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A Podbielski

Publications and source records attributed to A Podbielski.

At least 73 records · Page 4Linked to original sources
PubMed1994

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 DH5 alpha. 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 enn64/14 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 recombinant protein encoded by emmL9 was found to react with all four human IgG subclasses. About 30% of the 1152 bp open reading frame of emmL9 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 7 bp 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.

Bacterial Proteins↗
PubMed1994

Analysis of genes encoding two unique type IIa immunoglobulin G-binding proteins expressed by a single group A streptococcal isolate.

An emm-like gene (emmL) and a fcrA gene from group A streptococcal strain 64/14 (emmL64/14 and fcrA64/14) were amplified by PCR and force cloned into the heat-inducible expression vector pJLA 602. The emmL gene encoded a recombinant protein that bound human IgG1, IgG2, and IgG4 in a nonimmune fashion. This is the reactivity profile of a type IIa IgG-binding protein. The emmL64/14 gene product was antigenically similar to the previously identified high-molecular-weight type IIa IgG-binding protein of strain 64/14 and had an N-terminal sequence identical to that of the wild-type protein. The fcrA gene also encoded a recombinant protein with type IIa functional activity. This protein was similar to the lower-molecular-weight type IIa IgG-binding protein previously isolated from strain 64/14 and was antigenically distinct from the higher-molecular-weight type IIa protein encoded by the emmL64/14 gene. The sequences for both genes including the intervening regions are presented. The emmL gene demonstrates significant homology to other class I emm and emmL genes expressed by opacity factor-negative group A streptococcal isolates. The fcrA gene was found to be homologous to other fcrA genes normally present in opacity factor-positive group A isolates. The sequence upstream of the fcrA gene and the intervening sequence between the end of the fcrA gene and the start of the emmL gene were similar to those reported for other fcrA genes.

Amino Acid Sequence↗
PubMed1993

Type M12 protein from Streptococcus pyogenes is a receptor for IgG3.

Streptococcus pyogenes, serotype M12, was discovered to express a human IgG3 binding protein. Western blot analysis of partially purified M12 protein, exposed to IgG3 myeloma protein, showed that both M12 Ag and the receptor protein were the same apparent size. A lambda clone (lambda 4.1) containing the emm12 open reading frame expressed both the M12 Ag and the IgG3 binding protein. The emm12 open reading frame was amplified by the polymerase chain reaction and subcloned into the expression vector pJLA602. Based on Western blot analysis, one recombinant Escherichia coli (pD3) expressed M12 protein with IgG3 binding activity. This result confirmed that the M12 protein from strain CS24 is also an IgG3 receptor. Deletion analyses showed that a truncated M12 protein encoded by an internal PvuII fragment was sufficient for IgG3 binding activity. Further deletion studies suggested that the IgG3 binding domain was located in a 200 amino acid internal fragment containing two directly repeated sequences. Other experiments suggest that the receptor did not bind to the same IgG3 domain as that recognized by protein G. The M12 protein did not bind human IgG1, IgG2, IgG4, or Ig from several other animal species.

Antigens, Bacterial↗
PubMed1993

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

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.

Amino Acid Sequence↗
PubMed1993

Nucleotide substitutions and small-scale insertion produce size and antigenic variation in group A streptococcal M1 protein.

The presence of M protein on the surface of group A streptococci (GAS) confers the ability of the cell to resist phagocytosis in the absence of type-specific antibodies. It undergoes antigenic variation with more than 80 different serotypes having been defined. We have sequenced the M protein gene (emm1.1) from strain CS190 and present evidence that individual nucleotide substitutions are responsible for sequence variation in the N-terminal non-repeat region of emm1.1 and these substitutions have altered antibody recognition of opsonic epitopes. The N-terminal non-repeat domains of two other closely related strains, 71-155 and 76-088, were found to have sequence identical to emm1.1 with the addition of a 21 bp insert. This study provides the first evidence that nucleotide substitutions and small insertions are responsible for size and antigenic variation in the N-terminal non-repeat domain of the M protein of GAS.

Amino Acid Sequence↗
PubMed1992

The group A streptococcal M-type 3 protein gene exhibits a C terminus typical for class I M proteins.

The M protein gene (emm gene) from a reference group A streptococcal strain of serotype M3 was amplified by the polymerase chain reaction and partially sequenced. Hybridization assays using an oligonucleotide probe derived from the N-terminal sequence revealed that this gene segment is highly homologous among M-type 3 isolates. Of note, analysis of the nucleotide sequence data from the C terminus of the gene confirmed that the emm 3 gene exhibited all the features characteristic for group A streptococcal M-class I molecules. Recently published sequence data that were assigned to emm 3 resulted from a strain confusion and were shown to be the first one derived from an emm gene of an M-untypable isolate.

Amino Acid Sequence↗
PubMed1992

Ubiquitous occurrence of virR and scpA genes in group A streptococci.

Until now a few serotypes of M-class I group A streptococci (GAS) have been shown to encode VirR, a positive regulatory factor for the coordinate expression of the M protein (emm) and C5a peptidase (scpA) genes. The polymerase chain reaction technique has been applied to the genomic template of 36 GAS serotypes to demonstrate the general presence of VirR (virR) genes and scpA in GAS of both M classes. A virR gene region conserved in size was demonstrated for every strain investigated. Differences between virR genes from GAS of the two M classes were mainly confined to the 3' end of the gene and a region upstream of the gene's promoter. Every M-class II strain and some M-class I isolates were shown to possess a scpA gene of 4.6 kb, the rest of the M-class I GAS harbors a 3.5-kb scpA gene. The additional segment of 1.1 kb in the large-size scpA genes was located within a region of direct repeats at the 3' end of the gene. Among the serotypes encoding a large-size scpA gene a minority exhibits additional sequence variation downstream of the region of direct repeats.

Adhesins, Bacterial↗
PubMed1992

Surface protein-CAT reporter fusions demonstrate differential gene expression in the vir regulon of Streptococcus pyogenes.

Streptococcus pyogenes expresses at least two virulence factors, the anti-phagocytic M protein and an inhibitor of chemotaxis, the C5a peptidase (ScpA), under control of the virR locus. To facilitate studies of this regulatory unit, we constructed a new shuttle vector with a staphylococcal chloramphenicol acetyl transferase (CAT) reporter box which replicates in S. pyogenes. We cloned polymerase chain reaction (PCR)-derived potential promoter regions of the virR, M protein (emm12), and ScpA (scpA) genes from an M type 12 S. pyogenes, strain CS24. Promoter activity was assessed by measurements of specific mRNAs, transacetylase activity, and minimum inhibitory concentrations (MICs) for chloramphenicol resistance. We demonstrated that VirR is a necessary but not always sufficient positive trans-acting regulator of emm12 and scpA expression; however, virR is not autoregulated. A potential virR-binding consensus sequence is postulated for emm12, scpA and other M-like protein genes. Promoter activity of the structural genes was found to be dramatically influenced by growth conditions such as anaerobiosis. Levels of control, over and above the requirement for virR, are realized. The virR and scpA promoters were mapped for the first time using primer extension analysis. The observed mRNA start sites did not completely agree within the sequence predicted start sites. Data suggest that scpA could be subject to transcription attenuation.

Antigens, Bacterial↗
PubMed1992

Identical genes confer high-level resistance to gentamicin upon Enterococcus faecalis, Enterococcus faecium, and Streptococcus agalactiae.

The structural gene coding for the bifunctional aminoglycoside-modifying 6'-acetyltransferase-2''-phosphotransferase (6'AAC-2''APH) enzyme was specifically amplified by the polymerase chain reaction using template DNA of clinical isolates of enterococci (both Enterococcus faecalis and Enterococcus faecium) and the single high-level gentamicin-resistant Streptococcus agalactiae strain identified thus far. The results of the present study demonstrated that the genes encoding this antibiotic resistance trait are highly homologous in these species. In dot blot hybridization assays using nonradioactively labeled oligonucleotide probes, strains with and without high-level gentamicin resistance could be discerned unequivocally. The gene segment encoding 6'-acetylating activity and the gene segment encoding 2''-phosphorylating activity were simultaneously present in all isolates exhibiting high-level gentamicin resistance.

Acetyltransferases↗
PubMed1992

Similarity between the group B and A streptococcal C5a peptidase genes.

Group B streptococci (GBS) and group A streptococci (GAS) are known to have surface-associated peptidase activity which specifically cleaves C5a (C5a-ase), the primary chemotaxin for polymorphonuclear leukocytes. Amplification products were obtained from GBS genomic DNA template by using the polymerase chain reaction and primers corresponding to the C5a peptidase gene of GAS (scpA12). A restriction map of the GBS full-length amplified gene (scpB) was developed. The scpB restriction map was found to be highly similar to that of the analogous gene in GAS. A 50-bp deletion was located near the 5' end of scpB in a region where repeat sequences are found in scpA12. Hybridization experiments confirmed that other serotypes of GBS also carry an scpB-like gene. These results show that GBS contain a gene, scpB, which is very similar to that harbored by M12 GAS. The probability that scpB encodes the C5a-ase protein is discussed.

Adhesins, Bacterial↗
PubMed1992

M protein gene typing of Streptococcus pyogenes by nonradioactively labeled oligonucleotide probes.

A new approach for the typing of Streptococcus pyogenes is described. Oligonucleotide probes of 30 nucleotides in length were derived from currently known sequences of the N-terminal regions of M protein genes (emm genes). The oligonucleotides were labeled with digoxigenin-dUTP and hybridized to dot-blotted genomic DNA from 116 group A streptococcal strains of serotypes M-1, M-2, M-3, M-5, M-6, M-12, M-18, M-19, M-24, and M-49. Hybridization reactions were visualized with a chemiluminescent substrate. In comparison with conventional serological typing of expressed M proteins, the binding of the probes to the corresponding emm genes exhibited 100% sensitivity and specificity. The results emphasize the high degree of type-specific conservation of the N-terminal regions of emm genes from reference strains and epidemiologically unrelated U.S. and European clinical isolates. The existence of two distinct genetic subgroups among eight investigated M-49 strains was unequivocally shown by hybridization assays and further confirmed by nucleotide sequence data obtained from four selected M-49 strains. Because oligonucleotide probes are relatively easy to prepare, easy to handle, and known to give consistent interlaboratory results, the "oligotyping" technique appears to offer potential advantages over conventional serological typing methods.

Antigens, Bacterial↗
PubMed1992

[The vir-regulon of Streptococcus pyogenes: coordinate expression of important virulence factors].

Streptococcus pyogenes (group A streptococci; GAS) expresses important virulence factors like the antiphagocytic M protein, the complement factor-inactivating C5a peptidase and the immunoglobulin-Fc-binding proteins on its surface. The corresponding emm, scpA, and emm-related (fcrA, ennX) genes are adjacently encoded on the genome. They are coordinately in trans regulated by the positive regulatory VirR factor. The responsible virR gene is also located within this segment of the genome which was called vir-regulon. There are at least three different types of organization of the vir-regulon. A frequently encountered type is the "Large vir-regulon". It comprises from 5' to 3' the following genes: virR, fcrA, a relatively small emm, ennX, and a 4.6 kb version of scpA. Another common type is the "Small vir-regulon", which contains a virR deviating in its 3'-region, a relatively large emm, and a 3.5 kb version of scpA. The "Unusual vir-regulon" is less frequently detected. It closely resembles the small one, but harbors an additional 3 to 4 kb DNA fragment between emm and scpA, occasionally encoding an emm-related gene. The type of vir-regulon encoded by a GAS strain correlates to its serotype, its M class, and its expression of serum opacity factor. The structural genes of the vir-regulon are expressed at a high level during growth in exponential phase, under anaerobiosis, and at body temperature. The sensor molecule which modulates VirR activity according to these environmental conditions has not yet been detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesins, Bacterial↗
PubMed1992

[Infections by Streptococcus pyogenes: new aspects of diagnosis, epidemiology, clinical practice, and therapy].

Essential procedures for the cultural and serological laboratory diagnosis of Streptococcus pyogenes are described. A reliable and rapid species identification is achieved by serologic or biochemical tests. Direct antigen detection tests from throat swabs have broadened the diagnostic possibilities; however, they are often not satisfactory in terms of sensitivity. Typing of strains (either by classic serologic techniques or by molecular methods) is of high value for the study of clinical and therapeutic questions. In recent years, the epidemiology of the prevalent group A streptococcal strains has changed in Europe as well as in the U.S.: an increase of those M protein serotypes that are associated with higher virulence is being recognized. There is good evidence that the unexpected resurgence of rheumatic fever in the U.S. and the emerging number of life-threatening invasive and toxic manifestations of disease (including the toxic shock syndrome) in the U.S. and in Europe are related to the changing epidemiology. The existence of strains with rheumatogenic and nephritogenic potency has been established, but genetic host factors are a prerequisite for the sequelae as well. Although, based on experimental data, there are a lot of hypotheses concerning the nonsuppurative sequelae and the toxic shock syndrome, the exact pathogenesis of these diseases remains to be clarified. A thorough knowledge of the various clinical manifestations of group A streptococcal infections is necessary in order to apply the therapeutic and preventive measures in a rational and sophisticated manner. For most infections penicillins are considered the drug of choice, although no final conclusions regarding optimal dosages or the reasons for treatment failures can be made.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1991

Application of the polymerase chain reaction to study the M protein(-like) gene family in beta-hemolytic streptococci.

Evaluation of homologous regions of published M protein (emm) gene sequences from group A streptococci (GAS; Streptococcus pyogenes) was used to design three primer pairs for polymerase chain reaction (PCR) and three oligonucleotide probe sequences internal to the amplified products. One set of primers and corresponding probe should detect and lead to amplification of emm(-like) genes of virtually every type ("all M"), another ("SOR-M") should only amplify emm(-like) genes from GAS negative for serum opacity reaction (SOR) and the third ("SOR+ M") should expand only emm(-like) genes from SOR+ GAS. Using the "allM" primer pair for PCR on the genomic DNA from GAS of 29 different M types as well as from a group C and a group G streptococcal isolate, DNA fragments within the expected size range were amplified in every assay. All PCR products reacted with the "allM" probe. Related sequences were not detected in genomic DNA of an S. agalactiae and an Enterococcus faecalis isolate. Applying the "SOR-M" and "SOR+M" primers to identical assays led to mutually exclusive amplification products. The "SOR-M" and "SOR+M" probes hybridized only to their corresponding products. Exceptions to this exclusivity were the SOR- GAS of M types 3, 8, 27, 34, 42, 67, and 69, which consistently reacted only with the "SOR+M" primer/probe set. Analysis of sequence data from the amplified emm(-like) 2, 3, 18, and 19 genes revealed interesting specific features such as conserved gaps in the C-terminal sequence regions from SOR+ and the exceptional SOR- GAS strains. These data indicate the existence of a subgroup of strains among SOR- GAS and may advance our understanding of phylogenetic relationship between different serotypes of GAS.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and nucleotide sequence of the group B streptococcal hemolysin.

Genomic DNA from a Streptococcus agalactiae strain and pUC8 were used to clone the genetic determinant of the group B streptococcal hemolysin. The smallest recombinant plasmid, which enables its E. coli hosts to express beta-hemolysis on sheep blood agar or in blood broth, was found to contain a 3.9 kb insert of streptococcal DNA. A restriction endonuclease map was established for the enzymes Cla I, EcoRI, Hind II, Hind III and Sau I. Transposon mutagenesis experiments with Tn5 extinguished hemolytic activity when Tn5 was inserted at 1.7 kb or 1.9 kb downstream the 5' end of the streptococcal fragment between the Hind II and EcoRI sites. The complete 3.9 kb fragment was sequenced on both strands by the dideoxy method of Sanger. It was shown to contain 4 open reading frames; one of 690 bp, including the area of transposon insertion mentioned above, has a potential promoter region upstream. The deduced amino acid sequence of 230 residues shows a hydrophobic signal sequence of 19 amino acids at its N-terminus.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and nucleotide sequence of a new plasmid-coded Klebsiella pneumoniae beta-lactamase gene (SHV-2a) responsible for high-level cefotaxime resistance.

Patient specimen isolates of Klebsiella pneumoniae exhibiting an MIC of greater than 128 mg/l for cefotaxime were shown to produce a beta-lactamase with a pI of 7.6, which is encoded on a 66 kb conjugative plasmid. A 3.5 kb Bam HI fragment of this plasmid was cloned into pLG339 and totally sequenced. The nucleotide sequence of the beta-lactamase gene presented 99% homology to those of SHV-2 and SHV-3, the deduced amino acid sequence differed from both enzymes in one and two positions, respectively, leading to the denomination SHV-2a for the new enzyme. Since the kinetic data of SHV-2a and SHV-2 are similar, too, quantitative effects mediated by distinctly different promotor regions are thought to be responsible for the elevated MIC for cefotaxime induced by SHV-2a.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of a new plasmid-encoded SHV-type beta-lactamase (SHV-2 variant) conferring high-level cefotaxime resistance upon Klebsiella pneumoniae.

Between 1986 and 1988, multiresistant Klebsiella pneumoniae strains exhibiting high-level cefotaxime resistance were isolated from patient specimens particularly of the intensive care units of the Aachen Technical University Hospital. The resistance gene responsible was shown to be encoded on a conjugative 66 kb plasmid designated pZMP1. The MIC values for cefotaxime of the original isolates and the transconjugants were greater than 128 mg l-1 and 64 mg l-1, respectively. Isoelectric focusing of protein preparations from the transconjugants showed a beta-lactamase with a pI of 7.6. A 3.6 kb BamHI fragment containing the beta-lactamase gene was cloned into pLG339 resulting in the recombinant plasmid pZMP1-1. A restriction map of the cloned insert was established and PstI subfragments of the insert were further subcloned into pBGS18. The nucleotide sequence of the complete 3.6 kb fragment was determined. Within 3663 bp an open reading frame of 858 kb was found to show 99% homology to the SHV-2 and -3 nucleotide sequences. The deduced amino acid sequence differed in one and two positions, respectively, from these established SHV enzymes. The 3' noncoding sequence exhibited nearly perfect homology to that of SHV-2, but the 5' upstream sequence showed homology of less than 50% to the corresponding SHV-2 sequence, indicating an altered promoter region of the variant SHV-enzyme. Kinetic analysis of the beta-lactamase revealed a 50-100% elevated hydrolytic effectivity on cefotaxime in comparison to other SHV enzymes. Cefoxitin, ceftazidime, aztreonam and imipenem were not hydrolysed by the enzyme. The variant enzyme was inhibited by commonly available beta-lactamase inhibitors. Clavulanic acid had the highest affinity for the enzyme and the greatest effectivity in blocking its action. Based on the genetic and kinetic data we propose to classify the enzyme as a new variant beta-lactamase of the SHV-type and name it SHV-2a.

Base Sequence↗