Biological Characteristics of Listeria monocytogenes Following Deletion of TatD-like Protein Gene.

TatD is the subunit of the twin-arginine translocation (Tat) pathway. Members of TatD family are multifunctional, conserved and widely presented proteins in most prokaryotes. It has been reported that Tat can affect bacterial motility in some bacteria. This study was conducted to determine the contribution of the TatD protein (herein named LmTatD) to the regulation of flagella in Listeria monocytogenes. We constructed an LmTatD gene mutant in L. monocytogenes strain 10403 s and evaluated its biological characteristics. The results showed no difference in growth or morphology between the wild-type strain and the ΔLmTatD mutant. Intriguingly, the ΔLmTatD mutant showed impaired swimming motility and flagella structure but increased biofilm formation. Comparative proteomic analysis using tandem mass tag (TMT) combined with liquid chromatography-tandem mass spectrometry (LC‒MS/MS) was performed to determine differentially expressed proteins (DEPs). The results revealed that 134 proteins out of 2228 total proteins identified were differentially expressed, among which 18 proteins were upregulated and 116 proteins were downregulated in the ΔLmTatD mutant. Analysis of DEPs indicated that the reduced expression levels of the proteins related to flagellar assembly in the ΔLmTatD mutant correlate with its characteristics. Compared to the wild-type strain, the most downregulated proteins in the ΔLmTatD mutant included FlaA, FliD, FliR, FlgD, FlgL, and FlgG. Collectively, our data suggest that although LmTatD is not required for growth in L. monocytogenes, loss of LmTatD reduces flagellar production and motility by regulating flagellar assembly-related protein expression.

Effects of Salmonella enterica serovar typhimurium sseK1 on macrophage inflammation-related cytokines and glycolysis.

Salmonella enterica serovar Typhimurium (S. Typhimurium), an important virulent intracellular pathogen, causes inflammatory gastroenteritis or typhoid. Macrophages play a key role in innate immunity against Salmonella. Salmonella secreted effector K1 (SseK1) encoded by SPI2 has been identified a novel translocated protein. To investigate the role of Salmonella enterica serovar Typhimurium sseK1 about the inflammation and glycolysis in macrophages, the levels of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and Nitric Oxide in macrophages infected by S. Typhimurium SL1344 wild-type (WT) group, ΔsseK1 mutant group and sseK1-complemented group were measured. And the glycolysis level was determined in RAW 264.7 cells infected with these different Salmonella strains. The results showed that groups infected by wild-type strain, sseK1 mutant and sseK1-complemented strain upregulated the production of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and NO at 3 h, 6 h and 12 h, respectively. The production of IL-1ß, IL-2, IL-4, IL-6, IFN-γ and NO in wild-type strain group were significantly decreased compared with the ΔsseK1 mutant group, which suggested that sseK1 down-regulated the production of related inflammatory factors. Moreover, hexokinase, lactic acid and pyruvic acid levels significantly decreased by infection with sseK1 mutant compared to the wild-type strain. The ATP level of ΔsseK1 mutant group was remarkably increased than WT group and sseK1-complemented group. These indicated that the sseK1 enhanced the level of glycolysis of macrophages infected by S. Typhimurium. In summary, the results demonstrated that sseK1 can down-regulate the inflammation-related cytokines and enhance the glycolysis level in macrophages infected by S. Typhimurium, which may be beneficial for S. typhimurium survival in macrophages.

Salmonella enterica serovar Typhimurium gene sseK3 is required for intracellular proliferation and virulence.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most significant zoonotic pathogens that poses a threat to humans. Previous studies have identified that Salmonella-secreted effector K3 (SseK3) is a novel translated and secreted protein of S. Typhimurium. The objective of this study was to determine whether deletion of the sseK3 gene can attenuate the virulence of S. Typhimurium. To do this, we constructed an sseK3 deletion mutant using the double-exchange allele of the suicide plasmid pRE112ΔsseK3 and assessed the virulence and intracellular proliferation of the mutant. The sseK3 deletion mutant exhibited adhesion and invasion properties similar to those of wild-type (WT) S. Typhimurium, although the virulence and intracellular proliferation of the mutant were significantly reduced compared to that of the WT strain. Furthermore, the observed increase in the median lethal dose (LD50) reflects a decrease in the pathogenicity of the sseK3 deletion mutant in a murine model. In summary, we concluded that disruption of sseK3 can attenuate the intracellular proliferation and reduce the virulence of S. Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) est un des agents pathogènes zoonotiques les plus importants qui représente une menace pour les humains. Des études antérieures ont identifié que l'effecteur K3 secrété par Salmonella (SseK3) est une nouvelle protéine traduite et secrétée par S. Typhimurium. L'objectif de la présente étude était de déterminer si une délétion du gène sseK3 pouvait atténuer la virulence de S. Typhimurium. Nous avons construit un mutant avec la délétion de sseK3 en utilisant l'allèle d'échange double du plasmide suicide pRE112ΔsseK3 et avons évalué la virulence et la prolifération intracellulaire du mutant. Le mutant de délétion démontrait des propriétés d'adhésion et d'invasion similaires à celles du type sauvage (WT) de S. Typhimurium, bien que la virulence et la prolifération intracellulaire du mutant étaient considérablement réduites comparativement à celles de la souche WT. De plus, l'augmentation observée de la dose létale médiane (LD50) reflète une diminution dans la pathogénicité de ce mutant de délétion sseK3 dans un modèle murin. En résumé, nous concluons qu'une perturbation de sseK3 peut atténuer la prolifération intracellulaire et réduire la virulence de S. Typhimurium.(Traduit par Docteur Serge Messier).

Identification and Characterization of the Nuclease Activity of the Extracellular Proteins from Salmonella enterica Serovar Typhimurium.

Pathogens have evolved an array of strategies to establish a productive infection. The extracellular proteins secreted by pathogens are one of unique mechanisms to evade the host innate immune response. Many secretory proteins transported by the bacterial secretion systems have been widely investigated in Salmonella. Certain extracellular nucleases are essential for bacterial pathogenesis. However, there is no current data available for the enzymatic properties of the proteins secreted by Salmonella. Therefore, in the present study we have identified and characterized the nuclease activity of the extracellular proteins from Salmonella enterica serovar Typhimurium. It was demonstrated that the extracellular proteins from S. Typhimurium exhibited the deoxyribonucleases activity against λDNA by agarose gel electrophoresis and agar plate diffusion method. The activity was observed at 16 °C, 37 °C and 42 °C, and found to be highest at 42 °C and inhibited at temperatures over 60 °C. The nuclease activity was stable under alkaline conditions (pH 7-10) and the optimum pH was 9.0. The nuclease activity was promoted at high ionic strength of Ba2+, Ca2+, Mg2+, and Ni2+. Nuclease zymography analysis revealed that there were four activity bands in the extracellular proteins; followed by LC-ESI/MS/MS analysis seven proteins were identified. As demonstrated by nuclease zymography, the recombinant 5'-nucleotidase protein expressed in the prokaryotic expression system displayed the DNase activity. To our knowledge, the present findings represent the first direct and unambiguous demonstration of the nuclease activity of the extracellular proteins from S. Typhimurium, and it provides an important fundamental for further investigation of the role of the extracellular proteins in pathogenicity and immune evasion.

Salmonella enterica serovar Typhimurium sseK3 induces apoptosis and enhances glycolysis in macrophages.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important infectious disease pathogen that can survive and replicate in macrophages. Glycolysis is essential for immune responses against S. Typhimurium infection in macrophages, and is also associated with apoptosis. S. Typhimurium secreted effector K3 (SseK3) was recently identified as a novel translated and secreted protein. However, there is no study about the role of sseK3 in the relationship between apoptosis and glycolysis in cells infected with S. Typhimurium. It is unclear whether this protein exerts a significant role in the progress of apoptosis and glycolysis in S. Typhimurium-infected macrophages. RESULTS: Macrophages were infected with S. Typhimurium SL1344 wild-type (WT), ΔsseK3 mutant or sseK3-complemented strain, and the effects of sseK3 on apoptosis and glycolysis were determined. The adherence and invasion in the ΔsseK3 mutant group were similar to that in the WT and sseK3-complemented groups, indicating that SseK3 was not essential for the adherence and invasion of S. Typhimurium in macrophages. However, the percentage of apoptosis in the ΔsseK3 mutant group was much lower than that in the WT and sseK3-complemented groups. Caspase-3, caspase-8, and caspase-9 enzyme activity in the ΔsseK3 mutant group were significantly lower than in the WT group and sseK3-complemented groups, indicating that sseK3 could improve the caspase-3, caspase-8, and caspase-9 enzyme activity. We also found that there were no significant differences in pyruvic acid levels between the three groups, but the lactic acid level in the ΔsseK3 mutant group was much lower than that in the WT and sseK3-complemented groups. The ATP levels in the ΔsseK3 mutant group were remarkably higher than those in the WT and sseK3-complemented groups. These indicated that the sseK3 enhanced the level of glycolysis in macrophages infected by S. Typhimurium. CONCLUSIONS: S. Typhimurium sseK3 is likely involved in promoting macrophage apoptosis and modulating glycolysis in macrophages. Our results could improve our understanding of the relationship between apoptosis and glycolysis in macrophages induced by S. Typhimurium sseK3.

Effect of gga-miR-155 on cell proliferation, apoptosis and invasion of Marek's disease virus (MDV) transformed cell line MSB1 by targeting RORA.

BACKGROUND: Marek's disease (MD) is caused by the oncogenic Marek's disease virus (MDV), and is a highly contagious avian infection with a complex underlying pathology that involves lymphoproliferative neoplasm formation. MicroRNAs (miRNAs) act as oncogenes or tumor suppressors in most cancers. The gga-miR-155 is downregulated in the MDV-infected chicken tissues or lymphocyte lines, although its exact role in tumorigenesis remains unclear. The aim of this study was to analyze the effects of gga-miR-155 on the proliferation, apoptosis and invasiveness of an MDV-transformed lymphocyte line MSB1 and elucidate the underlying mechanisms. RESULTS: The expression level of gga-miR-155 was manipulated in MSB1 cells using specific mimics and inhibitors. While overexpression of gga-miR-155 increased proliferation, decreased the proportion of G1 phase cells relative to that in S and G2 phases, reduced apoptosis rates and increased invasiveness. However, its downregulation had the opposite effects. Furthermore, gga-miR-155 directly targeted the RORA gene and downregulated its expression in the MSB1 cells. CONCLUSION: The gga-miR-155 promotes the proliferation and invasiveness of the MDV-transformed lymphocyte line MSB1 and inhibits apoptosis by targeting the RORA gene.

The impact of sseK2 deletion on Salmonella enterica serovar typhimurium virulence in vivo and in vitro.

BACKGROUND: Salmonella enterica is regarded as a major public health threat worldwide. Salmonella secretes the novel translocated effector protein K2 (SseK2), but it is unclear whether this protein plays a significant role in Salmonella enterica Typhimurium virulence. RESULTS: A ΔsseK2 mutant of S. Typhimurium exhibited similar growth curves, adhesion and invasive ability compared with wild-type (WT) bacteria. However, deletion of sseK2 rendered Salmonella deficient in biofilm formation and the early proliferative capacity of the ΔsseK2 mutant was significantly lower than that of the WT strain. In vivo, the LD50 (median lethal dose) of the ΔsseK2 mutant strain was increased 1.62 × 103-fold compared with the WT strain. In addition, vaccinating mice with the ΔsseK2 mutant protected them against challenge with a lethal dose of the WT strain. The ability of the ΔsseK2 mutant strain to induce systemic infection was highly attenuated compared with the WT strain, and the bacterial load in the animals' internal organs was lower when they were infected with the ΔsseK2 mutant strain than when they were infected with the WT strain. CONCLUSIONS: We conclude that sseK2 is a virulence-associated gene that plays a vital role in Salmonella virulence.

cAMP Receptor Protein of Salmonella enterica Serovar Typhimurium Modulate Glycolysis in Macrophages to Induce Cell Apoptosis.

We studied the role of glycolysis in the mechanism of cAMP receptor protein-induced macrophage cell death of Salmonella enterica serovar Typhimurium (S. Typhimurium). Cell apoptosis, caspase-3, -8, -9 enzyme activity, and pyruvic acid, lactic acid, ATP, and hexokinase (HK) contents were determined after infection of macrophages with S. Typhimurium SL1344 wild-type and a cAMP receptor protein mutant strain. While cell apoptosis, caspase-3, -8, -9 enzyme activity, lactic acid, hexokinase, and ATP levels significantly changed by infection with crp mutants compared to the wild-type strain (P < 0.05). Our data suggest that the cAMP receptor protein of S. Typhimurium can modulate macrophage death by effecting glycolysis levels. This finding may help to elucidate the mechanisms of S. Typhimurium pathogenesis.

Roles of the crp and sipB genes of Salmonella enterica serovar Typhimurium in protective efficacy and immune responses to vaccination in mice.

Salmonella enterica serovar Typhimurium has a wide host range and is capable of causing infections ranging from severe gastroenteritis to systemic infection in humans. To determine if attenuated S. Typhimurium strains can serve as safe and effective oral vaccines to prevent typhoid fever, the biologic characteristics of crp and sipB deletion mutants were evaluated. Previous studies had found that the crp and sipB genes are related to Salmonella pathogenicity. In this study, cytotoxicity, protective efficacy, and immune responses of the host were analyzed. Our previous data had shown a significance decrease in virulence for the crp and sipB mutants compared with a wild-type strain. The current study confirmed this finding in HeLa cells and showed that the crp mutant was significantly less cytotoxic (P < 0.05) than the sipB mutant. Mice vaccinated with the crp mutant showed significantly better protection after challenge with the wild-type strain (P < 0.05) and significantly greater responses in serum IgG (P < 0.01) and secretory IgA (P < 0.05) compared with the mice vaccinated with the sipB mutant (P < 0.05). Our results indicate that the crp mutant has the potential to be a vaccine candidate and is safe in mice.

Salmonella enterica sérovar Typhimurium a un large spectre d'hôte et est capable de causer chez l'humain des infections allant d'une gastroentérite sévère à une infection systémique. Afin de déterminer si des souches atténuées de S. Typhimurium pourrait servir de vaccin oral sécuritaire et efficace afin de prévenir la fièvre typhoïde, les caractéristiques biologiques de mutants ayant subits des délétions dans les gènes crp et sipB furent évalués. Des études antérieures ont démontré que les gènes crp et sipB sont associés à la pathogénicité de Salmonella. Dans la présente étude, la cytotoxicité, l'efficacité protectrice, et les réponses immunitaires de l'hôte ont été analysées. Nos données antérieures avaient démontré une diminution significative de la virulence pour tous les mutants crp et sipB comparativement à une souche sauvage. Cette étude a confirmé ces données chez les cellules HeLa et démontré que le mutant crp était significativement moins cytotoxique (P < 0,05) que le mutant sipB. Après une infection défi avec une souche sauvage, les souris vaccinées avec le mutant crp étaient significativement mieux protégées (P < 0,05) et présentaient une plus grande réponse en IgG sérique (P < 0,01) et IgA sécrétoire (P < 0,05) comparativement aux souris vaccinées avec le mutant sipB. Nos résultats indiquent que le mutant crp possède le potentiel pour être un vaccin candidat et est sécuritaire chez la souris.(Traduit par Docteur Serge Messier).

Role of the sseK1 gene in the pathogenicity of Salmonella enterica serovar enteritidis in vitro and in vivo.

Salmonella enteritidis is a common food-borne pathogen associated with consumption of contaminated poultry meat and eggs, which frequently causes gastroenteritis in humans. Salmonella secreted effector K1 (SseK1), as a translocated and secreted protein has been identified to be essential for the virulence of Salmonella typhimurium in host cells. However, the role of the sseK1 gene in the pathogenicity of S. enteritidis remain unclear. In this study, a sseK1 deletion mutant of S. enteritidis was constructed and its biological characteristics were examined. It was found that the sseK1 deletion mutant did not affect the growth, adherence and invasion of Salmonella enteritidis when compared to the wild-type S. enteritidis. However, the mutant showed decreased formation of biofilm and significantly reduced intracellular survival of bacteria in activated mouse peritoneal macrophages, as well as showed reduced pathogenicity to a murine model by increasing the lethal dose 50% (LD50) value and decreasing the proliferation ratio of bacteria in vivo. Taken together, this study determined an important role for SseK1 in the pathogenicity of S. enteritidis in vitro and in vivo.

Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus (NDV) protects chickens against NDV and Salmonella Pullorum challenge.

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

Trichinella spiralis and Tumors: Cause, Coincidence or Treatment?

BACKGROUND: Conventional therapeutic strategies for tumors have had limited success, and innovative and more effective approaches to treatment are urgently required. The ancient idea that various biological, bacterial, yeast, viral, and parasitic agents can be used as cancer therapeutics has gradually attracted considerable interest. Certain parasites have been widely discussed in association with human and animal tumors. The purpose of this review was to examine previous literatures which investigates the relations between Trichinella spiralis (T. spiralis) and tumors. METHODS: Using PubMed, articles published before 2018 in the whole world have been searched and comprehensively reviewed. RESULTS: Many researches have provided proofs that T. spiralis possesses antitumor activities. The antitumor effect of T. spiralis was first described in the 1970s. However, its research has been inconsistent, and little progress has been made in this field. Therefore, the mechanisms underlying these inhibitory effects are still unclear, and convincing evidence of the links between T. spiralis and the prevention or treatment of tumors from clinical trials is absent. Meanwhile, some other researches also suggested that T. spiralis may cause or contribute to coinfection with a tumors. CONCLUSION: The review has highlighted the scientific literature focussing on evidence for T. spiralis to act as a pro- or antitumorigenic agent is summarized and discussed, in hope of contributing to a better understanding of the relations between T. spiralis and tumors.

Genetic Characterization of Fowl Adenovirus Strains Isolated from Poultry in China.

Fowl adenoviruses (FAdVs) infect chickens worldwide, resulting in global economic losses in the poultry industry. We examined the strains present in chickens in regions of China where infections are particularly prevalent. Fifteen FAdV strains were successfully isolated in the field. The L1 loop region of the hexon gene was sequenced to genetically identify the FAdV isolates. By comparing these sequences to adenovirus reference strain sequences using phylogenetics, 15 adenovirus strains were found to cluster into two distinct species. One cluster containing 12 strains belonged to the fowl adenoviruses C species and serotyped as FAdV-4. The other cluster containing three strains belonged to the fowl adenoviruses E species and serotyped as FAdV-10. To our knowledge, this is the first report of the existence of fowl adenoviruses E in China. Furthermore, at least two types of fowl adenovirus strains are predominant among poultry in China. Cumulatively, this study helps lays the groundwork for future research on the pathogenicity and potential treatment measures for FAdV infections in chickens.

Isolation, molecular characterization and an artificial infection model for a variant porcine epidemic diarrhea virus strain from Jiangsu Province, China.

Porcine epidemic diarrhea virus (PEDV) is a causative agent of porcine intestinal disease, which causes vomiting, diarrhea, and dehydration in piglets. PEDV is associated with the most severe pathogenesis in one-week-old piglets, with mortality rates reaching 100%. A PEDV strain was isolated from the intestinal tract of diarrheic piglets from a pig farm in Jiangsu Province in March 2016, termed the JS201603 isolate. The isolated virus was confirmed to be PEDV via RT-PCR, electron microscopy, a cytopathic effect assay and sequence analysis. The S and ORF3 genes of the JS201603 isolate were sequenced, revealing that the S gene was associated with a 15-base insertion at 167 nt, 176 - 186 nt, and 427 - 429 nt, as well as a six-base deletion in 487 - 492 nt, indicating that it was a current epidemic variant compared with the classical strain, CV777. No deletion occurred between 245 - 293 nt of the ORF3 gene in the JS201603 isolate compared with the vaccine isolates YY2013 and SQ2014. An experimental infection model indicated that the piglets in the challenge group successively developed diarrhea, exhibiting yellow-colored loose stools with a foul odor. The piglets in the JS201603 isolate challenge group displayed reduced food consumption, lost weight, and in severe cases even died. No abnormalities were observed in the control group. The JS201603 variant isolated in this study contributes to the evolutionary analysis of diarrhea virus. The experimental infection model has established a foundation for further studies on vaccine development.

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Identification of genes involved in Mycoplasma gallisepticum biofilm formation using mini-Tn4001-SGM transposon mutagenesis.

Mycoplasma gallisepticum (MG) is an important pathogen that can cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. MG has the ability to form biofilms. The molecular mechanisms underlying MG biofilm formation are complex and poorly understood. To better understand the mechanisms involved in biofilm formation, mini-Tn4001-SGM, a novel transposon vector containing the gentamicin gene was constructed and electroporated into MG strain Rlow. Of the 738 mutants obtained, 12 had significantly reduced capacity to form biofilms in a polystyrene microtiter-plate biofilm assay. Ten different genes were identified as disrupted in these mutants using genomic walking from the transposon insertion sites and Southern bolt hybridization with a transposon-based probe. Four genes were associated with cellular processes, especially synthesis of extracellular polysaccharide and several lipoproteins encoded. Other genes were associated with translation, metabolism and gene regulation, and one had unknown function. Seven genes identified in this study have been previously associated with biofilm formation in MG or other bacterial species. The other three have not been previously reported to play a role in biofilm formation in MG. In conclusion, a new transposon vector was shown to be a powerful tool for future studies of MG pathogenesis. This study adds to our understanding of the molecular mechanisms involved in MG biofilm formation and may shed light on the persistence of MG infections.

[Construction and characterization of type III secretion system of attenuated Salmonella typhimurium].

In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD50) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×104 times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.

[Construction of host-vector balanced lethal system of Salmonella typhimurium SL1344Δcya mutant and immune protection test of chickling].

OBJECTIVE: To develop a host-vector balanced lethal system of Salmonella typhimurium adenylate cyclase mutant, and detect its biological characteristics. METHODS: We constructed SL1344ΔcyaΔasd mutant strain by recombinant suicide plasmid (pREAasd), and screened by two-step method, transformed pYA3493 plasmid containing the asd gene without resistance electric into the lack of SL1344 AcyaΔasd, then the recombinant strains SL1344 ΔcyaΔasd (pYA3493) was constructed successfully. RESULTS: The biochemical characteristics and growth rate of the mutant were different from that of the wild strain SL1344, but almost the same as that of the parent strain SL1344Δcya. The mutant strain could neither ferment maltose, lactose, and sorbitol, nor decompose H2S, galactose and rat lee sugar, but still retained the ability to use glucose. The one-day chicken lethal test showed that SL1344ΔcyaΔasd (pYA3493) mutant was at least 104 times lower than SL1344 strain. The protection rate induced by the SL1344ΔcyaΔasd (pYA3493) mutant was 62. 5%. CONCLUSION: The SL1344ΔcyaΔasd (pYA3493) mutant was successfully constructed, and had good immune protection, it laid a foundation for developing potential oral vaccines.

Deletion of Invasion Protein B in Salmonella enterica Serovar Typhimurium Influences Bacterial Invasion and Virulence.

Salmonella enterica serovar Typhimurium (S. Typhimurium) has a wide host range and causes infections ranging from severe gastroenteritis to systemic infections in human, as well as causing typhoid-like disease in murine models of infection. S. Typhimurium translocates its effector proteins through the Salmonella pathogenicity island-I (SPI-I)-encoded T3SS-I needle complex. This study focuses on invasion protein B (SipB) of S. Typhimurium, which plays an active role in SPI-I invasion efficiency. To test our hypothesis, a sipB deletion mutant was constructed through double-crossover allelic using the suicide vector pRE112ΔsipB, and its biological characteristics were analyzed. The results showed that the SipB does not affect the growth of Salmonella, but the adherence, invasion, and virulence of the mutant were significantly decreased compared with wild-type S. Typhimurium (SL1344). This research indicates that SipB is an important virulence factor in the pathogenicity of S. Typhimurium.

Histamine levels in embryonic chicken livers infected with very virulent infectious bursal disease virus.

Histamine is an endogenous nitrogenous compound with extensive effects on immunologic cells and involved in many physiological functions. The current aim was to determine histamine levels in embryonic liver and its association with the pathogenicity of a very virulent infectious bursal disease virus (vvIBDV) isolate serially passaged in chicken embryos. A vvIBDV isolate and the passaged viruses were inoculated into SPF embryonated chicken eggs (0.2 ml per egg) via the chorioallantoic membrane. Embryonic livers were collected at 24, 48, 72, 96, and 120 h post-inoculation and histamine contents were quantified by fluorescence spectrophotometry analyses. Results showed that the histamine content in embryonic livers infected with the original vvIBDV isolate and the early passaged viruses significantly increased 48 h post-inoculation, as compared with the adapted IBDV isolate (p<0.01) and controls (p<0.01), with the concentration peaking from 72 h to 96 h. Most of the infected chicken embryos died from 48 h to 96 h post-inoculation. Moreover, the histamine content in dead embryos was markedly increased compared with live embryos (p<0.05), peaking at 72 h post-inoculation (p<0.01). There was an association between histamine content in embryonic livers and an elevation in histidine decarboxylase activity. Taken together, our results suggest that an excess of histamine correlates with inflammatory responses during vvIBDV infection. This study provides an incremental step in the understanding of the pathogenesis of vvIBDV.