Identification of an Escherichia coli gene homologous to virR, a regulator of Shigella virulence.

Virulence in Shigella spp., as well as in strains of enteroinvasive Escherichia coli, is regulated by growth temperature. Previously, virR had been identified as the gene controlling the temperature-regulated expression of Shigella virulence. Since Shigella spp. and E. coli are also known to share greater than 90% DNA sequence homology, we sought to determine if nonpathogenic E. coli K-12 C600 contains a gene homologous to the Shigella flexneri 2a gene virR. Through the use of transduction and molecular cloning of strain C600 chromosomal DNA we have shown that E. coli K-12 does indeed contain a gene functionally homologous to the virR of S. flexneri.     

Fatty acid composition of the lipopolysaccharides of bacteria in the genera Escherichia, Shigella and Salmonella as a taxonomic trait

The comparative study of the fatty acid composition of bacterial lipopolysaccharides (LPS) in the genera Escherichia, Shigella, Salmonella has been carried out under identical experimental conditions. The LPS of the bacteria under study have been found to contain a number of saturated fatty acids and oxyacids which could not be previously detected in these bacteria in other studies. In all bacterial strains under study LPS include mainly 3-oxytetradecanoic, tetradecanoic and dodecanoic fatty acids. The essential feature of the fatty acid composition of Salmonella is the presence of 2-oxytetradecanoic acid; this acid is absent in Escherichia and Shigella, which can thus be used as a differentiating criterion. The content of other fatty acids in Salmonella is similar to that in Escherichia and Shigella. These data confirm that the genera Escherichia, Shigella and Salmonella are phylogenetically related, the relationship between Escherichia and Shigella being more close.     

Specific antigens of the causative agents and their antibodies in the circulating immune complexes in acute dysentery

The level of circulating immune complexes has been determined in 53 patients in the dynamics of the disease. For the first time circulating immune complexes have been found to contain Shigella sonnei K-antigen and Shigella flexneri O-antigen, as well as IgA, IgG and IgM to Shigella. Shigella antigens can be detected from the first week of the disease, and their occurrence does not depend on the level of circulating complexes in patients blood serum.     

Frequency of IS1-mediated molecular events in different members of the family Enterobacteriaceae.

The numbers of chromosomal copies of the insertion sequence IS1 in strains of Salmonella typhimurium (0 to 8 copies), Shigella sonnei (56 copies), and Shigella flexneri (41 copies) isolated in Mexico City, Mexico, were similar to those reported for these genera isolated in other countries. Of the 11 Shigella strains studied, all carried several small plasmids; however, in only one of these strains did a small plasmid contain IS1, IS1 recombination, cointegrate formation mediated by IS1 or by the IS1-flanked transposon Tn9, and transposition of Tn9 occurred at a higher frequency in S. typhimurium than in either Escherichia coli or S. sonnei strains. The frequencies of IS1 recombination in S. typhimurium strains containing either zero or eight copies of IS1 were similar.     

Location of the ss--mutation of bacteriophage T7 in genes 10, the structural gene for the major capsid protein.

T7+ phage are unable to plate on a strain of Shigella sonnei D2 371-48. Spontaneous phage mutants arise (ss--mutants) that are able to plate on this strain of Shigella. We have shown by complementation studies and genetic crosses that the ss--mutation maps in gene 10, the structural gene for the major protein of the capsid. This finding implies that the gene 10 protein may interact with a host protein during phage development and that the abortive infection of T7 observed in S. sonnei D2 371-48 with T7+ phage may be a defect in head morphogenesis. Our studies also reveal that various T7 strains commonly contain deletions in nonessential regions. T7 ss--mutants selected after growth of T7+ on Shigella D2 371-48 often acquire a deletion in the 0.7 gene that is not necessary for the ss--phenotype. Finally, we have found a new nonessential region of the T7 chromosome that is located between 33 and 35.5% of the T7 genome length.     

Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes

The genetic differences between the human pathogen, Shigella flexneri, and the non-pathogenic Escherichia coli were investigated in an attempt to identify pathogenicity islands (PAIs) in the S. flexneri genome. Genomic subtraction identified a large unique region of DNA which was present in S. flexneri serotype 2a but absent from E. coli K-12. This 42-kb DNA segment was localised to the S. flexneri chromosome and was found to contain a number of elements often associated with PAIs including: insertion sequence elements, bacteriophage genes, and a previously identified Shigella virulence gene (criR). These findings indicate that this region may form a new PAI in the S. flexneri genome.     

Structural and molecular characterization of Shigella boydii type 16 O antigen

Shigella is a well-known human pathogen causing dysentery and their typing is solely based on the O antigens. We investigated the chemical structure and gene cluster of Shigella boydii type 16 O antigen. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen has an O-acetylated branched pentasaccharide repeating O unit, which consists of two D-mannose residues (D-Man), one residue each of d-glucuronic acid (D-GlcA), N-acetylglucosamine (D-GlcNAc) and D-galactose (D-Gal), and the structure of the O unit was established. The O antigen gene cluster of S. boydii type 16 was identified and shown to contain putative genes for the synthesis of GDP-D-Man, genes encoding sugar transferases, O unit flippase (Wzx) and O antigen polymerase (Wzy) as expected. The function of the wzy gene was characterized by mutation test. Genes specific to S. boydii type 16 O antigen gene cluster were identified by screening 186 Escherichia coli and Shigella type strains, and can be used to develop PCR assays for detection of type 16 strains.     

Nucleotide sequence of the ipaBCD structural genes of Shigella dysenteriae

A 9 kb EcoRI and two PstI fragments from the virulence plasmid of Shigella dysenteriae CG097 were shown to contain all ipa genes by probing with Shigella flexneri ipaB, -C, -D and -A gene probes. The DNA sequences of S. dysenteriae ipaBC genes were very similar to those of S. flexneri M90T and S. flexneri YSH6000, but ipaD differed by 22 codons from that of S. flexneri. The differences in ipaD may account for the different in vitro host specificities shown by S. dysenteriae and S. flexneri. The nucleotide composition of ipa genes revealed an unusually large number of codons that are rarely used in Escherichia coli chromosomal genes, indicating a different origin.     

The role of oxyR and soxRS in oxidative stress survival in Shigella flexneri

Shigella flexneri, a facultative intracellular pathogen, is exposed to a variety of environments inside and outside of the human host. Some of these environments may contain significant oxidative stress. S. flexneri mutants were generated with deletions in the major oxidative stress regulators oxyR and/or soxRS to test their importance in Shigella biology. Strains that contained a deletion of oxyR had reduced growth and survival during aerobic growth, but not microaerobic growth. The mutants were also defective in surviving exposure to oxidative stress: oxyR mutants were sensitive to hydrogen peroxide, while soxRS mutants were sensitive to superoxide. Although the ΔsoxRS, ΔoxyR, and ΔoxyR/ΔsoxRS mutant Shigellae survived similarly to the parental strains within macrophages, the mutants formed plaques on Henle cell monolayers that were slightly smaller than the plaques formed by the wildtype strain.     

The immunochemistry of Shigella flexneri lipopolysaccharides. A quantitative analysis of their monosaccharide constituents

1. The lipopolysaccharides of a representative selection of Shigella flexneri serotypes all contain the same constituents as Salmonella chemotype VII, namely, aldoheptose phosphate, 3-deoxy-2-oxo-octonate, O-phosphorylethanolamine, d-galactose, d-glucose, N-acetyl-d-glucosamine and l-rhamnose. 2. The presence of all the Salmonella basal sugars in Sh. flexneri lipopolysaccharides is consistent with the view that the latter contain a basal structure or core which is similar to the common basal structure of Salmonella lipopolysaccharides. 3. Although the Sh. flexneri lipopolysaccharides belong to one chemotype, there appear to be quantitative differences in the composition of their O-specific side chains. The repeating units of Sh. flexneri serotypes 1a, 2a, 3a, 4a, and variant X contain d-glucose, N-acetyl-d-glucosamine and l-rhamnose in the proportions 1:1:2 respectively. The analogous repeating units of serotypes 5a and 6 contain an additional mole of d-glucose and d-galactose respectively and that of variant Y 1 mole of d-glucose less.     

Study of the methylation process and DNA methylase specificity in Shigella]

The nature and content of minor bases in DNA of 3 Shigella strains are investigated. DNAs from Shigella stutzeri 2, Sh. sonnei 1188 and Sh. sonnei 311 are found to contain 0.43, 0.56 and 0.45 mol.% of N6-methyladenine respectively. 5-methylcytosine (0.16 mol.%) is discovered in Sh. sonnei 311. Substrate specificity of adenine methylase from Sh. sonnei 1188 with respect to phage DNAs of different host modification is investigated. Recognition sites for guanine methylase of DDVI phage and for adenine methylase of Sh. sonnei 1188 turned to be different. DNA of DDII phage grown in Sh. stutzeri 2 cells does not accept methyl groups under the treatment with Sh. sonnei 1188 extracts, but it is methylated by Escherichia coli extract. Adenine methylases of Sh. sonnei 1188 and Sh. stutzeri 2 are suggested to be either the same enzyme, or enzymes, which recognition sites are partially overlapped.     

Structural studies of the Shigella flexneri variant X, type 5 a and type 5 b O-antigens.

Previous studies of different Shigella flexneri O-antigens indicate that their O-specific region is composed of oligosaccharide repeating units containing a basic tetrasaccharide structure, to which alpha-D-glucopyranosyl groups and O-acetyl groups may be attached to different positions. Structural studies of O-antigens from variant X, type 5a and type 5b lend further support to this assumption. These antigens contain terminal alpha-D-glucopyranosyl groups, one each per repeating unit in X and 5a, two in 5b. The location of these groups in the repeating unit has been determined.     

大肠杆菌O54 O-抗原基因簇的破译及进化分析

破译了大肠杆菌O5 4O 抗原基因簇的序列 ,序列全长 1 4 0 6 2bp。用生物信息学方法分析序列并鉴定基因 ,共确定 1 0个基因 ,包括鼠李糖合成酶基因BDA和C(rmlBDA和rmlC) ,糖基转移酶基因 ,O 抗原转运酶基因 ,O 抗原聚合酶基因和合成磷酸丝氨酸侧链的基因及 1个不能确定功能的开放阅读框。对rmlC的 (G C) %含量 ,稀有密码子含量及进化分析都表明大肠杆菌O5 4O 抗原基因簇是在近期通过rmlC介导的重组形成 ,而且大肠杆菌O5 4和鲍氏志贺氏菌 9型的亲缘关系很近。对UTP 葡萄糖 1 磷酸 尿苷转移酶基因 (galF)和 6 磷酸葡萄糖脱氢酶基因(gnd)的进化分析揭示志贺氏菌属与大肠杆菌属在进化上属于同一个属。用PCR方法筛选出了针对大肠杆菌O5 4的特异基因 ,用于基因芯片或PCR方法对大肠杆菌O5 4的快速检测。     

Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteins

Shigella move through the cytosol of infected cells by assembly of a propulsive actin tail at one end of the bacterium. Vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins, is important in cellular actin dynamics and is present on intracellular Shigella. VASP binds both profilin, an actin monomer-binding protein, and vinculin, a component of intercellular contacts that also binds the Shigella actin assembly protein IcsA. It has been postulated that VASP might serve as a linker between vinculin and profilin on intracellular Shigella, thereby delivering profilin to the Shigella actin assembly machinery. We show that Shigella actin-based motility is unaltered in cells that are deficient for the Ena/VASP family of proteins. In these cells, Shigella form normal-appearing actin tails and move at rates that are comparable to the rates of bacterial movement in Ena/VASP-deficient cells complemented with the Ena/VASP family member Mena. Finally, whereas vinculin can bind the Arp2/3 complex, we show that Arp2/3 recruitment to Shigella is not correlated with vinculin recruitment, indicating that the role of vinculin in Shigella motility is not recruitment of Arp2/3. Thus, although VASP is recruited to the surface of intracellular Shigella, it is not essential for Shigella actin-based motility.     

核桃楸树皮提取物对志贺氏菌抑菌效果研究

目的：观察核桃楸树皮提取物对志贺氏茵的抑菌效果。方法：采用两倍稀释法和纸片琼脂扩散法考察桃楸树皮提取物对志贺氏茵的抑茵作用。结果：核桃楸树皮提取物对痢疾志贺氏Ⅰ型茵、痢疾志贺氏Ⅱ型菌、鲍氏志贺Ⅰ型茵、宋内氏志贺茵均为0．0313g／mL,福氏志贺Ⅱ型菌为O．0625g／mL。结论：核桃楸树皮提取物对志贺氏茵均有良好的抑菌作用,各菌对药物的敏感强弱顺序为：宋内氏志贺菌〉鲍氏志贺Ⅰ型菌〉痢疾志贺Ⅰ型菌〉痢疾志贺菌Ⅱ型菌〉福氏志贺Ⅱ型茵。     

Cytosine DNA-methylase of a SsoII-type from Shigella sonnei 47 cells

Shigella sonnei 47 cells were found to contain DNA-methylase SsoII which is a modifying component of the system of host specificity of SsoII. The recognition sequence (RS) of methylase SsoII is represented by a five-member palyndromic structure--5'...CCNGG...3'--with a degenerated central nucleotide. Modification of SsoII affords protection of acceptor DNA not only from SsoII type restriction, but also from other restrictases, e. g., Eco RII having an analogous RS but with a less degenerated central nucleotide pair. A simple and rapid procedure for isolation and purification of DNA-methylase ScoII, which employs hydrophobic chromatography on phenyl-Sepharose, has been developed. The enzyme preparation does not contain trace amounts of specific and nonspecific endonucleases and keeps stable on storage in 30% glycerol over a period of one year.     

Anti-bacterial and anti-endotoxic immunity, induced by the administration of an enterobacterial vaccine

The work demonstrates that the sera of animals immunized with enterobacterial vaccine, when adsorbed on sheep red blood cells sensitized with glycolipid Re, lose their capacity of decreasing the lethal effect of Shigella sonnei endotoxin, which is indicative of the antiendotoxic action of antibodies. At the same time, immune sera obtained after immunization with enterobacterial vaccine contain antibodies having also other specificity and thus ensuring antibacterial immunity.     

The type III secretion system needle tip complex mediates host cell sensing and translocon insertion

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.     

Cdc42 facilitates invasion but not the actin-based motility of Shigella

The enteric pathogen Shigella utilizes host-encoded proteins to invade the gastrointestinal tract. Efficient invasion of host cells requires the stimulation of Rho-family GTPases and cytoskeletal alterations by Shigella-encoded IpaC. Following invasion and lysis of the phagosome, Shigella exploits the host's actin-based polymerization machinery to assemble an actin tail that serves as the propulsive force required for spreading within and between cells. The Shigella surface protein IcsA stimulates actin-tail formation by recruiting host-encoded N-WASP to drive Arp2/3-mediated actin assembly. N-WASP is absolutely required for Shigella motility, but not for Shigella invasion. Although Rho-family GTPases have been implicated in both the invasion and motility of Shigella, the role of Cdc42, an N-WASP activator, in this process has been controversial. In these studies, we have examined the role of Cdc42 in Shigella invasion and actin-based motility using Cdc42-deficient cells. We demonstrate that Cdc42 is required for efficient Shigella invasion but reveal a minor Cdc42-independent pathway that can permit Shigella invasion. However, the actin-based motility of Shigella, as well as vaccinia, proceeds unperturbed in the absence of Cdc42. These data further support the involvement of distinct host-encoded proteins in the steps regulating invasion and intercellular spread of Shigella.