高毒力A组链球菌致病机制研究进展

A组链球菌(Group A Streptococcus,GAS)常导致咽炎和皮肤感染,也能引起严重侵袭性感染.根据其表面M蛋白编码基因emm可将GAS分为200多型,严重侵袭性感染多由高毒力株引起,以emm1、emm3、emm12、emm28和emm89型常见.研究发现高毒力GAS株中CovRS基因突变可导致细菌逃逸固...     

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.Abbreviations bis-Tris bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - pfu plaque-forming units     

A group A streptococcal Enn protein potentially resulting from intergenomic recombination exhibits atypical immunoglobulin-binding characteristics

The gene encoding the Enn protein (enn) of the M untypeable group A streptococcal (GAS) strain 64/14 was amplified by polymerase chain reaction, cloned into the expression vector pJLA602 and expressed in Escherichia coli DH5α. Unlike other GAS–Enn proteins, which exhibit IgA-binding activity, the recombinant Enn enn64/14 protein reacted preferentially with human IgG₃. The 1050 bp open reading frame comprising the enn64/14 gene was completely sequenced. The region of the gene encoding the signal peptide and the C-terminus exhibited >95% homology to corresponding sections of other enn genes. The region of enn64/74 encoding the N-terminus of the mature Enn protein was found to be highly homologous to the corresponding section of the gene encoding the M-like protein of GAS serotype M9 (emmL9). The reoombinant protein encoded by emmL9 was found to react with all four human IgG subclasses. About 30% of the 1152bp open reading frame of emmiL9 encoding the N-terminus was found to display >90% homology to the corresponding section of enn64/14 but was <50% homologous in the remainder of the gene sequence. The functional analysis of the subcloned N-terminal section of emmL9 demonstrated a polypeptide exhibiting selective binding to human IgG₃. These findings suggested that enn64/14 was a hybrid gene formed by recombination of an enn gene and an emmL9 gene. The putative recombinational event could have Involved a set of flanking 7bp direct repeats. Since enn64/14 and emmL9 are genes from different phylogenetic lineages of GAS, this report provides evidence that intergenomic recombinations between different types of GAS genes can occur and could lead to hybrid proteins with unique Ig-binding characteristics.     

Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein

SUP35is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1α and a unique N-terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF-1α-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi⁺] strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.     

Domain structure and conserved epitopes of Sfb protein, the fibronectin-binding adhesin of Streptococcus pyogenes

Streptococcus pyogenes expresses a fibronectin-binding surface protein (Sfb protein) which mediates adherence to human epithelial cells. The nucleotide sequence of the sfb gene was determined and the primary sequence of the Sfb protein was analysed. The protein consists of 638 amino acids and comprises five structurally distinct domains. The protein starts with an N-terminal signal peptide followed by an aromatic domain. The central part of the protein is formed by four proline-rich repeats which are flanked by non-repetitive spacer sequences. A second repeat region, consisting of four repeats that are distinct from the proline repeats and have been shown to form the fibronectin-binding domain, is located in the Cterminal part of the protein. The protein ends with a typical cell wall and membrane anchor region. Comparative sequence analysis of the N-terminal aromatic domain revealed similarities with carbohydrate-binding sites of other proteins. The proline repeat region of the Sfb protein shares characteristic features with proline-rich repeats of functionally distinct surface proteins from pathogenic Gram-positive cocci. Immunoelectron microscopy revealed an even distribution of the fibronectin-binding domain of Sfb protein on the surface of streptococcal cells. Analyses of 38 sfb genes originating from different S. pyogenes isolates revealed primary sequence variability in regions coding for the N-termini of mature Sfb proteins, whereas sequences coding for the central and C-terminal repeats were highly conserved. The repeat sequences are postulated to act as target sites for intragenic recombination events that result in variable numbers of repeats within the different sfb genes. A model of the Sfb protein is presented.     

Identification of IS elements in <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains grown in a medium with ferrous iron or adapted to elemental sulfur

IS elements were identified in the genomes of five Acidithiobacillus ferrooxidans strains isolated from various media. IST2 elements were revealed in all the strains grown in a medium with ferrous iron, ISAfe1 elements were detected in four strains (TFBk, TFL-2, TFV-1 and TFO). Three strains (TFV-1, TFN-d and TFO) were found to contain IS elements, ~600 bp long. These were named preliminary as ISAfe600. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of an ISAfe1 element in TFBk and TFL-2 strains and complete sequencing of the ISAfe1 element in the TFBk strain has revealed nucleotide substitutions as compared to the prototype, i.e., the ISAfe1 element of an ATCC 19859 strain. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of the IST2 elements in TFO, TFBk and TFL-2 strains has shown numerous nucleotide substitutions when compared to the IST2 element of an ATCC 19859 strain. Complete sequencing of the IST2 element in the TFBk strain has revealed: the divergence between the IST2 elements in the TFBk strain and the prototype was 21.2%. Southern hybridization of EcoRI fragments of the chromosomal DNA from five A. ferrooxidans strains grown in a medium with ferrous iron using an internal region of ISAfe1, a full-length ISAfe1 or a full-length IST2 as probes has shown them to differ in the number of copies of IS elements and their localization on the chromosomes. Adaptation to elemental sulfur in A. ferrooxidans strains caused changes in the number, intensity and localization of hybridization bands. The authors discuss the role of IS elements in the adaptation of A. ferrooxidans to the new energy substrate. The nucleotide sequence data reported in this paper were deposited in GenBank under accession numbers: AY823401, the ISAfe1 from A. ferrooxidans TFBk; AY825254, the IST2 from TFBk; DQ002894, the 5′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ002895, the 3′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ005952, the 5′-terminal nucleotide sequence of IST2 from TFV-1; DQ005953, the 3′-terminal nucleotide sequence of IST2 from TFV-1.     

The Precursor of Differentiation Factor HLDF and Ribosomal Protein RPS21 Have a Common N-Terminal Sequence

The mature differentiation factor HLDF, isolated from cultural medium, comprises 54 aa, whereas the open reading frame of mRNA encodes a 97-aa protein. We presumed that the protein translation begins from the first ATG codon, whose environment mostly meets the requirements for the initiation point. Two more ATG triplets are localized in positions 48–50 and 100–102 (numbering according to the structure of S21), i.e., in the area preceding the cDNA fragment that encodes the N-terminal fragment of the mature protein. The mRNAs of HLDF and S21 ribosomal protein have previously been shown to be highly homologous, and, therefore, their differences appear to be derived from two point deletions in the cDNA of the HLDF-encoding sequence (a G residue in position 112 and a C residue in position 224). As a result, the mature differentiation factor and RPS21 may be the products of translation from different open reading frames, the differentiation factor may be synthesized in the cell as a precursor, and its N-terminal sequence may be identical to that of RPS21. To test this hypothesis, we prepared recombinant RPS21 and the polyclonal antibodies to HLDF, full-size RPS21, and the C-terminal RPS21 peptide. Immunochemical staining by specially produced antibodies of native HL-60 cells and the same cells brought into apoptosis or differentiation confirmed that the precursor of the differentiation factor and the ribosomal S21 protein have a common N-terminal sequence and different cellular localizations. Neither an intron-containing gene nor a pseudogene with the nucleotide sequence corresponding to the HLDF cDNA was detected in the human genome or in the HL-60 cell line genome. On the basis of these facts, we propose a hypothesis of the molecular mechanism of the HLDF mRNA biosynthesis by means of posttranslational modifications of pre-mRNA of RPS21.     

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea

The extrinsic 33 kDa polypeptide of the water-oxidizing complex has been extracted from pea photosystem II particles by washing with alkaline-Tris and purified by ion-exchange chromatography. The N-terminal amino acid sequence has been determined, and specific antisera have been raised in rabbits and used to screen a pea leaf cDNA library in gt11. Determination of the nucleotide sequence of positive clones revealed an essentially full-length cDNA for the 33 kDa polypeptide, the deduced amino acid sequence showing it to code for a mature protein of 248 amino acids with an N-terminal transit peptide of 81 amino acids. The protein showed a high degree of conservation with previously reported sequences for the 33 kDa protein from other species and the sequence contained a putative Ca²⁺-binding site with homology to mammalian intestinal calcium-binding proteins. Northern analysis of total pea RNA indicated a message of approximately 1.4 kb, in good agreement with the size of the cDNA obtained at 1.3 kbp. Southern blots of genomic DNA probed with the labelled cDNA give rise to several bands suggesting that the 33 kDa polypeptide is coded by a multi-gene family.Abbreviations ATZ - anilinothiazolinone - DITC - p-phenylenediisothiocyanate - PTH - phenylthiohydantoin - TFA - trifluoroacetic acid - Tris - tris (hydroxymethyl) aminomethane - bis-Tris - bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - p.f.u. - plaque-forming units     

Catalytic analysis of a recombinant D-hydantoinase from Agrobacterium tumefaciens

The d-hydantoinase gene of a wild strain of Agrobacterium tumefaciens BQL9 had 99.78% nucleotide sequence identity with other available Agrobacterium genes. The resulting amino acid sequence showed two important substitutions affecting two -helixes in the secondary structure of the protein. The union of Mn²⁺ to the protein was essential for activating the enzyme and was independent of the temperature. d-Hydantoinase only was inactivated in the presence of 70 mM EDTA and at over 40 °C. The enzyme showed both hydantoinase and pyrimidinase activities, but only with the d-enantiomers of the substrates. Activity was greater for substrates with apolar groups in the number 5 carbon atom of the hydantoin. The native structure of the N-terminal end of this d-hydantoinase proved to be indispensable to its enzymatic activity.     

M-related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsono-phagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp 4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co-incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.     

New actinoporins from sea anemone <Emphasis Type="Italic">Heteractis crispa</Emphasis>: Cloning and functional expression

A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10–27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins.     

Diversifying Selection Governs Sequence Polymorphism in the Major Adhesin Proteins FimA, PapA, and SfaA of Escherichia coli

Fimbriae or pili are essential adherence factors usually found in pathogenic bacteria to aid colonization of host cells. Three major structural pilin genes, fimA, sfaA, and papA, from Escherichia coli natural isolates were examined and nucleotide sequence data revealed elevated levels of both synonymous and nonsynonymous site variation at these loci. Examination of synonymous site variation shows a fivefold increase in fimA sites, relative to the housekeeping gene mdh; and similarly the sfaA and papA genes have increased synonymous sites variation relative to fimA. Nonsynonymous site variation is also elevated at all three loci but, in particular, at the papA locus (k _N= 0.44). The k _N/k _S ratio for the three genes are among the highest yet reported for E. coli genes. Regional variation in nucleotide polymorphism within each of the genes reveal hypervariable segments where nonsynonymous substitutions exceed synonymous substitutions. We propose that at the fimA, papA, and sfaA genes, diversifying selection has brought about the increase levels of polymorphism. Received: 7 August 1997 / Accepted: 8 March 1998     

A sequence-specific function for the N-terminal signal-like sequence of the TonB protein

TonB is a proline-rich protein which provides a functional link between the inner and outer membranes of Gram-negative bacteria. TonB is anchored to the inner membrane via an N-terminal signal-like sequence and spans the periplasm, interacting with transport receptors in the outer membrane. We have investigated the role of the N-terminal signal-like peptide in TonB function. Replacement of the N-terminal sequence with heterologous sequences indicates that it has at least three distinct rotes in TonB function: (i) to facilitate translocation of TonB across the cytoplasmic membrane; (ii) to anchor TonB to the cytoplasmic membrane; (iii) a sequence-specific functional interaction with the ExbBD proteins.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.      

The human leucocyte surface antigen CD53 is a protein structurally similar to the CD37 and MRC OX-44 antigens

CD53 is an N-glycosylated pan-leucocyte antigen of 35–42 000 M _r. The sequence of the CD53 polypeptide deduced from a cDNA clone is 219 amino acids in length. It appears to lack a conventional leader sequence because the deduced NH₂-terminal amino acid sequence is very similar to the rat MRC OX-44 and human CD37 antigens. The CD53 molecule is likely to consist of four transmembrane regions and a major extracellular hydrophilic loop containing two potential N-glycosylation sites. It is suggested that the CD53 glycoprotein is the true human homologue of the rat OX-44 antigen, rather than the CD37 antigen of more restricted expression and lower NH₂-terminal sequence similarity to OX-44.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M37033. Offprint requests to: P. Angelisová.     

Three different types of organization of the vir regulon in group A streptococci

Summary The DNA of group A streptococci (GAS) encodes several important virulence factors such as the antiphagocytic M protein, the Ig-Fc-binding M-related proteins (FcrA-like and EnnX-like) and the complement factor-inactivating C5a peptidase. The corresponding genes emm, fcrA, ennX, and scpA, respectively, were assumed to be located close together in the GAS genome. Additionally, emm and scpA have been found to be under the positive, coordinate control of the virR locus, which led to the designation vir regulon for the corresponding genomic segment. In order to map the vir regulons of many GAS serotypes and to analyse any correlation between the organization of vir regulons and circumscribed heterogeneities within the emm, virR, and scpA genes, an approach using several distinct sets of polymerase chain reaction (PCR) experiments was chosen. By examination of the genomic DNA of 42 GAS isolates from 36 different M serotypes three patterns of vir regulon topography were found. The first, designated large vir regulon (LVR), consists of virR -fcrA(-like) emm - ennX(-like) - scpA. The second, designated small vir regulon (SVR), contains virR - emm- scpA, and the last, designated unusual vir regulon (UVR), resembles SVR but contains additional heterogeneous sequences between emm and scpA. The patterns correlate with heterogeneities at the 3 ends of the virR and scpA genes, with the M classification system and the occurrence of specific non-coding intervening sequences within the vir regulons. The potential impact of these patterns on models to account for generation of vir regulons is discussed.     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.Revisions requested 30 September 2004; Revisions received 22 October 2004     

Synthesis and Antibacterial Activity of Analogues of the N-Terminal Fragment of the Sarcotoxin IA Antimicrobial Peptide

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1–18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.