Construction of a trivalent candidate Shigella vaccine strain with host-vector balanced-lethal system

A trivalent live Shigella vaccine candidate FSD01 against S. flexneri 2a, S. sonnei and S. dysen-teriae I was constructed. This candidate strain was based on the S. flexneri 2a vaccine T32. By homologous recombi-nation exchange, the chromosomal asd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while another asd gene of S. mutans was employed to construct an Asd complementary vector. This combination of asd 'host/ Asd vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S. sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S, sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey     

Genomic compositions and phylogenetic analysis of Shigella boydii subgroup

Comparative Genomic Hybridization (CGH) microarray analysis was used to compare the genomic compositions of all eighteen Shigella boydii serotype representative strains. The results indicated the genomic “backbone” of this subgroup contained 2552 ORFs homologous to nonpatho-genic E. coli K12. Compared with the genome of K12199 ORFs were found to be absent in all S. boydii serotype representatives, including mainly outer membrane protein genes and O-antigen biosynthesis genes. Yet the specific ORFs of S. boydii subgroup contained basically bacteriophage genes and the function unknown (FUN) genes. Some iron metabolism, transport and type II secretion system related genes were found in most representative strains. According to the CGH phylogenetic analysis, the eighteen S. boydii serotype representatives were divided into four groups, in which serotype C13 strain was remarkably distinguished from the other serotype strains. This grouping result corresponded to the distribution of some metabolism related genes. Furthermore, the analysis of genome backbone genes, specific genes, and the phylogenetic trees allowed us to discover the evolution laws of S. boydii and to find out important clues to pathogenesis research, vaccination and the therapeutic medicine development.     

Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1(DHAV-1) Isolates in Shandong Province of China

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD 50 s) and the median lethal doses(LD 50 s),respectively.The results showed that the ELD 50 s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10 6 /mL to 1.44 × 10 7 /mL,while the LD 50 s were 2.39 × 10 5 /mL to 6.15 × 10 6 /mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(aa 158-160,180-193 and 205-219) and other variable points in VP1 protein,but which didn’t cause virulence of DHAV-1 change.     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow＇s Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow＇s milk, as screened in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Corresponding virulence genes （iap, prfA, picA, hly, mpl, actA, plcB, InlA and lnlB） were compared with L. monocytogenes reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S in mouse and chicken embryo models （50% lethal dose： 10^8.14 VS. 10^5.49 and 10^6.73 VS. 10^1.9, respectively）. The genes prfA, picA and mpl were homologous among L. monocytogenes strains H4, 10403S and EGD （〉98%）. Genes iap, hly, plcB, lnlA and lnlB of L. monocytogenes 10403S had higher homology to those of strain EGD （〉98%） than isolate H4. The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level, but 98.7% at the amino acid level. The actA gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acid deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.     

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogens serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD 18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow's Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow's milk,as screened in mouseand chicken embryonated egg models,was examined for virulence-related phenotypic traits.Correspondingvirulence genes (iap,prfA,plcA,hly,mpl,actA,plcB,InlA and InlB) were compared with L.monocytogenesreference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence.Al-though L.monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity,invitro growth and invasiveness and even had higher adhesiveness,faster intracellular growth and higherphospholipase activity in vitro,it was substantially less virulent than the strain 10403S in mouse and chickenembryo models (50% lethal dose:10~(8.14) vs.10~(5.49) and 10~(6.73) vs.10~(1.9),respectively).The genes prfA,plcA andmpl were homologous among L.monocytogenes strains H4,10403S and EGD (>98%).Genes iap,hly,plcB,InlA and InIB of L.monocytogenes 10403S had higher homology to those of strain EGD (>98%) than isolateH4.The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level,but 98.7% at the amino acid level.The actA gene of isolate H4 had deletions of 105 nucleotides correspondingto 35 amino acid deletions falling Within the proline-rich region.Taken together,this study presents someclues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletionmutations of actA.     

Listeria monocytogenes Mutants Carrying Newcastle Disease Virus F Gene Fused to its actA and plcB： In vitro Expression and Immunogenicity in Chickens

Recombinant Listeria monocytogenes mutants carrying Newcastle disease virus (NDV) fusionprotein gene F were constructed by homologous recombination.NDV F or its truncated fragment Fa wasused as the model heterologous gene to be integrated into actA or plcB downstream of their signal sequences.Correct orientation of the inserted genes was verified by polymerase chain reaction amplification of F or Fa.The inserted F and Fa were expressed in the two recombinants Lm-AactA-F and Lm-AplcB-Fa as shown bysodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Both recombinantsexhibited reduced virulence to embryonated eggs and mice by about 1.5-2.5 logs as compared with theparent wild strain 10403S. They were also less invasive than strain 10403S (P<0.05). Chickens receiving therecombinant strains orally or intraperitoneally were partially protected from virulent NDV challenge possiblydue to enhancement of non-specific immunity because the antibody titers against the homologous virus strainor the recombinant truncated fusion protein were marginal. Further research is needed in other animal modelsto see if the low antibody response results from insufficient expression of the heterologous genes as a resultof failure of L. monocytogenes or its recombinants to persist or replicate in chickens.     

福氏2a志贺氏菌△aroA突变减毒株的构建

志贺氏菌芳香族氨基酸合成酶基因缺陷能够使菌体明显减毒,并有可能成为新一代痢疾疫苗．用ＰＣＲ技术从野生型福氏２ａ志贺氏菌２４５７Ｔ中克隆出ａｒｏＡ基因,在体外进行精确的缺失突变,并通过体内同源重组,构建成△ａｒｏＡ突变体ＲＳ４２６．实验结果表明,这种突变体仍保持了侵袭能力和保护性Ｏ抗原的表达,但其毒力已明显降低,不能产生豚鼠角结膜炎,小鼠半数致死量明显提高．免疫保护试验显示,ＲＳ４２６可在小鼠中产生对福氏２ａ野生菌１００％的保护作用．     

弗氏志贺菌毒力大质粒导入大肠杆菌MG1655

目的:将弗氏2a志贺菌2457T的毒力大质粒pSF导入大肠杆菌MG1655。方法:通过诱动转移技术,将弗氏2a志贺菌2457T的毒力大质粒导入大肠杆菌MG1655。结果:构建了MG1655/pSF:pXL275-virG的毒力大质粒导入突变株,双向电泳初步比较分析表明在重组MG1655中有志贺菌毒力的表达。结论:成功地将弗氏2a志贺菌2457T毒力大质粒pSF导入了大肠杆菌MG1655。     

New animal model of shigellosis in the Guinea pig: its usefulness for protective efficacy studies

It has been difficult to evaluate the protective efficacy of vaccine candidates against shigellosis, a major form of bacillary dysentery caused by Shigella spp. infection, because of the lack of suitable animal models. To develop a proper animal model representing human bacillary dysentery, guinea pigs were challenged with virulent Shigella flexneri serotype 2a (strains 2457T or YSH6000) or S. flexneri 5a (strain M90T) by the intrarectal (i.r.) route. Interestingly, all guinea pigs administered these Shigella strains developed severe and acute rectocolitis. They lost approximately 20% of their body weight and developed tenesmus by 24 h after Shigella infection. Shigella invasion and colonization of the distal colon were seen at 24 h but disappeared by 48 h following i.r. infection. Histopathological approaches demonstrated significant damage and destruction of mucosal and submucosal layers, thickened intestinal wall, edema, erosion, infiltration of neutrophils, and depletion of goblet cells in the distal colon. Furthermore, robust expression of IL-8, IL-1beta, and inducible NO synthase mRNA was detected in the colon from 6 to 24 h following Shigella infection. Most importantly, in our new shigellosis model, guinea pigs vaccinated with an attenuated S. flexneri 2a SC602 strain possessing high levels of mucosal IgA Abs showed milder symptoms of bacillary dysentery than did animals receiving PBS alone after Shigella infection. In the guinea pig, administration of Shigella by i.r. route induces acute inflammation, making this animal model useful for assessing the protective efficacy of Shigella vaccine candidates.     

驱除痢疾杆菌侵袭大质粒的新方法

用质粒不相容性原理驱除痢疾杆菌福氏 2a 2 4 5 7T和宋内S7的侵袭大质粒 ,先从福氏2a侵袭大质粒分别扩增ori和inc基因 ,将它们克隆至 pMD18 T载体 ,得重组质粒pMDori和 pMDinc ,然后转化 2 4 5 7T和S7,不管是pMDori还是 pMDinc都能竞争驱除痢疾杆菌福氏 2a 2 4 5 7T和宋内S7的侵袭大质粒。     

Shigella enterotoxin-2 is a type III effector that participates in Shigella-induced interleukin 8 secretion by epithelial cells

We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short-circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study, we show that ShET-2 secretion into the extracellular space requires the T3SS in Shigella flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is cotranscribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Although other phenotypes were not different compared with the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells.     

福氏2a志贺氏菌2457T HtpG蛋白诱导小鼠炎性反应

[目的]构建福氏2a志贺氏菌2457T株的htpG缺失突变株和回复株,对HtpG蛋白的功能进行初步研究.[方法]采用X-Red重组系统对htpG基因进行缺失突变,构建了福氏2a志贺氏菌2457T株的htpG缺失突变株,并利用低拷贝质粒构建了htpG突变株的回复株.在此基础上,对野生株、突变株和回复株的生长曲线、生化反应、豚鼠角膜试验进行了比较分析,并考察了野生株、突变株和回复株腹腔注射引起小鼠炎症反应的强弱.[结果]HtpG蛋白功能与福氏志贺氏菌的基本生化代谢无关,也不影响细菌穿透上皮细胞的能力,但腹腔注射后能够引起小鼠强烈的炎症反应.[结论]HtpG蛋白功能可能与细菌的免疫致病性相关.     

Construction and characterization of a live attenuated Shigella flexneri 2a vaccine strain, sf301 Delta virG and dsbA33G]

Construction and characterization of a live attenuated Shigella flexneria 2a sf301 vaccine strain to prevent the endemic of shigellosis. Using Chinese majority epidemic Shigella flexneri 2a serotype sf301 as the target, p Delta virG, a deletion derivation of the virG gene in the SacB suicide vector pCVD442 and pDsbA33G, an mutant of a disulfide bond catalyst DsbA, replaced its 33 amiano acid Cystine by Glycerin in pCVD442, were used to generate a attenuated mutant strain sf301: Delta virG: DsbA33 G. Its virulence was evaluated by Sereny test, the invasive ability was detected by HeLa cell invasive assay, immunogenicity was detected by immunized Guinea pigs through inoculated guinea pigs' conjunctive sac. Sereny test was negative and HeLa invasive assay showed sf301: Delta virG: DsbA33 G retained partial invasive ability. In contrast to control group, sf301: Delta virG: DsbA33 G could induced significantly high antibody levels of IgA and IgG against sf301 LPS in animal's mucosal lavage fluids and sera in both primary immunization protocol and boosting protocol. The numbers of ASCs in local draining lymph nodes and spleens were significantly higher than control group. The immune response to sf301: Delta virG: DsbA33 G could provide completely protection from the challenge of wild type sf301. sf301: Delta virG: DsbA33 G is a safe and effective oral candidate vaccine to prevent the infection of Shigella strains.     

痢疾杆菌酸抗性系统相关基因缺失突变体的构建

依赖于谷氨酸脱羧酶的酸抗性系统对痢疾杆菌在宿主细胞内的生存和繁殖至关重要,hdeA、hdeB、yhiE和yhiF是其中几个重要的酸抗性基因。借助Red系统的重组功能,PCR扩增两翼与目的基因上下游同源的抗性基因片段,电击转化痢疾杆菌2457T,对筛选到的阳性转化子再导入编码FLP位点特异性重组酶的质粒pCP20以去除抗性基因。结果成功的敲除了hdeA、hdeB、yhiE和yhiF等4个酸抗性系统相关基因,为深入研究痢疾杆菌酸抗性基因的调控网络奠定了基础.     

福氏2a志贺氏菌2457T株argT基因突变体的构建

目的：构建福氏2a志贺氏菌2457T株argT基因缺失突变体和ArgT蛋白非降解突变体,以进行后续ArgT功能研究。方法：根据福氏2a志贺氏菌2457T株基因组全序列,采用λ-Red重组系统对argT基因进行缺失,并经PCR验证;采用定点突变的方法构建ArgT非降解株,并经SDS-PAGE验证;对野生株、argT缺失突变株和ArgT非降解突变株37℃时的生长曲线及生化反应进行比较研究。结果：构建了2457T的argT缺失突变株和ArgT非降解突变株;2种突变株初始生长均较慢,但最终和野生株状态一致;2种突变株利用甘露醇的能力都比野生株强,而利用葡萄糖的能力降低。结论：获得了福氏2a志贺氏菌2457T株argT基因缺失突变体和ArgT蛋白非降解突变体。     

志贺菌福氏2a信号标签诱变突变体文库的构建

以合成的单链序列特异性标签为模板,通过PCR得到双链DNA标签并将其克隆到自杀质粒pUT-Tn5 Km2的转座子中,转化大肠杆菌S17-1λpir;然后用经转化的S17-1λpir与福氏志贺菌2a 2457T交配,挑出对氨苄青霉素敏感,对卡那霉素和萘啶酮酸抗性的菌落,结果表明构建了包含4376个福氏志贺菌突变体信号标签诱变库,为进一步鉴定该病原体的毒力基因打下了基础。