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V Kapur

Publications and source records attributed to V Kapur.

At least 73 records · Page 4Linked to original sources
PubMed1995

Identification, cloning, and sequencing of DNA essential for encapsulation of Streptococcus pneumoniae.

This paper reports the cloning and sequencing of a region of DNA from Streptococcus pneumoniae serotype 3 surrounding transposon Tn916, insertion of which was previously shown to result in lack of expression of the extracellular capsule. Sequence analysis revealed that the transposon inserted into a consensus insertion site 71 bp from the 5' end of the cloned fragment. Within the clone, 3' downstream regions from two different pneumococcal lytA genes were identified, as well as a putative 194 AA open reading frame (ORF1). Moreover, two copies of the repeat element BOX, oriented in opposite directions, were located immediately 3' of orf1. Within the region bounded by the first pair of internal sequencing primers, analysis revealed that the fragment amplified by PCR was always of the same size. Moreover, Southern blotting showed that for all serotypes examined to date, homology exists with the cloned fragment. These results indicate that this region of the chromosome is highly conserved and, taken together with other independently derived data, suggest that interruptions or deletions within this DNA lead to unencapsulation.

Bacterial Capsules↗
PubMed1995

Molecular population genetic analysis of the enn subdivision of group A streptococcal emm-like genes: horizontal gene transfer and restricted variation among enn genes.

The group A streptococcal emm-like genes, which encode the cell-surface M and M-like proteins, are divided into distinct mrp, emm and enn subdivisions and are clustered together in a region of the chromosome called the vir regulon. In order to understand the mechanisms involved in the evolution of emm-like genes, a 180 bp fragment of the 5' variable region of the enn gene was characterized in 31 strains for which emm sequences and multilocus enzyme electrophoretic profiles have been previously determined. The results demonstrate that nucleotide polymorphisms at the enn locus are generated predominantly by point mutations and short deletions or insertions, and that variation among enn and emm genes has arisen by similar mechanisms. However, diversity at the enn locus is restricted in comparison to the emm locus. Moreover, there is strong evidence for intragenic recombination at the enn locus and the pattern of distribution of emm and enn alleles among strains suggests that these genes may be independently acquired by horizontal transfer and recombination from distinct donor strains, thereby generating a mosaic structure for the vir regulon. The results add to a growing body of evidence that horizontal gene transfer has played a major role in the evolution of Streptococcus pyogenes vir regulons.

Amino Acid Sequence↗
PubMed1995

Molecular population genetic analysis of the streptokinase gene of Streptococcus pyogenes: mosaic alleles generated by recombination.

To understand the mechanisms governing molecular evolution of the streptokinase gene (skn), a 384 bp DNA fragment encoding two variable regions of the molecule was characterized in 47 isolates of Streptococcus pyogenes. The results reveal that alleles of the streptokinase gene have a mosaic structure, and provide strong evidence for intragenic recombination. Moreover, organisms that are well differentiated in overall chromosomal character have identical skn alleles, which suggests that horizontal gene transfer and recombination have participated in the evolution of this locus. No simple relationship between skn allele and serum opacity factor production or specific disease was identified. The predicted amino acid sequences of highly divergent skn alleles are strikingly similar in hydrophilicity and hydrophobicity profiles, distribution of amphipathic and flexible regions, surface probability plots, and antigenic indices, indicating that despite extensive nucleotide polymorphism in the two skn variable regions, selective pressure has constrained overall structural divergence. These results add to an important emerging theme that intragenic recombination plays a critical role in diversifying genes coding for streptococcal virulence factors.

Alleles↗
PubMed1995

Genetic diversity and relationships among Streptococcus pyogenes strains expressing serotype M1 protein: recent intercontinental spread of a subclone causing episodes of invasive disease.

Chromosomal diversity and relationships among 126 Streptococcus pyogenes strains expressing M1 protein from 13 countries on five continents were analyzed by multilocus enzyme electrophoresis and restriction fragment profiling by pulsed-field gel electrophoresis. All isolates were studied for the presence of the gene encoding streptococcal pyrogenic exotoxin A by PCR. Strain subsets were also examined by automated DNA sequencing for allelic polymorphism in genes encoding M protein (emm), streptococcal pyrogenic exotoxin A (speA), streptokinase (ska), pyrogenic exotoxin B (interleukin-1 beta convertase) (speB), and C5a peptidase (scp). Seven distinct emm1 alleles that encode M proteins differing at one or more amino acids in the N-terminal variable region were identified. Although substantial levels of genetic diversity exist among M1-expressing organisms, most invasive disease episodes are caused by two subclones marked by distinctive multilocus enzyme electrophoretic profiles and pulsed-field gel electrophoresis restriction fragment length polymorphism (RFLP) types. One of these subclones (ET 1/RFLP pattern 1a) has the speA gene and was recovered worldwide. Identity of speA, emm1, speB, and ska alleles in virtually all isolates of ET 1/RFLP type 1a means that these organisms share a common ancestor and that global dispersion of this M1-expressing subclone has occurred very recently. The occurrence of the same emm and ska alleles in strains that are well differentiated in overall chromosomal character demonstrates that horizontal transfer and recombination play a fundamental role in diversifying natural populations of S. pyogenes.

Amino Acid Sequence↗
PubMed1995

Different forms of streptolysin O produced by Streptococcus pyogenes and by Escherichia coli expressing recombinant toxin: cleavage by streptococcal cysteine protease.

To resolve apparent discrepancies in the literature, N-terminal sequences of the active high- and low-molecular-weight (high- and low-M(r)) forms of native streptolysin O (nSLO) purified from Streptococcus pyogenes culture supernatants and of the similar-size high- and low-M(r) forms of recombinant SLO (rSLO) found in the periplasm of Escherichia coli expressing a cloned slo gene were determined. The high-M(r) forms of nSLO and rSLO are identical, reflecting removal of a 31-residue signal peptide, but the similar-size low-M(r) forms are very different. Removal of C-terminal sequences by proteases in the E. coli periplasm produces an inactive low-M(r) form of rSLO. In contrast, an active low-M(r) form of nSLO is produced by proteolytic cleavage between the N-terminal residues Lys-77 and Leu-78, which was shown to correspond to an extremely sensitive cleavage site for the pyrogenic exotoxin B-derived streptococcal cysteine protease.

Amino Acid Sequence↗
PubMed1995

Molecular population genetic analysis of Staphylococcus aureus recovered from cows.

Staphylococcus aureus is one of the most common causes of bovine mastitis. To estimate genetic relationships among S. aureus strains recovered from cows, 357 isolates from milk samples from worldwide localities were examined for electrophoretic variation at 13 metabolic-enzyme loci. Thirty-nine electrophoretic types which represented distinctive multilocus enzyme genotypes were identified, and nearly 90% of all isolates were assigned to one of eight clones. Genetic heterogeneity was found among organisms recovered from dairy herds from which multiple isolates were obtained, indicating that the S. aureus population in a single herd can be multiclonal. Although humans and cows shared 7 of the 39 S. aureus clones, each clone was predominantly associated with one of these host species. These results are consistent with the concept of host specialization among S. aureus clones and imply that successful transfer of bacteria between humans and cows is limited.

Alleles↗
PubMed1995

DNA fingerprinting of pathogenic bacteria by fluorophore-enhanced repetitive sequence-based polymerase chain reaction.

Fluorophore-labeled oligonucleotide primers complementary to defined interspersed repetitive sequences conserved in diverse bacteria were used in the polymerase chain reaction to generate DNA fingerprint patterns from selected pathogenic bacteria. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction allowed discrimination between unrelated isolates of penicillin-resistant Streptococcus pneumoniae recovered from pediatric patients and Mycobacterium avium cultured from patients with acquired immunodeficiency syndrome. Combinations of oligonucleotide primers labeled with distinct fluorescent dyes enabled simultaneous DNA fingerprinting and Shiga-like toxin gene detection in enterohemorrhagic Escherichia coli isolates. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction was performed with either purified DNA or intact cells that were lysed during the polymerase chain reaction. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction successfully combines polymerase chain reaction amplification and fluorescent label detection for DNA fingerprinting of cultured bacterial pathogens.

Base Sequence↗
PubMed1995

Rapid Mycobacterium species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing.

OBJECTIVE: To develop and demonstrate the utility of automated DNA sequencing strategies for rapid and unambiguous identification of Mycobacterium species and mutations associated with antimicrobial resistance in Mycobacterium tuberculosis. DESIGN AND SPECIMENS: A 360-base pair segment of the gene (hsp65) encoding a 65-kd heat shock protein was characterized from 91 isolates assigned to 24 Mycobacterium species by traditional biochemical techniques. Areas of seven genes recently shown to contain mutations associated with antimicrobial resistance in M tuberculosis strains were also sequenced in a sample of 128 resistant organisms. Early positive BACTEC 460 cultures and acid-fast, bacterium-positive sputum specimens from patients with tuberculosis were also studied. RESULTS: Automated DNA sequencing identified species-specific polymorphism in the target segment of hsp65, successfully identified organisms to the species level in smear-positive sputum samples, and unambiguously characterized seven genes associated with antimicrobial resistance in M tuberculosis. CONCLUSIONS: Rapid identification of M tuberculosis and other Mycobacterium species is possible by automated DNA sequencing of a portion of hsp65. The technique is also feasible for analysis of some smear-positive sputum specimens. Unambiguous characterization of target segments of genes harboring mutations associated with antimicrobial resistance in M tuberculosis is possible from primary patient specimens. Taken together, the data demonstrate the feasibility of mycobacterial species identification and potential to identify mutations associated with antimicrobial resistance in less than 48 hours.

Alleles↗
PubMed1994

Characterization and distribution of insertion sequence IS1239 in Streptococcus pyogenes.

The human pathogenic bacterium Streptococcus pyogenes causes pharyngitis, acute rheumatic fever, glomerulonephritis and toxic-shock-like syndrome. The bacterium synthesizes several extracellular products, including the recently described streptococcal superantigen SSA, a molecule that shares considerable homology with several Staphylococcus aureus enterotoxins. While studying allelic variation at the ssa locus, six isolates expressing serotypes M4, M23, M33, M41, M43, and provisional type PT4854, were identified that had PCR products about 40-bp larger than expected, and one isolate (M15) had an amplified fragment that was more than 1-kb larger than expected. All six isolates have a 34-bp insert located 103 bp 5' of the ssa start codon. The larger product is a result of a 1110-bp insertion at the analogous location. The complementary strand of this insert has a 981-bp open reading frame that potentially encodes a 326-amino-acid polypeptide with substantial homology to the Escherichia coli IS30 transposase. Results of Southern blot analysis showed that at least twelve copies of the sequence are present in the serotype M15 S. pyogenes isolate. This element, designated IS1239, is the first simple insertion sequence described in group-A streptococci. Results of PCR screening showed that 26 of 78 (33%) S. pyogenes isolates expressing distinct M protein serotypes contained sequences with homology to IS1239, which means that the element is widely distributed in the species.

Amino Acid Sequence↗
PubMed1994

Proteolytically active streptococcal pyrogenic exotoxin B cleaves monocytic cell urokinase receptor and releases an active fragment of the receptor from the cell surface.

Urokinase plasminogen activator (u-PA) receptor (u-PAR) is a glycosyl-phosphatidylinositol-anchored membrane protein that promotes pericellular proteolysis and cellular migration. This investigation demonstrates that u-PAR is a substrate for the proteolytically active form of streptococcal pyrogenic exotoxin B (SPE B), a potent virulence factor secreted by Streptococcus pyogenes. Treatment of U937 monocyte-like cells with SPE B decreased specific 125I-labeled single-chain u-PA binding by up to 85%. Cysteine proteinase inhibitors neutralized SPE B without affecting the activity of phosphatidylinositol-specific phospholipase C. Due to decreased u-PA binding, SPE B-treated U937 cells expressed decreased activity against a u-PA-specific fluorogenic substrate and plasminogen. SPE B released single-chain u-PA that was noncovalently bound to U937 cells or cross-linked to cellular receptors with bis(sulfosuccinimidyl) suberate. The mass of the released u-PA-receptor complex was 100 kDa. Western blot analysis confirmed that the u-PA receptor that was cleaved by SPE B is u-PAR. After deglycosylation, the mass of SPE B-released u-PAR was 35 kDa, slightly smaller than the phosphatidylinositol-specific phospholipase C-derived form of this receptor. SPE B-released u-PAR retained the ability to bind u-PA, as determined by u-PA affinity chromatography. We conclude that SPE B may inhibit u-PA binding to monocytic cells by at least two mechanisms: (i) by decreasing the level of functional cell surface u-PAR and (ii) by releasing a soluble form of u-PAR that competes with the cellular receptor for ligand.

Bacterial Proteins↗
PubMed1994

Vaccination with streptococcal extracellular cysteine protease (interleukin-1 beta convertase) protects mice against challenge with heterologous group A streptococci.

Virtually all clinical isolates of group A streptococci secrete a highly conserved extracellular cysteine protease that cleaves human fibronectin and vitronectin, and converts IL-1 beta precursor to biologically active IL-1 beta. Based on the high degree of gene conservation within the species and its role in host pathogenicity, it was postulated that antibodies to the cysteine protease would confer protective immunity against S. pyogenes infection. To test this hypothesis, Swiss CD1 mice were intraperitoneally administered either saline, rabbit IgG, or IgG from rabbits immunized with the protease, and challenged with a highly virulent (minimum lethal dose approximately 10 cfu) clinical isolate of S. pyogenes expressing a heterologous cysteine protease. The results indicate that mice administered IgG from rabbits immunized with purified cysteine protease had significantly enhanced survival when compared with mice given either non-specific rabbit IgG (log rank test; chi 2; p = 0.0195) or saline (log rank test; chi 2; p = 0.0002). Moreover, mice actively immunized with the cysteine protease had a significantly longer time to death than the control group (log rank test; chi 2; p = 0.0418). The results show that the cysteine protease elicits non-type-specific immunity to challenge with heterologous S. pyogenes.

Animals↗
PubMed1994

Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci.

To examine the molecular population genetics of the M protein family of Streptococcus pyogenes (group A Streptococcus), the 5' regions of polymerase chain reaction-amplified emm products from 79 M serotypes were sequenced and the phylogeny was compared to estimates of overall genetic relationships among strains determined by multilocus enzyme electrophoresis. Although the 5' emm sequences from several strains designated as distinct M types were identical or almost identical, the overall pattern is characterized by very extensive variation. The composition of distinct emm sequence clusters generally parallels the ability of strains to express serum opacity factor and in some cases historical associations of certain M types with acute rheumatic fever, but not with M types classified as nephritogenic. For many strains there is a lack of congruency between variation in 5' emm sequences and estimates of overall chromosomal relationships, which is undoubtedly due to horizontal transfer and recombination of emm sequences. The results of these studies provide insights into the nature and extent of emm sequence variation and describe how this variation 'maps' onto the population genetic structure of extant S. pyogenes lineages. The complexity of emm sequence and streptococcal cell lineage relationships revealed by this analysis has significant implications for understanding evolutionary events generating strain diversity and the epidemiology of S. pyogenes diseases.

Amino Acid Sequence↗
PubMed1994

Molecular characterization and phylogenetic distribution of the streptococcal superantigen gene (ssa) from Streptococcus pyogenes.

A striking increase in the frequency and severity of invasive infections caused by Streptococcus pyogenes has occurred in recent years. Among these diseases is streptococcal toxic-shock-like syndrome (TSLS), a condition characterized by fulminant soft-tissue destruction and multiorgan failure. Streptococcal superantigen (SSA), a superantigen isolated from a TSLS-inducing, serotype M3 S. pyogenes strain, has recently been identified. We here describe the cloning, sequencing, and phylogenetic distribution of the SSA structural gene. The 783-bp open reading frame encodes a predicted 260-amino-acid protein that is similar in size to several other bacterial superantigens. The deduced sequence of the mature protein is 60.2% identical to that of staphylococcal enterotoxin B but only 49% identical to that of streptococcal pyrogenic exotoxin A. Southern blot and PCR analysis of 138 group A streptococcal strains representing 65 M protein serotypes and 15 nontypeable isolates identified ssa in 68 strains from 10 distinct clonal lineages. All ssa-positive clones expressed SSA. Of the two clones associated with TSLS, the ET 2-M3 lineage, but not the ET 1-M1 lineage, carried the SSA gene. Further analysis of the ET 2-M3 lineage found evidence for temporal variation in ssa association. Contemporary ET 2-M3 disease isolates had ssa, but two older isolates of this clone recovered in 1910 and 1920 lacked the gene. The clonal and temporal distribution patterns of ssa suggest a relatively recent acquisition of this superantigen-encoding gene by the ET 2-M3 lineage, perhaps by horizontal transfer and recombination.

Amino Acid Sequence↗
PubMed1994

Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.

Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level.

Amino Acid Sequence↗