Relatedness between the coagulase gene 3'-end region and coagulase serotypes among Staphylococcus aureus strains

The 3'-end region of the coagulase gene from 22 strains of Staphylococcus aureus including 10 standard serotype strains was sequenced, and five subgroups with 4-8 tandem repeating units were distinguished among the tested strains. Phylogenetic analysis of the 3'-end region of the coagulase gene indicated that strains belonging to the same serotype were clustered in the same branch. A phylogenetic tree of the deduced amino acid sequences revealed that the C-terminal region might not be responsible for the epitope of the coagulase protein.     

Study on the polymorphism of the coagulase gene by the method of sequencing in methicillin-resistant Staphylococcus aureus strains, isolated in hospitals in different regions of Russia and Belarus

This method was used for typing of 31 Staphylococcus aureus methicillin-resistant (MRSA) strains; of these, 27 were clinical isolates obtained in hospitals of different cities of Russia and Belarus and 4 were international epidemic strains EMRSA-1, -2, -3, -12. The sequencing of the variable area, located in the middle part of the coagulase gene between nucleotides 979-1355 and detected with the use of information technologies, was carried out. The results of this sequencing were compared with those of the earlier study on the polymorphism of the area of the same gene between nucleotides 1513-2188, carried out by the method of PCR-restrictive fragment length polymorphism. The sequencing of the part of the coagulase gene made it possible to confirm the presence of essential differences in the nucleotide sequences of the coagulase gene in international strains EMRSA-1, -3, -12, grounds for classifying clinical isolates of MRSA strains with two groups (4 and 5), as well as the genetic relationship of different phage types, isolated in different clinics. The study revealed considerable similarity in the nucleotide composition of strains EMRSA-2 and EMRSA-12 despite the fact that, according to the results of Cfol restriction of the 3'-end, they were classified with different groups; the study also revealed the identity of the nucleotide sequences of the coagulase gene in the cultures of group 5, isolated in hospitals of Moscow, St. Petersburg, Orenburg, and strain EMRSA-2, as well as methicillin-sensitive S. aureus strain 8325-4; in addition, in clinical isolates of group 4 and strain EMRSA-1 a considerable degree of homology was revealed. The study of two different loci made it possible to find out the strain with the recombinant form of the coagulase gene. The approach used in this study permitted the differentiation of the international epidemic strains EMRSA-1, -2, -3 and -12 into individual groups, which coincided with the results of Enright et al. (2002) who used multilocus sequencing.     

Protein fingerprinting profiles in different strains of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> isolated from diseased freshwater fish

Summary Aeromonas hydrophila(Ah) strains isolated from diseased fish in India were studied for protein profiling using the SDS-PAGE protein fingerprinting profile pattern of whole cells of 12 local strains of A. hydrophilaand one reference strain (MTCC 646). Variability among the strains was observed. The average similarity between the 12 strains of A. hydrophila ranged from 0.272 to 0.916. Proteins with molecular mass of 55.6 and 14.67 kDa in Ah1, Ah2 and Ah3, 28.5 and 27.9 kDa in Ah4, Ah5 and Ah6, 21.4 and 19.5 kDa in Ah7, Ah8, Ah9 and 72.9, 91.5 and 71.3 kDa in Ah10, Ah11 and Ah12 were common. The protein polypeptide bands from 19.5 to 86.2 kDa were common in both local strains and reference strain of A. hydrophila. The protein fingerprinting study showed that there is genetic similarity between strains of A. hydrophila and reference strain (MTCC 646). These protein markers may be useful for further strain differentiation in epidemiological study.     

Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization

The B-domain, which is one of IgG-binding domains of staphylococcal protein A, was repeated five times and a cysteine residue was introduced at its C-terminus by a genetic engineering technique. The resulting protein, designated B5C1, retained the same IgG-binding activity as native protein A. The B5C1 was assembled on a gold plate surface by utilizing a strong affinity between thiol of cysteine and a gold surface. IgG-binding activity of B5C1 on a gold surface was much higher than that of physically adsorbed B5, which lacks cysteine residue. Furthermore, antigen-binding activity of immobilized antibody molecules through the use of assembled B5C1 on a gold surface was about 4.3 times higher than that of physically adsorbed antibody molecules. Immobilization of highly oriented antibody molecules was realized with the engineered IgG-binding protein.     

Biochemical and genetic heterogeneity of staphylococcal protein A

Abstract We investigated the biochemical and genetic heterogeneity of protein A from Staphylococcus aureus . SpA genes ( spas ) of various strains were heterogeneous when detected as Dra I and Eco RV fragments of chromosomal DNA. Polymerase chain reaction using primers to detect DNA encoding the IgG-binding domains in spa revealed that they numbered between 2 and 5. Protein A from several S. aureus strains showed two types of reactivities to immunoglobulins in normal canine serum according to the number of active domains.     

A synthetic IgG-binding domain based on staphylococcal protein A

A synthetic IgG-binding domain based on staphylococcal protein A was designed with the aid of sequence comparisons and computer graphic analysis. A strategy, utilizing non-palindromic restriction sites, was used to overcome the difficulties of introducing site-specific changes into the repetitive gene. A single mutagenized gene fragment was polymerized to different multiplicities, and the different gene products were expressed in Escherichia coli. Using this scheme, protein A-like proteins composed of different numbers of IgG-binding domains were produced. These domains were changed to lack asparagine--glycine dipeptide sequences as well as methionine residues and are thus, in contrast to native protein A, resistant to treatment with hydroxylamine and cyanogen bromide.     

Horizontal gene transfer in the evolution of group A streptococcal emm-like genes: gene mosaics and variation in Vir regulons

Most M type 5 group A streptococcal strains were found to contain a single emm-like gene between virR and scpA (the Vir reguion), but two distinct emm-like genes were identified in the Vir regulon of the MS strain NCTC8193. The complete sequences of both of these genes were determined. One, called emm5.8193, was shown to be a minor variant of the previously described emm5 gene from strain Manfredo. The second, designated enn5.8193, expresses an IgG-binding protein when cloned in Escherichia coli. A comparison of enn5.8193 with emm-like gene sequences from other strains indicated that it has a mosaic structure, consisting of distinct segments originating from emm-like genes in different OF⁺ and OF⁻strains. These data provide the first clear evidence that the horizontal transfer of emm-like sequences between distinct strains contributes to the evolution of group A streptococcal emm-like genes and Vir regulons.     

Synthesis,Expression and Purification of a Type of Chlorotoxin-like Peptide from the Scorpion, <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch,and its Acute Toxicity Analysis

A gene, rBmK Cta, encoding a chlorotoxin-like peptide from the scorpion, Buthus martensii Karsch, was synthesized according to the sequence optimized for codon usage in Escherichia coli and was expressed in E.␣coli BL21 (DE3) using a pExSecI expression system in which the IgG-binding domain-ZZ of protein A is fused to the N-terminal of rBmK CTa. The fusion protein, ZZ-rBmK CTa, was expressed in soluble form (7.8 mg l⁻¹) and was purified to give a single band on SDS-PAGE. The domain-ZZ of fusion protein ZZ-rBmK CTa was removed by cleavage of an Asn–Gly peptide bond with hydroxylamine. The rBmK CTa was separated from the IgG-binding moiety by a second passage through the IgG affinity column. Western blot analysis demonstrated that this protein was rBmK CTa. Acute toxicity assay in mice demonstrated that the rBmK CTa had an LD₅₀ value of 4.3 mg kg⁻¹.     

Analysis of protein A encoded by a mutated gene of Staphylococcus aureus Cowan I

The protein A (spa) genes from Staphylococcus aureus Cowan I and a mutant strain of Cowan I called V-1 earlier suggested to produce a monovalent IgG-binding protein A have been cloned in Escherichia coli. The DNA sequences coding for the IgG-binding part of the spa genes from both strains have been determined and compared with each other and with a partial amino acid sequence of purified protein A from strain V-1. The nucleotide sequence of the spa gene from strain V-1 reveals an NH2-terminally located IgG-binding region homologous to region E first reported for strain 8325-4, region D and the major portion of region A. The amino acid sequence analysis of the purified protein A from this strain also shows the presence of regions E and D but only a minor part of region A. Reversed-phase high-performance liquid chromatography fractionation of purified protein A from strain V-1 revealed that the preparation was heterogeneous, containing mainly two peptides with different abilities to bind IgG molecules. A shuttle vector containing the cloned protein A gene from V-1 was constructed and transformed into different strains of S. aureus and the produced protein A was purified and analysed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcal protein A, designated domain B, was chemically synthesized. This gene was tandemly repeated to give dimeric and tetrameric domain B genes by the use of two restriction enzymes which gave blunt ends. The genes were highly expressed in Escherichia coli to afford a large amount of dimeric and tetrameric domain B proteins. The single domain B protein was efficiently produced as a fusion protein with a salmon growth hormone fragment. The fusion protein was converted to monomeric domain B by cyanogen bromide cleavage. The CD spectra of the monomeric, dimeric and tetrameric domain B proteins were essentially the same as that of native form protein A, showing that their secondary structures were very similar. The dimeric and tetrameric domain B proteins formed precipitates with IgG as protein A. This system permits the efficient production of mutated single and multiple IgG-binding domains which can be used to study structural changes and protein A-immunoglobulin interactions.     

Evidence for functional heterogeneity in IgG Fc-binding proteins associated with group A streptococci

A number of group A streptococcal isolates have been compared for their nonimmune reactivity with each human IgG subclass, and rabbit, pig, or horse IgG. The results obtained demonstrate considerable heterogeneity in the expression of type II IgG-binding proteins among and within group A isolates. Extraction and analysis of type II IgG-binding proteins from selected strains demonstrate the existence of five functionally distinct IgG-binding proteins. The type IIo IgG binding protein displayed the greatest range of reactivities, binding to all four human IgG subclasses, and rabbit, pig, and horse IgG. A variant of this protein, designated type II'o, bound all four human subclasses and rabbit IgG, but failed to react with pig or horse IgG. A type IIa protein was recovered from certain group A strains which bound human IgG1, IgG2, IgG4, as well as reacting with rabbit, pig, and horse IgG. A functionally related type IIc activity that displayed all of the reactivities of the type IIa protein but did not bind with human IgG2 was also identified. The final functional form of group A IgG-binding protein, the type IIb protein, bound exclusively to human IgG3. Comparison of these functionally different type II IgG-binding proteins demonstrated no simple structure-function relationship. These studies underscore the heterogeneity of type II Ig-binding proteins expressed by different group A streptococci and document that a single strain can change its pattern of expression of type II IgG-binding protein both quantitatively and qualitatively.     

Synthesis and expression of an artificial gene of an IgG-binding fragment of protein A from Staphylococcus aureus

The chemical-enzymatic synthesis of a gene for IgG-binding fragment of the staphylococcal protein A has been carried out. The design of the gene, which consists of signal peptide and modified E and B domains, and strategy of the synthesis provided possibility of various degrees of polymerization of the gene fragment coding for B domain and of the whole gene. Several protein A-like polypeptides composed of the leader sequence, E domain and 1 to 4 copies of B domain were produced in E. coli cells under the lac promoter control.     

Streptococcal protein G. Gene structure and protein binding properties

Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.     

Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups.     

Fc-mediated nonspecific binding between fibronectin-binding protein I of Streptococcus pyogenes and human immunoglobulins.

Fibronectin-binding protein I (SfbI) from Streptococcus pyogenes plays a key role in bacterial adhesion to, and invasion of, eukaryotic cells. In addition, SfbI exhibits a considerable potential as mucosal adjuvant and can trigger polyclonal activation of B cells. Here, we report that SfbI is also capable of binding human IgG in a nonimmune fashion. SfbI was reactive with IgG1, IgG2, IgG3, and IgG4 isotypes (type IIo IgG-binding profile). The affinity constant (Kd) of the SfbI-IgG interaction was in the range of 1-2 x 10(-5) M. Further studies demonstrated that the SfbI binding was mediated by the Fc component of the IgG molecule. Experiments performed using purified recombinant proteins spanning different domains of SfbI showed that the IgG-binding activity was restricted to the fibronectin-binding domains, and in particular to the fibronectin-binding repeats. Finally, the presence of recombinant SfbI resulted in an impairment of both phagocytosis of IgG-coated RBCs and Ab-dependent cell cytotoxicity by macrophages. These results demonstrated for the first time that, in addition to its major role during the colonization process, SfbI may also favor bacterial immune evasion after the onset of the infection by interfering with host clearance mechanisms.     

Structure of the IgG-binding regions of streptococcal protein G.

The gene encoding the IgG-binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5-kb insert gave a functional product in Escherichia coli. Protein analysis of affinity-purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG-binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N-terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C-terminal part of streptococcal protein G consists of three IgG-binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made.     

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein a and rabbit IgG

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A fromStaphylococcus aureus (termed SpA¹) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA¹ and IgG-Fc were examined by the individual substitution of the residues in SpA¹ involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA¹ proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbant assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG₁-Fc (Fc fragment of human IgG₁) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.     

The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagulase-deficient mutants

The sequence of the coagulase gene (coa) from Staphylococcus aureus strain 8325-4 is reported. The deduced amino acid sequence of the coagulase protein is compared with previously reported sequences of coagulases from strains 213 and BB. The secreted mature forms of coagulase proteins are composed of three distinct segments: (i) the N-terminal 150-270 residues, which are c. 50% identical, (ii) a central region with high (greater than 90%) residue identities, and (iii) a C-terminal region composed of repeated 27-amino-acid residue sequences. The variable N-terminal sequences are probably responsible for antigenic differences among coagulases of different serotype. The region of coagulase which binds to prothrombin and activates it to form staphylothrombin is also located in the N-terminal half of the protein. A site-specific substitution mutation in the coa gene, which abolished plasma clotting activity, was isolated by recombinational allele-replacement in strains 8325-4 and M60. The Coa- mutants did not show diminished virulence in subcutaneous and intramammary infections of mice. No evidence for a role for coagulase in virulence of toxigenic or nontoxigenic strains was obtained. This contradicts findings of several groups using Coa- mutants generated by chemical mutagenesis and suggests that the earlier results were obtained with strains that had suffered additional mutations in virulence-related genes.     

Analysis of Bacterial Immunoglobulin-Binding Proteins by X-Ray Crystallography

Protein crystallography offers a powerful means of analyzing the molecular mechanisms that underlie the action of bacterial immunoglobulin-binding proteins. Successful approaches used to date involve the isolation of individual IgG-binding domains from the immunoglobulin-binding protein under study and the crystallization of these on their own or in complex with Fc or Fab fragments. Two structures of complexes that have been determined to high resolution by protein crystallography are compared. A single IgG-binding domain from protein A (from Staphylococcus) binds to a human Fc fragment through formation of two α-helices, which bind in the cleft between the CH2 and the CH3 domains. Recognition is mediated by side chains on protein A which interact with conserved side chains on the surface of the antibody, ensuring binding to IgG molecules from different subclasses and species. A similar analysis of the complex of a single IgG-binding domain from protein G (from Streptococcus) with an Fab fragment from mouse IgG1 reveals that the same problem in molecular recognition is tackled in a different way. Protein G binds via an antiparallel alignment of β-strands from the IgG-binding domain and the CH1 domain in Fab: this main chain-main chain interaction is supported by a number of specific hydrogen bonds between the side chains in both proteins. By recognition of a high proportion of main-chain atoms, protein G minimizes the effects of IgG sequence variability in a way that is distinct from that adopted by protein A.     

Comparative characteristics of protein G gene in Streptococci of groups C and G isolated from humans

The treatment of streptococci, groups C and G, with bromocyanogen made it possible to isolate surface G protein, capable of binding human serum albumin (HSA) and polyclonal IgG. In this work the presence of G protein in all staphylococcal strains, groups C and G, is shown. The differences between the strains by the level of expression, molecular weight and functional activity of G protein, extracted from streptococci of groups C and G, permitted the identification of 3 groups of strains, containing the molecules of G protein with different numbers of IgG- and HSA-binding domains: with 3 IgG- and HSA-binding domains, with 2 IgG- and HSA-binding domains and with only 2 IgG-binding domains. Each strain under study expressed only one of the molecule of G protein. The work shows the possibility of the identification of streptococci, groups C and G, by the molecular characteristics of G proteins themselves and their respective coding genes.