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Contribution of the Lipopolysaccharide to Resistance of Shigella flexneri 2a to Extreme Acidity  [PDF]
Mara Martini?,Anilei Hoare,Inés Contreras,Sergio A. álvarez
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0025557
Abstract: Shigella flexneri is endemic in most underdeveloped countries, causing diarrheal disease and dysentery among young children. In order to reach its target site, the colon, Shigella must overcome the acid environment of the stomach. Shigella is able to persist in this stressful environment and, because of this ability it can initiate infection following the ingestion of very small inocula. Thus, acid resistance is considered an important virulence trait of this bacterium. It has been reported that moderate acid conditions regulate the expression of numerous components of the bacterial envelope. Because the lipopolysaccharide (LPS) is the major component of the bacterial surface, here we have addressed the role of LPS in acid resistance of S. flexneri 2a. Defined deletion mutants in genes encoding proteins involved in the synthesis, assembly and length regulation of the LPS O antigen were constructed and assayed for resistance to pH 2.5 after adaptation to pH 5.5. The results showed that a mutant lacking O antigen was significantly more sensitive to extreme acid conditions than the wild type. Not only the presence of polymerized O antigen, but also a particular polymer length (S-OAg) was required for acid resistance. Glucosylation of the O antigen also contributed to this property. In addition, a moderate acidic pH induced changes in the composition of the lipid A domain of LPS. The main modification was the addition of phosphoethanolamine to the 1′ phosphate of lipid A. This modification increased resistance of S. flexneri to extreme acid conditions, provide that O antigen was produced. Overall, the results of this work point out to an important role of LPS in resistance of Shigella flexneri to acid stress.
Construction of Stable and Non-resistant Bivalent Vaccine Candidate Strain Against Shigella flexneri 2a and Shigella sonnei
稳定、无抗药的痢疾福氏2a和宋内双价菌苗候选株的构建

Rui Xianliang Xu Yongqiang Wang Hong Su Guofu Huang Cuifen,
芮贤良
,徐永强,王红,苏国富,黄翠芬

生物工程学报 , 1996,
Abstract: The E. coli cs3 gene coding for CFA/11 antigen was site-specifically integrated into the asd gene locus of S. flexneri 2a vaccine strain T32, resulting in the asd gene inactivated. Meanwhile, the S. sonnei O antigen gene was cloned into a non-resistant expression vector pXL378 to construct the plasmid pXL390. By transforming the asd minus T32 strain with pXL390, the bivalent vaccine candidate strain FS01 against S. flexneri 2a and S. sonnei was finally obtained. Experiments showed that the recombinant plasmid pXL390 was very stable in the asd minus T32 strain without the use of any antibiotics; FS01 strain was genetically stable and expressed two kinds of Shigella LPS-O antigens with no enhancement of its toxicity. Animal test demonstrated that FS01 strain, when administered subcutaneously in mice,could confer 100% protection against the intraperi-toneal challenges of virulent S. flexneri 2a and S. sonnei.
Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a
Huan Nie, Fan Yang, Xiaobing Zhang, Jian Yang, Lihong Chen, Jing Wang, Zhaohui Xiong, Junping Peng, Lilian Sun, Jie Dong, Ying Xue, Xingye Xu, Shuxia Chen, Zhijian Yao, Yan Shen, Qi Jin
BMC Genomics , 2006, DOI: 10.1186/1471-2164-7-173
Abstract: The Sf8401 chromosome is 4.5-Mb in size, a little smaller than that of Sf301, mainly because the former lacks the SHI-1 pathogenicity island (PAI). Compared with Sf301, there are 6 inversions and one translocation in Sf8401, which are probably mediated by insertion sequences (IS). There are clear differences in the known PAIs between these two genomes. The bacteriophage SfV segment remaining in SHI-O of Sf8401 is clearly larger than the remnants of bacteriophage SfII in Sf301. SHI-1 is absent from Sf8401 but a specific related protein is found next to the pheV locus. SHI-2 is involved in one intra-replichore inversion near the origin of replication, which may change the expression of iut/iuc genes. Moreover, genes related to the glycine-betaine biosynthesis pathway are present only in Sf8401 among the known Shigella genomes.Our data show that the two S. flexneri genomes are very similar, which suggests a high level of structural and functional conservation between the two serotypes. The differences reflect different selection pressures during evolution. The ancestor of S. flexneri probably acquired SHI-1 and SHI-2 before SHI-O was integrated and the serotypes diverged. SHI-1 was subsequently deleted from the S. flexneri 5b genome by recombination, but stabilized in the S. flexneri 2a genome. These events may have contributed to the differences in pathogenicity and epidemicity between the two serotypes of S. flexneri.Shigella species that cause bacillary dysentery or shigellosis are Gram-negative, non-sporulating, facultative anaerobes, and the disease remains a major worldwide health problem. An estimated annual infection of 160 million individuals, with 1.1 million deaths, most of them children under 5 years old in developing countries, occurs with shigellosis [1]. The poor sanitary conditions prevalent in these areas contribute to the spread of the bacteria, and the expense of antibiotics and increasing antibiotic resistance complicate treatment [2].Shigella was r
Dipstick for Rapid Diagnosis of Shigella flexneri 2a in Stool  [PDF]
Faridabano Nato, Armelle Phalipon, Lan Phuong Thi Nguyen, Tai The Diep, Philippe Sansonetti, Yves Germani
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0000361
Abstract: Background Shigellosis or bacillary dysentery, an acute bloody diarrhoea, is a major public health burden in developing countries. In the absence of prompt and appropriate treatment, the infection is often fatal, particularly in young malnourished children. Here, we describe a new diagnostic test for rapid detection, in stool, at the bedside of patients, of Shigella flexneri 2a, the most predominant agent of the endemic form of the disease. Methodology/Principal Findings The test is based on the detection of S.flexneri 2a lipopolysaccharide (LPS) using serotype 2a-specific monoclonal antibodies coupled to gold particles and displayed on one-step immunochromatographic dipstick. A concentration as low as 20 ng/ml of LPS is detected in distilled water and in reconstituted stools in under 15 minutes. The threshold of detection corresponds to a concentration of 5×107 CFU/ml of S. flexneri 2a, which provides an unequivocal positive reaction in three minutes in distilled water and reconstituted stools. The specificity is 100% when tested with a battery of Shigella and unrelated strains, in culture. When tested in Vietnam, on clinical samples, the specificity and sensitivity were 99.2 and 91.5%, respectively. A decrease of the sensitivity during the evaluation on stool samples was observed after five weeks at room temperature and was due to moistening of the dipsticks caused by the humidity of the air during the fifth week of the evaluation. This drawback is now overcome by improving the packaging and providing dipsticks individually wrapped in waterproof bags. Conclusion This simple dipstick-bases test represents a powerful tool for case management and epidemiological surveys.
Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a
Jiyu Zhang,Hong Liu,Xiaobing Zhang,Jian Yang,Fan Yang,Guowei Yang,Yan Shen,Yunde Hou,Qi Jin
Science China Life Sciences , 2003, DOI: 10.1360/02yc0060
Abstract: The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.
Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

ZHANG Jiyu,LIU Hong ZHANG Xiaobing YANG Jian YANG Fan YANG Guowei SHEN Yan HOU Yunde JIN Qi,

中国科学C辑(英文版) , 2003,
Abstract: The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.
Outer Membrane Protein A (OmpA) of Shigella flexneri 2a, Induces Protective Immune Response in a Mouse Model  [PDF]
Debasis Pore, Nibedita Mahata, Amit Pal, Manoj K. Chakrabarti
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0022663
Abstract: Background In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. Key Results In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPO? vector and expressed using induction with isopropyl thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. Conclusion These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection.
Engineered and construction of pDS132:: virG as suicide vector for targeted gene deletion of virG from Shigella flexneri 2a in order to generation a live attenuated Shigella vaccine  [cached]
,Mojtaba Saadati,Sayed Mostafa Hosseini
Journal of Fasa University of Medical Sciences , 2012,
Abstract: Background & Objective: Shigella are Gram negative bacteria capable of inducing their entry into non-phagocytic cells via secretion of various effector proteins called invasion plasmid antigens (Ipas). The most important of them is VirG protein. Live attenuated Shigella vaccines have indicated promise in inducing protective immune responses in human clinical trials. In current situation, constructions of Shigella vaccine candidate strains based on classical allelic exchange systems are considered. The aim of this research was engineered and constructed pDS132:: virG as a suicide plasmid for targeted deletion regions of virG gene by using allele exchange method in Shigella flexneri 2a. Method & Materials: In this applied study, species and serotype of shigella was confirmed by using serological and Polymerase Chain Reaction (PCR) tests. Detection primers of virG gene were designed and cloned to pGEM-5zf vector and finally, sequencing was done. According to virG restriction enzyme map, 1751 bp of virG gene was removed by using of HincП restriction enzyme and the virG was successfully constructed. The pGEM virG vector was digested by use of SphI and SalI enzymes and then cloned to pSD132 as suicide vector. Precision of process were verified through phenotype and genotype experiment. Results: The Shigella flexneri type 2a strain was verified by serological and PCR tests. Sequence of the virG gene in native strain was sequentially identical with the strains submitted in the Gene-Bank database. Since the pDS132:: virG contains 1484 bp which derived from virG gene, therefore, it can be utilized for interference in virG gene as specific suicide vector in shigella flexneri 2a. Conclusion: Application of suicide systems facilitated mutant construction in more specific and effective method in comparison with the other early techniques such as serial passage.
A novel anti-virulence gene revealed by proteomic analysis in Shigella flexneri 2a
Ge Zhao, Li Zhu, Erling Feng, Xiaoyu Cao, Na Shang, Xiankai Liu, Xiang Liao, Tianyi Ying, Jie Wang, Huipeng Chen, Hengliang Wang
Proteome Science , 2010, DOI: 10.1186/1477-5956-8-30
Abstract: The ArgT protein was dramatically down-regulated at 37 degrees C. In contrast, the ArgT from the non-pathogenic E. coli did not show this differential expression as in S. flexneri, which suggested that argT might be a potential anti-virulence gene. Competitive invasion assays in HeLa cells and in BALB/c mice with argT mutants were performed, and the results indicated that the over-expression of ArgTY225D would attenuate the virulence of S. flexneri. A comparative proteomic analysis was subsequently performed to investigate the effects of ArgT in S. flexneri at the molecular level. We show that HtrA is differentially expressed among different derivative strains.Gene argT is a novel anti-virulence gene that may interfere with the virulence of S. flexneri via the transport of specific amino acids or by affecting the expression of the virulence factor, HtrA.Shigella flexneri is a gram-negative, facultative pathogen that is the leading cause of communicable bacterial dysenteries in developing countries. The virulence genes of S. flexneri are located on a large plasmid that was acquired by horizontal gene transfer. These genes encode a type III secretion system (TTSS) apparatus that enables S. flexneri to invade epithelial cells in the lower gut of humans [1].The expression of virulence genes is induced under growth conditions similar to those found at the site of invasion. For example, a temperature of 37°C is a particularly important environmental signal. Maurelli et al. [2] found that S. flexneri cultivated at 37°C produced kerato-conjunctivitis in guinea pigs and were able to penetrate and replicate in intestinal epithelial cells, but that bacteria grown at 30°C were phenotypically avirulent and noninvasive. Other studies in S. flexneri have focused on regulation of the expression of these virulence genes at different temperatures [1,3]. However, we expected that, in addition to known virulence genes, a large number of other genes would be differentially expressed in
Screening and identification of Shigella flexneri 2a virulence-related genes induced after invasion of epithelial cells
Zhaoxing Shi,Hengliang Wang,Kun Hu,Erling Feng,Xiao Yao,Liuyu Huang,Guofu Su,Peitang Hunag,Cuifen Huang
Science China Life Sciences , 2004, DOI: 10.1360/03yc0208
Abstract: An in vivo expression technology (IVET) was applied to screen S. flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three appeared to be antisense to genes involved in the transmethylation, biosynthesis, and phosphotransferase system; and three were predicted to encode polypeptides with unknown functions. Intracellular survival assays showed that the mutants of alkA, citC and wcaJ genes had lower capability of intracellular replication or survival than the wild-type strain. The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intracellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.
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