Identification of essential loops and residues of glucosyltransferase V (GtrV) of Shigella flexneri

Lipopolysaccharide (LPS), particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen modification is mediated by glucosyltransferase (gtr) genes encoded by temperate serotype-converting bacteriophages. The gtrV and gtrX genes encode the GtrV and GtrX glucosyltransferases, respectively. These are integral membrane proteins, which catalyze the transfer of a glucosyl residue via an α1,3 linkage to rhamnose II and rhamnose I of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a and X, respectively. Essential regions in the topology of GtrV protein were identified by in vivo recombination and a PCR-mediated approach. A series of GtrX-GtrV and GtrV-GtrX chimeric proteins were constructed based on the fact that GtrV and GtrX share sequence similarity. Analysis of their respective serotype conversion abilities led to the identification of two important periplasmic loops: loops No 2 and No 10 located in the N- and C-termini, respectively. Within these two loops, three conserved motifs were identified; two in loop No 2 and one in loop No 10. These conserved motifs contain acidic residues which were shown to be critical for GtrV function.     

Identification of essential loops and residues of glucosyltransferase V (GtrV) of Shigella flexneri

Lipopolysaccharide (LPS), particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen modification is mediated by glucosyltransferase (gtr) genes encoded by temperate serotype-converting bacteriophages. The gtrV and gtrX genes encode the GtrV and GtrX glucosyltransferases, respectively. These are integral membrane proteins, which catalyze the transfer of a glucosyl residue via an alpha1,3 linkage to rhamnose II and rhamnose I of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a and X, respectively. Essential regions in the topology of GtrV protein were identified by in vivo recombination and a PCR-mediated approach. A series of GtrX-GtrV and GtrV-GtrX chimeric proteins were constructed based on the fact that GtrV and GtrX share sequence similarity. Analysis of their respective serotype conversion abilities led to the identification of two important periplasmic loops: loops No 2 and No 10 located in the N- and C-termini, respectively. Within these two loops, three conserved motifs were identified; two in loop No 2 and one in loop No 10. These conserved motifs contain acidic residues which were shown to be critical for GtrV function.     

双向BN/SDS-PAGE分离鉴定痢疾杆菌福氏5型M90T株毒力相关膜蛋白复合物

通过蓝色非变性凝胶电泳(BN-PAGE)比较痢疾杆菌福氏5型野生株M90T和大质粒缺失株M90T△T的膜蛋白复合物,发现一个野生株特有的复合物,其分子量的为290 kD,命名为M90T-290.通过第二向SDS-PAGE分离M90T-290得到6个蛋白亚基,质谱鉴定为:一个由大质粒编码的毒力蛋白Apyrase(ATP-二磷酸水解酶)和5个染色体编码的蛋白,这些蛋白可能以膜复合物的形式影响毒力蛋白IcsA的单极性分布和痢疾杆菌在细胞间的扩散.这个新发现的毒力相关膜复合物在痢疾杆菌的致病过程中可能发挥重要作用.     

Topological analysis of glucosyltransferase GtrV of Shigella flexneri by a dual reporter system and identification of a unique reentrant loop

Lipopolysaccharide, particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-Antigen modification is mediated by glucosyltransferase genes (gtr) encoded by temperate serotype-converting bacteriophages. The gtrV gene encodes the GtrV glucosyltransferase, an integral membrane protein that catalyzes the transfer of a glucosyl residue via an alpha1,3 linkage to rhamnose II of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a. Analysis of the GtrV amino acid sequence using computer prediction programs indicated that GtrV had 9-11 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of GtrV to a dual reporter system composed of alkaline phosphatase and beta-galactosidase. Sandwiched GtrV-PhoA/LacZ fusions were also constructed at predetermined positions. The enzyme activities of cells with the GtrV-PhoA/LacZ fusions and the particular location of the fusions in the gtrV indicated that GtrV has nine transmembrane segments and one large N-terminal periplasmic loop with the N and C termini located on the cytoplasmic and periplasmic sides of the membrane, respectively. The existence of a unique reentrant loop was discovered after transmembrane segment IV, a feature not documented in other bacterial glycosyltransferases. Its potential role in mediating serotype conversion in S. flexneri is discussed.     

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.     

Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure

Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nanomachine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation. The bile salt deoxycholate (DOC) binds to IpaD to induce recruitment of the translocator protein IpaB into the maturing tip complex. We recently used spectroscopic analyses to show that IpaD undergoes a structural rearrangement that accompanies binding to DOC. Here, we report a crystal structure of IpaD with DOC bound and test the importance of the residues that make up the DOC binding pocket on IpaD function. IpaD binds DOC at the interface between helices α3 and α7, with concomitant movement in the orientation of helix α7 relative to its position in unbound IpaD. When the IpaD residues involved in DOC binding are mutated, some are found to lead to altered invasion and secretion phenotypes. These findings suggest that adoption of a DOC bound structural state for IpaD primes the Shigella TTSA for contact with host cells. The data presented here and in the studies leading up to this work provide the foundation for developing a model of the first step in Shigella TTS activation.     

弗氏志贺菌磷酸化蛋白的检测

目的:检测弗氏志贺菌全菌蛋白中被磷酸化修饰的蛋白。方法:制取弗氏2a志贺菌2457T野生株全菌磷酸化蛋白样品时加入磷酸化酶抑制剂,随后对样品进行双向电泳,以抗磷酸丝氨酸/苏氨酸/酪氨酸抗体为免疫探针,通过Western印迹找到被磷酸化的蛋白,并进行胶内酶解及MALDI-TOF质谱分析。结果与结论:共检测到13个磷酸化蛋白,其中9个为代谢途径中的酶。     

弗氏2a志贺菌2457T株碱性蛋白质组图谱的建立

目的:建立弗氏2a志贺菌2457T株的碱性蛋白质组图谱。方法:首先采用双向电泳技术对弗氏2a志贺菌2457T株表达的全部碱性菌体蛋白及碱性膜蛋白进行分离,再通过基质辅助激光解析/电离串联飞行时间质谱进行鉴定。结果:共鉴定到46个蛋白点,对应于38种蛋白质。结论:首次完成了弗氏2a志贺菌2457T株的碱性蛋白质组图谱。     

Immune response to rotavirus VP4 expressed in an attenuated strain of Shigella flexneri

An attenuated strain of Shigella flexneri was utilised to express viral protein (VP) 4 of rotavirus and the immunogenicity of the recombinant constructs was studied in BALB/c mice. VP4 was expressed as a fusion with maltose binding protein (MBP) in both the cytoplasm and periplasm, with a much higher level of expression occurring in the former. While all constructs induced a Shigella-specific response in mice, only the construct expressing MBP-VP4 in the cytoplasm of Shigella stimulated an immune response specific to rotavirus. This study demonstrates that Shigella can be used to deliver rotavirus antigens and induces an immune response directed towards both rotavirus and Shigella.     

Assay of IgA to Shigella flexneri during shigellosis

Abstract An enzyme-linked immunosorbent assay (ELISA) to Shigella flexneri 2a whole bacterium was used to determine IgM, IgG and IgA serum titers in 50 acute-phase shigellosis patients and 37 controls, i.e., hospital patients without known recent infections. Compared to controls, the shigellosis patients displayed statistically raised average serum titers to S. flexneri in all 3 above immunoglobulin classes, most notably IgA, which displayed an average 42-fold increase. Specific IgM and IgG were 5- and 16-fold higher, respectively. All sera displayed statistically raised titers in at least one immunoglobulin class. A Widal agglutination detected a 7-fold increase in serum titers; this was comparable to the IgM ELISA. Statistical analysis showed that the intra-assay error of the ELISA varied from 5 to 14%, depending on the absorbance from which titers were calculated. A second ELISA was performed on the above shigellosis sera to determine titers to purified lipopolysaccharide (LPS): a statistical correlation was found between these and the above values for all 3 immunoglobulin classes. We conclude that the use of S. flexneri whole bacterium as an antigen in an IgA ELISA is a statistically valid and convenient parameter for monitoring shigellosis, comparable to the use of LPS as antigen, and more sensitive than IgM or IgG ELISAs or agglutinations.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

弗氏2a志贺氏菌2457T株YciD蛋白的融合表达和纯化

目的：原核表达重组弗氏2a志贺氏菌2457T株YciD蛋白,为其功能研究奠定基础。方法：用PCR方法从弗氏2a志贺氏菌2457T株染色体中扩增YciD蛋白编码序列,经过纯化、酶切后克隆到原核表达载体pET32a中,构建重组载体pET32a-yciD,转化大肠杆菌BL21（DE3）菌株获得工程菌株,对其表达和纯化条件进行优化;利用Western Blot检测融合蛋白的表达。结果：构建了YciD蛋白的融合表达载体,并在大肠杆菌中获得高效表达;经Ni-NTA亲和层析柱纯化获得了高纯度的YciD蛋白;Western Blot表明,此蛋白可与His标签抗体反应,表明获得了目的蛋白。结论：在原核表达系统中表达、纯化弗氏2a志贺氏菌2457T株YciD蛋白,为进一步对其进行功能研究奠定了基础。     

Gene expression profiling of the pH response in Shigella flexneri 2a

The pH response of Shigella flexneri 2a 301 was identified by gene expression profiling. Gene expression profiles of cells grown in pH 4.5 or 8.6 were compared with the profiles of cells grown at pH 7.0. Differential expression was observed for 307 genes: 97 were acid up-regulated, 102 were acid down-regulated, 91 were base up-regulated, and 86 were base down-regulated. Twenty-seven genes were found to be both acid and base up-regulated, and 29 genes were both acid and base down-regulated. This study showed that (1) the most pH-dependent genes regulate energy metabolism; (2) the RpoS-dependent acid-resistance system is induced, while the glutamate-dependent acid resistance system is not; (3) high pH up-regulates some virulence genes, while low pH down-regulates them, consistent with Shigella infection of the low gut; and (4) several cross-stress response genes are induced by pH changes. These results also illustrate that many unknown genes are significantly regulated under acid or basic conditions, providing researchers with important information to characterize their function.     

Influence of the conserved active site residues of histidyl tRNA synthetase on the mechanism of aminoacylation reaction

The relation between the conservation of active site residues and the molecular mechanism of aminoacylation reaction is an unexplored problem. In the present paper, the influences of the conserved active site residues on the reaction mechanism as well as the electrostatic potential near the reaction center are analyzed for Histidyl tRNA synthetase from Escherichia coli, Thermus thermophilus and Staphylococcus aureus. While the primary structures show both convergence as well as divergence, the secondary level structures of the active sites of the three species show considerable conservation in the respective structural organizations. The conserved active site residues near the reaction center, which have a major role in the reaction mechanism and catalysis, retain their specific position and orientation relative to the substrate in the three species. In order to understand the influence of different conserved and nonconserved residues near the reaction center, two different models are considered. First, a large model of active site with the substrates, Mg²⁺ ions and water is constructed in which the first shell residues (including both conserved as well as nonconserved) near the reaction center are studied. From the large model, a smaller model is constructed for reaction path modeling individually for three species. Validation of the smaller model is carried out by comparing the energy surfaces of large and small models as a function of reaction coordinates. Further, the electrostatic potential near the reaction center for the large and small model are compared. The transition state structures of the activation step of aminoacylation reaction for E. coli, T. thermophilus and S. aureus are calculated using the combined ab-initio/semi-empirical calculation. The similarity of the energy profiles as a function of the relevant reaction coordinate and the orientation of the catalytic residue, Arg259, indicate that the reaction mechanisms are identical which are guided by the strikingly similar structural pattern formed by conserved residues for three species. The energy surfaces have close resemblance in three species and present a clear perspective that how the reaction proceeds with the aid of different conserved residues. The study of electrostatic potential confirms this view. The present study provides an understanding of the relationship between the conservation of residues and the efficient reaction mechanism of aminoacylation reaction.     

Topological role of cytochrome P450 2D6 active site residues

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.     

5-Enolpyruvylshikimate-3-phosphate synthase: determination of the protonation state of active site residues by the semiempirical method

EPSP synthase (EPSPS) catalyzes the addition of shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (TI) that is converted to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. A semiempirical molecular modeling study of the EPSPS active site containing the TI was implemented for the assignment of the protonation states of four basic residues, Lys22, Lys340, His385, and Lys411, based on the evaluation of 16 different protonation states and comparison of the resulting energy minimized heavy atoms coordinates with available X-ray crystallographic data of the D313A mutant of EPSPS. The results, employing both gas phase and continuum solvent models, are indicative that after the TI formation the histidine residue is most probably in neutral form (N^ε-protonated) and the lysine residues are in protonated form, which suggests that none of the presently proposed assignments of aminoacid residues involved in the reaction mechanism could be completely correct. The protonated state of Lys22 in the presence of the TI supports the proposal that this residue is a general acid catalyst for TI breakdown. Modeling of the native enzyme active site suggests that Asp313 residue has only minor effects on the definition of the TI position inside the active site. Hydrogen-bonds distances suggest that, in order to act as a base, Asp313 needs the intermediacy of a hydroxyl group of the TI for effecting the attack on the TI methyl group in the elimination step leading to EPSP, as suggested previously in the literature.     

弗氏2a志贺菌htrA基因突变体的构建

目的:构建弗氏2a志贺菌2457T的htrA基因缺失突变株及HtrA酶失活突变株,以便进一步研究HtrA蛋白的功能。方法:用PCR扩增htrA基因上下游同源臂,构建含有kan基因的打靶片段,采用λ-Red重组系统对htrA基因进行缺失,用PCR进行验证;通过定点突变的方法构建HtrA酶失活突变株,并测序验证。结果与结论:构建了2457T htrA缺失突变株和2457T/htrAFSA酶失活突变株。     

Structural analysis of the active site architecture of the VapC toxin from Shigella flexneri

The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg²⁺‐dependent ribonuclease and has been shown to target initiator tRNA^fMet in vivo. Here, we present crystal structures of active site catalytic triad mutants D7A, D7N, and D98N of the VapC toxin in absence of antitoxin. In all structures, as well as in solution, VapC forms a dimer. In the D98N structure, a Hepes molecule occupies both active sites of the dimer and comparison with the structure of RNase H bound to a DNA/RNA hybrid suggests that the Hepes molecule mimics the position of an RNA nucleotide in the VapC active site. Proteins 2016; 84:892–899. © 2016 Wiley Periodicals, Inc.     

The distribution of insertion sequences in the genome of Shigella flexneri strain 2457T

Shigella flexneri, which causes shigellosis in humans, evolved from Escherichia coli. The sequencing of Shigella genomes has revealed that a large number of insertion sequence (IS) elements (over 200 elements) reside in the genome. Although the presence of these elements has been noted previously and summarized, more detailed analyses are required to understand their evolutionary significance. Here, the genome of S. flexneri strain 2457T is used to investigate the spatial distribution of IS copies around the chromosome and the location of elements with respect to genes. It is found that most IS isoforms occur essentially randomly around the genome. Two exceptions are IS91 and IS911, which appear to cluster due to local hopping. The location of IS elements with respect to genes is biased, however, revealing the action of natural selection. The non-coding regions of the genome (no more than 21%) carry disproportionally more IS elements (at least 28%) than the coding regions, implying that selection acts against insertion into genes. Of the genes disrupted by ISs, those involved in signal transduction, intracellular trafficking, and cell motility are most commonly targeted, suggesting selection against genes in these categories.     

On the active site of hydrogenase from Chromatium vinosum

Isotope substitution of ⁵⁷Fe ($\text{I =}\text{1}\text{2}$) for ⁵⁶Fe has a pronounced effect on the two EPR signals of hydrogenase of Chromatium vinosum. It is proposed that signal 1, the intensity of which is increased several-fold by a deoxygenation-oxygenation cycle with a simultaneous increase of a signal from Fe³⁺, is due to a [3Fe-xS] cluster. It is further proposed that signal 2 is caused by a magnetic interaction of a [4Fe-4S]³⁺ cluster with an unidentified paramagnet. The addition of 10 μM Ni to the culture medium (already containing 1 μM Ni) increased the enzyme activity 3–6-fold, without effect on the growth of the bacterium. Addition of ⁶¹Ni ($\text{I =}\text{3}\text{2}$) to the medium did not change the EPR spectrum of hydrogenase. From a comparison of the EPR signal intensities and the enzyme activities it is concluded that, in the hydrogenase preparation as isolated, molecules containing a [3Fe-xS) cluster are not active, and that active molecules have a [4Fe-4S]^3+(3+,2+) cluster plus an as yet unidentified paramagnetic redox component. The latter is thought to be the primary site of interaction of the enzyme with H₂. Ni is considered as a possible candidate for this component.