharide (LPS) biosynthesis, which changed from the smooth type to rough type. Also, the RfbN protein lacking any of the three-segment amino acid (aa) sequences (90-120 aa, 121-158 aa, and 159-194 aa) produces the same result. Transmission electron microscopy and confocal microscopy assays demonstrated that phage S55 dramatically reduced adsorption to the rfbN deletion strain as compared to the wild strain D6. After co-incubation of the S55 with the purified smooth LPS, D6 could not be lysed, indicating that the smooth LPS binds to the S55 in vitro and then inhibits the cleavage activity of the S55. To sum up, the rfbN gene affects phage adsorption by regulating LPS synthesis. Furthermore, the functioning of the RfbN protein requires the involvement of multiple structures. To the best of our knowledge, this study is the first report of the involvement of the bacterial rfbN gene involved in the phage-adsorption process.

Isolation, Characterization, and Application in Poultry Products of a Salmonella-Specific Bacteriophage, S55.

ABSTRACT: Salmonellosis occurs frequently worldwide, causing serious threats to public health. The abuse of antibiotics is increasing antibiotic resistance in bacteria, thereby making the prevention and control of Salmonella more difficult. A phage can help control the spread of bacteria. In this study, the lytic phage S55, whose host bacterium is Salmonella Pullorum, was isolated from fecal samples obtained from poultry farms. This phage belongs to the Siphoviridae and has a polyhedral head and a retraction-free tail. S55 lysed most cells of Salmonella Pullorum (58 of 60 strains, 96.67%) and Salmonella Enteritidis (97 of 104 strains, 93.27%). One-step growth kinetics revealed that the latent period was 10 min, the burst period was 80 min, and the burst size was 40 PFU per cell. The optimal multiplicity of infection was 0.01, and the phage was able to survive at pH values of 4 to 11 and temperatures of 40 to 60°C for 60 min. Complete genome sequence analysis revealed that the S55 genome consists of 42,781 bp (50.28% GC content) and 58 open reading frames, including 25 frames with known or assumed functions without tRNA genes. S55 does not carry genes that encode virulence or resistance factors. At 4 and 25°C, S55 reduced the populations of Salmonella Pullorum and Salmonella Enteritidis on chicken skin surfaces. S55 may be useful as a biological agent for the prevention and control of Salmonella infections.

Salmonella Pullorum spiC mutant is a desirable LASV candidate with proper virulence, high immune protection and easy-to-use oral administration.

Live attenuated Salmonellavaccine (LASV) is considered to be an effective contributory measure during the control of Salmonella infection. A Salmonella Pullorum spiC mutant was evaluated comprehensively as a LASV candidate (LASV-p) for broilers in terms of safety and immunogenicity. LASV-p was adminstered to 3-day broilers by intramuscular injection. The LD50 increased 126 fold, and no tissue lesions were observed in the liver, spleen and cecum, in comparison with the control group inoculated with PBS and a passive group by wild-type Salmonella. Growth rates of all broilers were normal and not affected. LASV-p persisted in vivo until 21 days in liver, 28 days in spleen and 35 days in feces, and induced high levels of humoral IgG and mucosal IgA. Cellular immunity was also stimulated in the form of antigen-specific lymphocyte proliferation and higher counts of CD3+CD8+ T cells and increased expression of mRNA of Th1 cytokines, IFN-γ and IL-2, in the early stage, and Th2 cytokines, IL-4 and IL-10, in the later stages. LASV-p provided at least 90% immuneprotection against a wild-type Salmonella Pullorum and cross-protection in different degree against other Salmonella searovars. Oral vaccine could also offer high immune protection of 87.5%. These results indicated that LASV-p vaccine candidate had a high level of safety and immune protection and it might be developed as a novel easy-to-use oral vaccine to improve poultry health in the future.

WbaP is required for swarm motility and intramacrophage multiplication of Salmonella Enteritidis spiC mutant by glucose use ability.

Salmonella spp. can survive and replicate in macrophage cells to cause persistent infection, SpiC is a necessary T3SS effector, but its pathogenic mechanism is still not known completely. In our study, Salmonella Enteritidis spiC mutant (SEΔspiC) was found to have stronger swarming motility and intramacrophage hyperproliferation which was closely related to glucose metabolism. SEΔspiC wbaP::Tn5 mutant was screened out by transposon mutagenesis, which had weaker swarming motility and intramacrophage replication ability than SEΔspiC in the presence of glucose. Bioinformatics displayed that undecaprenyl-phosphate galactose phosphotransferase (Wbap), encoded by wbaP gene, was a key enzyme for glucose metabolism and Lipopolysaccharide(LPS) synthesis, which confirmed our outcome that Wbap was involved in intramacrophage replication ability by glucose use in addition to swarming motility based on SEΔspiC. This discovery will further promote the understanding of the interaction between wbaP gene and spiC gene and the intracellular Salmonella replication mechanism.

Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence.

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpAL10A ) did not alter its translocation to the nucleus but abolished the effector's capacity to interact with EBP2. VgpA-EBP2 interaction led to the re-localization of c-Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA-EBP2 interaction elevated EBP2's affinity for c-Myc and prolonged the oncoprotein's half-life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re-localization of c-Myc. Moreover, the in vivo VgpA-EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus' colonization and virulence. These observations suggest that direct effector stimulation of a c-Myc controlled host cell growth program can contribute to pathogenesis.

ATG7-dependent and independent autophagy determine the type of treatment in lung cancer.

Based on the role of ATG7 in the initiation of autophagy, autophagy can be divided into ATG7-dependent selective autophagy and ATG7-independent alternative autophagy. However, the detailed roles of two different types of autophagy in antitumor therapy have not been fully elucidated so far. Here, we for the first time demonstrated an investigational inducer, w09, could induce both selective autophagy and alternative autophagy in NSCLC, but the phenotypes of these two kinds of autophagy are different：(1) w09-induced selective autophagy mainly promoted cell apoptosis, while w09-triggered alternative autophagy markedly induced autophagic cell death in NSCLC；(2) w09-induced ATG7 dependent autophagy mainly promoted the accumulation of SQSTM1/p62, while w09-triggered ATG7 independent autophagy markedly accelerated the degradation of SQSTM1/p62. These above results were further confirmed by knockout ATG7 gene in A549 cells or restoration of ATG7 function in H1650 cells. Deletion of ATG7 gene markedly attenuated the effect of w09-induced autophagy or apoptosis on A549 cells, while restoration of functional ATG7 markedly enhanced the effect of w09-induced autophagy and apoptosis on H1650 cells. Mechanistically, we further revealed that w09 induced two different types of autophagy through inhibiting PI3K/AKT/mTOR signaling pathway. Notably, compared with A549WT xenograft model, the in vivo antitumor effect of w09 or Taxel on the ATG7-deficient A549 xenograft model was significantly attenuated. Therefore, a special attention must be paid to distinguish which kinds of autophagy have been induced by autophagy inducers with antitumor agents by targeting PI3K/AKT/mTOR signaling pathway.

Cholesterol-Rich Lipid Rafts in the Cellular Membrane Play an Essential Role in Avian Reovirus Replication.

Cholesterol is an essential component of lipid rafts in cellular plasma membranes. Although lipid rafts have been reported to have several functions in multiple stages of the life cycles of many different enveloped viruses, the mechanisms by which non-enveloped viruses, which lack outer lipid membranes, infect host cells remain unclear. In this study, to investigate the dependence of non-enveloped avian reovirus (ARV) infection on the integrity of cholesterol-rich membrane rafts, methyl-ß-cyclodextrin (MßCD) was used to deplete cellular membrane cholesterol at the ARV attachment, entry, and post-entry stages. Treatment with MßCD significantly inhibited ARV replication at both the entry and post-entry stages in a dose-dependent manner, but MßCD had a statistically insignificant effect when it was added at the attachment stage. Moreover, MßCD treatment markedly reduced syncytium formation, which occurs at a relatively late stage of the ARV life cycle and is involved in cell-cell transmission and release. Furthermore, the addition of exogenous cholesterol reversed the effects mentioned above. Colocalization data also showed that the ARV proteins σC, µNS, and p10 prefer to localize to cholesterol-rich lipid raft regions during ARV infection. Altogether, these results suggest that cellular cholesterol in lipid rafts plays a critical role in ARV replication.

Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus.

The adsorption of phages to hosts is the first step of phage infection. Studies have shown that tailed phages use tail fibres or spikes to recognize bacterial receptors and mediate adsorption. However, whether other phage tail components can also recognize host receptors is unknown. To identify potential receptors, we screened a transposon mutagenesis library of the marine pathogen Vibrio parahaemolyticus and discovered that a vp0980 mutant (vp0980 encodes a predicted transmembrane protein) could not be lysed by phage OWB. Complementation of this mutant with wild-type vp0980 in trans restored phage-mediated lysis. Phage adsorption and confocal microscopy assays demonstrated that phage OWB had dramatically reduced adsorption to the vp0980 mutant compared to that to the wild type. Pulldown assays showed that phage tail tubular proteins A and B (TTPA and TTPB) interact with Vp0980, suggesting that Vp0980 is a TTPA and TTPB receptor. Vp0980 lacking the outer membrane region (aa 114-127) could not bind to TTPA and TTPB, resulting in reduced phage adsorption. These results strongly indicated that TTPA and TTPB binding with their receptor Vp0980 mediates phage adsorption and subsequent bacterial lysis. To the best of our knowledge, this study is the first report of a bacterial receptor for phage tail tubular proteins.

In situ melt electrospun polycaprolactone/Fe3O4 nanofibers for magnetic hyperthermia.

Magnetic fibrous membrane used to generate heat under the alternating magnetic field (AMF) has attracted wide attention due to their application in magnetic hyperthermia. However, there is not magnetic fibrous membrane prepared by melt electrospinning (e-spinning) which is a solvent-free, bio-friendly technology. In this work, polycaprolactone (PCL)/Fe3O4 fiber membrane was prepared by melt e-spinning and using homemade self-powered portable melt e-spinning apparatus. The hand-held melt e-spinning apparatus has a weight of about 450 g and a precise size of 24 cm in length, 6 cm in thickness and 13 cm in height, which is more portable for widely using in the medical field. The PCL/Fe3O4 composite fibers with diameters of 4-17 µm, are very uniform. In addition, the magnetic composite fiber membrane has excellent heating efficiency and thermal cycling characteristics. The results indicated that self-powered portable melt e-spinning apparatus and PCL/Fe3O4 fiber membrane may provide an attractive way for hyperthermia therapy.

SspH2 as anti-inflammatory candidate effector and its contribution in Salmonella Enteritidis virulence.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a facultative intracellular pathogen deploying the type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) to transfer effector proteins into host cells to modify its functions and accomplish intracellular replication. To study the effect of SspH2 on immune response induced by S. Enteritidis, we generated a deletion mutant of the effector gene sspH2 and a plasmid mediated complementary strain in S. Enteritidis C50336. The results of LD50 showed that SspH2 has no obvious effect on the virulence of S. Enteritidis. However, deletion of sspH2 decreased the invasion and intercellular colonization of the bacteria in Caco2 BBE cells. Using bacteriological counts from tissue homogenates the result of colonization in internal organs showed that in spleen and liver tissues, at 3rd and 4th day p.i. there is a significance decreased number of C50336-ΔsspH2 compared to the C50336-WT and C50336-ΔsspH2-psspH2, respectively. The qRT-PCR analysis results of both in vivo and in vitro experiments clearly showed that the mutant strain C50336ΔsspH2 significantly promoted expression of IL-1ß, INF-γ, IL-12, and iNOS cytokines compared to the groups infected with the wild type or complementary strains, while the IL-8 synthesis was decreased in the mutant strain infected group. All of these findings revealed that SspH2 promotes the colonization of S. Enteritidis in host cells, and it is an important anti-inflammatory biased effector in Salmonella.

The "fighting wisdom and bravery" of tailed phage and host in the process of adsorption.

In the process of bacteriophage and bacteria struggle, adsorption is the key factor to determine who is the winner. In this paper, the molecular mechanism of tailed bacteriophage recognition and adsorption to host and the strategy of "fighting wisdom and courage" between them are reviewed.

Enhanced Th1/Th2 mixed immune responses elicited by polyethyleneimine adjuvanted influenza A (H7N9) antigen HA1-2 in chickens.

Influenza A (H7N9) viruses have caused severe human infections and deaths every year in China since 2013. To reduce the risk of human infection and prevent a new influenza pandemic, there is a pressing need to develop safe and effective anti-H7N9 vaccines for poultry. Polyethyleneimine (PEI) is an organic polycation used extensively as a transfection reagent for decades. Although the adjuvant potential of PEI is well studied in mammals, its applicability and immune characteristics to avian species are still very rare. Here, to investigate the adjuvant activity of PEI, we analyzed the immune responses in chicken peripheral blood mononuclear cells in vitro. PEI significantly upregulated the expression of immune-related cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-18, and IL-1ß) and chemokines (CXCLi1, CXCLi2, MIP-1ß, and MCP-3), suggesting that PEI promoted immune responses of avian cells. We also assessed the in vivo immune responses to PEI in a chicken model. After the second and third vaccinations, significantly higher IgG titers were observed in the chickens immunized with HA1-2+PEI than that of HA1-2 alone. The HA1-2+PEI group also increased percentages of CD4+ and CD8+ T cells and improved PBMC proliferation. The significantly upregulated IFN-γ and IL-4 levels of splenocytes from HA1-2+PEI vaccinated chickens further indicated that PEI promoted a Th1/Th2 mixed immune responses. This study not only demonstrates the adjuvant potential of PEI when co-administered with influenza H7N9 antigen HA1-2 in chickens, but also supports the use of PEI as a versatile systemic adjuvant platform in poultry.

Immunization with recombinant Salmonella expressing SspH2-EscI protects mice against wild type Salmonella infection.

BACKGROUND: Enhancing caspase-1 activation in macrophages is helpful for the clearance of intracellular bacteria in mice. Our previous studies have shown that EscI, an inner rod protein of type III system in E. coli can enhance caspase-1 activation. The purpose of this study was to further analyze the prospect of EscI in the vaccine design. RESULTS: A recombinant Salmonella expressing SspH2-EscI fusion protein using the promotor of Salmonella effector SspH2, X4550(pYA3334-P-SspH2-EscI), was constructed. A control recombinant Salmonella expressing SspH2 only X4550(pYA3334-P-SspH2) was also constructed. In the early stage of in vitro infection of mouse peritoneal macrophages, X4550(pYA3334-P-SspH2-EscI) could significantly (P < 0.05) enhance intracellular caspase-1 activation and pyroptotic cell death of macrophages, when compared with X4550(pYA3334-P-SspH2). Except for the intracellular pH value, the levels of reactive oxygen species, intracellular concentration of calcium ions, nitric oxide and mitochondrial membrane potential in macrophages were not significantly different between the cells infected with X4550(pYA3334-P-SspH2-EscI) and those infected with X4550(pYA3334-P-SspH2). Besides, only lower inflammatory cytokines secretion was induced by X4550(pYA3334-P-SspH2-EscI) than X4550(pYA3334-P-SspH2). After intravenous immunization of mice (1 × 106 cfu/mouse), the colonization of X4550(pYA3334-P-SspH2-EscI) in mice was significantly limited at one week post immunization (wpi), when compared with X4550(pYA3334-P-SspH2) (P < 0.05). The population of activated CD8+T lymphocytes in mouse spleens induced by X4550(pYA3334-P-SspH2-EscI) was lower than that induced by X4550(pYA3334-P-SspH2) at 2-3 wpi, and the ratio of CD4+T cells to CD8+T cells decreased. The blood coagulation assay indicated that no significant difference was found between X4550(pYA3334-P-SspH2-EscI) and uninfected control, while X4550(pYA3334-P-SspH2) could induce the quick coagulation. Notably, immunization of X4550(pYA3334-P-SspH2-EscI) could limit the colonization of challenged Salmonella strains in the early stage of infection and provide more effective protection. CONCLUSION: The activation of caspase-1 in macrophages by EscI can be used in the design of live attenuated Salmonella vaccine candidate.

A Promising Listeria-Vectored Vaccine Induces Th1-Type Immune Responses and Confers Protection Against Tuberculosis.

Deaths associated with tuberculosis (TB) is rising and accounted for 1.4 million deaths in 2015 many of which were due to drug-resistant bacteria. Vaccines represent an important medical intervention, but the current Bacilli Calmette-Guerin (BCG) vaccine is not ideal for the protection of teenagers and adults. Therefore, a safe and effective vaccine is urgently needed. In this study, we designed a novel vaccine using an attenuated Listeria monocytogenes strain carrying fusion antigen FbpB-ESAT-6 (rLM) and characterized its safety and protective efficacy against Mycobacterium tuberculosis (M.tb) infection in mice. Compared to the wild type strain yzuLM4 and parental strain LMΔactA/plcB (LM1-2), the virulence of rLM was significantly reduced as judged by its infectious kinetics and LD50 dose. Further characterization of intravenous immunization showed that prime-boost vaccination significantly increased the levels of Th1 cytokines (IFN-γ, IL-17, and IL-6), and enhanced cytotoxic T lymphocyte (CTL) CTLs activity, suggesting that rLM could elicit potent Th1/Th17 responses. More importantly, rLM significantly conferred the protection against M.tb H37Rv challenge. Collectively, our findings indicated that rLM is a novel and useful tool to prevent M.tb infection, and can be potentially be used to boost BCG-primed immunity.

[Expression and assembly of chimeric flagellins in Escherichia coli BL21(DE3) and Salmonella].

Flagellin can be expressed in monomeric or polymeric form based on assembly. The difference of these two forms of flagellin is less studied. In this experiment, recombinant plasmid pET-fliC/M2e2 was transferred into Escherichia coli BL21(DE3) and Salmonella SL5928 to express chimeric flagellin, mfliC/M and pfliC/M, respectively, and then their assembly characteristics were analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated that the two recombinant bacteria could successfully express chimeric flagellin. The transmission electronic microscope observation showed that no flagella were found on the surface of recombinant E. coli, whereas it was found for recombinant Salmonella. After purification, distinct circular dichroism spectra between them were found and pfliC/M showed the similar structure as wild-type flagellin, but not for mfliC/M. The dynamic light scattering assay also indicated that the polymerization of mfliC/M was much lower than that for pfliC/M. Three hours after transfection into mouse peritoneal macrophages, both could induce interleukin 1ß secretion, but mfliC/M is stronger than pfliC/M. These data will be helpful for the selection of expression form of flagellin.