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A Podbielski

Publications and source records attributed to A Podbielski.

At least 37 records · Page 2Linked to original sources
PubMed1999

Characterization of nra, a global negative regulator gene in group A streptococci.

During sequencing of an 11.5 kb genomic region of a serotype M49 group A streptococcal (GAS) strain, a series of genes were identified including nra(negative regulator of GAS). Transcriptional analysis of the region revealed that nra was primarily monocistronically transcribed. Polycistronic expression was found for the three open reading frames (ORFs) downstream and for the four ORFs upstream of nra. The deduced Nra protein sequence exhibited 62% homology to the GAS RofA positive regulator. In contrast to RofA, Nra was found to be a negative regulator of its own expression and that of the two adjacent operons by analysis of insertional inactivation mutants. By polymerase chain reaction and hybridization assays of 10 different GAS serotypes, the genomic presence of nra, rofA or both was demonstrated. Nra-regulated genes include the fibronectin-binding protein F2 gene (prtF2) and a novel collagen-binding protein (cpa). The Cpa polypeptide was purified as a recombinant maltose-binding protein fusion and shown to bind type I collagen but not fibronectin. In accordance with nra acting as a negative regulator of prtF2 and cpa, levels of attachment of the nra mutant strain to immobilized collagen and fibronectin was increased above wild-type levels. In addition, nra was also found to regulate negatively (four- to 16-fold) the global positive regulator gene, mga. Using a strain carrying a chromosomally integrated duplication of the nra 3' end and an nra-luciferase reporter gene transcriptional fusion, nra expression was observed to reach its maximum during late logarithmic growth phase, while no significant influence of atmospheric conditions could be distinguished clearly.

Adhesins, Bacterial↗
PubMed1999

Lmb, a protein with similarities to the LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin.

Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of the common serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiae wild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.

Adhesins, Bacterial↗
PubMed1999

Identification of pel, a Streptococcus pyogenes locus that affects both surface and secreted proteins.

A Tn917 insertion mutant of an M49 serotype, opacity factor-positive Streptococcus pyogenes, was isolated. It had the following phenotypes: decreased beta-hemolysis mediated by streptolysin S, reduction in the activity of a secreted cysteine protease and streptokinase, and an altered immunoglobulin and fibrinogen-binding phenotype. The site of insertion of Tn917 into the chromosome and the surrounding sequence, the pel region (pleiotropic effect locus), was determined. Phage A25 transduction confirmed that the pleiotropic changes in phenotype could be cotransduced with Tn917. The pel region was cloned and sequenced, and the transposon was found to be inserted upstream of a single open reading frame which led to a failure to transcribe a 500-base mRNA. The loss of this transcript decreased the transcription of emm and speB genes and reduced the secretion of streptokinase. Enhanced Pel expression from a nisin-inducible plasmid resulted in increased message levels for emm in a wild-type organism. Characterization of the pel mutant provides evidence for the coordinated regulation of secreted and surface proteins and suggests the existence of a new global regulatory factor in S. pyogenes.

Antigens, Bacterial↗
PubMed1998

An ectoprotein kinase of group C streptococci binds hyaluronan and regulates capsule formation.

A 56-kDa protein had been isolated and cloned from protoplast membranes of group C streptococci that had erroneously been identified as hyaluronan synthase. The function of this protein was reexamined. When streptococcal membranes were separated on an SDS-polyacrylamide gel and renatured, a 56-kDa protein was detected that had kinase activity for a casein substrate. When this recombinant protein was expressed in Escherichia coli and incubated in the presence of [32P]ATP, it was responsible for phosphorylation of two proteins with 30 and 56 kDa that were not present in the control lysate. The 56-kDa protein was specifically phosphorylated in an immunoprecipitate of a detergent extract of the recombinant E. coli lysate with antibodies against the 56-kDa protein, indicating that it was autophosphorylated. The E. coli lysate containing the recombinant protein could bind hyaluronan, and hyaluronan binding was abolished by the addition of ATP. Kinetic analysis of hyaluronan synthesis and release from isolated protoplast membranes indicated that phosphorylation by ATP stimulated hyaluronan release and synthesis. Incubation of membranes with antibodies to the 56-kDa protein increased hyaluronan release. The addition of [32P]ATP to intact streptococci led to rapid phosphorylation of two proteins, 56 and 75 kDa each at threonine residues. This phosphorylation was neither observed with [32P]phosphate nor in the presence of trypsin, indicating that the kinase was localized extracellularly. The addition of ATP to growing group C streptococci led to increased hyaluronan synthesis and release. However marked differences were found between group A and group C streptococci. Antibodies against the 56-kDa protein from group C streptococci did not recognize proteins from group A strains, and a homologous DNA sequence could not be detected by polymerase chain reaction or Southern blotting. In addition, Group A streptococci did not retain a large hyaluronan capsule like group C strains. These results indicated that the 56-kDa protein is an ectoprotein kinase specific for group C streptococci that regulates hyaluronan capsule shedding by phosphorylation.

Adenosine Triphosphate↗
PubMed1998

Genetic inactivation of the extracellular cysteine protease enhances in vitro internalization of group A streptococci by human epithelial and endothelial cells.

Group A Streptococcus (GAS) produces an extracellular cysteine protease (streptococcal pyrogenic exotoxin B) that participates in virulence. We examined two pairs of isogenic GAS strains (serotype M2 and M3) for ability to be internalized by human umbilical vein endothelial cells and A549 human lung fibroblasts. For both host cell types, the level of internalization by the cysteine protease-negative mutant strains was significantly greater than the wild type parent organisms. The data suggest that expression of the cysteine protease contributes to extracellular survival, an observation consistent with recent results from mouse infection studies (Lukomski et al., Infect immun 1998; 66: 771-6).

Bacterial Proteins↗
PubMed1998

A secreted streptococcal cysteine protease can cleave a surface-expressed M1 protein and alter the immunoglobulin binding properties.

Previous studies of recent clinical isolates of serotype M1 group A streptococci indicated that they display two patterns of non-immune human IgG subclass binding reactivity associated with their M1 protein. One group reacted with all four IgG subclasses (type IIo), while the second group expressed an M1 protein reacting preferentially with human IgG3 (type IIb). In this study, we have demonstrated that a cysteine protease, SpeB, present in culture supernatants of M1 serotype group A streptococcal isolates expressing type IIb IgG binding protein, can convert a recombinant Emm1 protein from a type IIo functional profile to a type IIb profile by removal of 24 amino acids from the N-terminus of the mature M1 protein. Furthermore, SpeB can convert bacteria expressing IgG binding proteins of the type IIo phenotype into those expressing type IIb proteins. The role of the cysteine protease as the central bacterial enzyme in this posttranslational modification event was confirmed by generation of an isogenic SpeB-negative mutant.

Amino Acid Sequence↗
PubMed1998

The group A streptococcal dipeptide permease (Dpp) is involved in the uptake of essential amino acids and affects the expression of cysteine protease.

The majority of characterized bacterial dipeptide permeases (Dpp) are membrane-associated complexes of five proteins belonging to the ABC-transporter family. They have been found to be involved in the uptake of essential amino acids, haem production, chemotaxis and sporulation. A 5.8 kb genomic DNA fragment of the serotype M49 group A streptococcal (GAS) strain CS101 was sequenced and found to contain five putative GAS Dpp genes (dppA to dppE). Deduced amino acid sequences exhibited 17-54% similarity to corresponding ABC-transporter sequences. The operon organization of the five genes was confirmed by transcriptional analysis, and a shorter, more abundant, dppA-only transcript was detected similar to that found in the GAS oligopeptide permease (Opp) system. Insertional inactivation was used to create serotype M2 and M49 strains that did not express the dppD and dppEATPase genes or nearly the entire operon. In feeding experiments with di- to hexapeptides, the wild-type strain grew with each peptide tested. The dpp mutants were unable to grow on dipeptides, whereas hexapeptides did not sustain the growth of opp mutants. Expression of the dpp operon was induced approximately fourfold in late exponential growth phase. In addition, a striking increase in the dppA to dppA-E ratio from 5:1 to more than 20:1 occurred during late exponential growth phase in complex medium. Growth in chemically defined medium (CDM) supplemented with various dipeptides specifically induced the expression of dpp and reduced both the dppA to dppA-E and oppA to oppA-F mRNA ratios. Expression of the virulence factor SpeB (major cysteine protease) was reduced eightfold in dpp mutants, whereas dpp expression was decreased about fourfold in a Mga virulence regulator mutant. Taken together, these data indicate a correlation between levels of intracellular essential amino acids and the regulation of virulence factor expression.

Amino Acids, Essential↗
PubMed1998

Role of emm and mrp genes in the virulence of group A streptococcal isolate 64/14 in a mouse model of skin infection.

The virulence of group A streptococcal isolate 64/14 and paired isogenic mutants in which either the emm or mrp gene had been insertionally inactivated was compared in mice. Loss of expression of the emm gene product resulted in a significant loss of virulence when the isolate was injected into the skin but had no significant difference when injected intraperitoneally. By contrast, inactivation of the mrp gene caused the organism to be more virulent in the skin, while having no significant effect intraperitoneally. An isogenic mutant, in which the mga gene was inactivated and neither the emm gene nor the mrp gene was expressed, demonstrated no significant difference in virulence from the wild type organism. Organisms recovered from the spleen of mice lethally infected with the mga mutant expressed all Mga-regulated IgG-binding gene products despite the presence of the spectinomycin-resistance cassette, which was used to inactivate the mga gene, in its original position.

Animals↗
PubMed1998

Genetic inactivation of an extracellular cysteine protease (SpeB) expressed by Streptococcus pyogenes decreases resistance to phagocytosis and dissemination to organs.

Streptococcal pyrogenic exotoxin B (SpeB), a conserved cysteine protease expressed by virtually all Streptococcus pyogenes strains, has recently been shown to be an important virulence factor (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574-2580, 1997). Genetic inactivation of SpeB significantly decreased the lethality of a serotype M49 strain for mice and abolished the lethality of a serotype M3 strain after intraperitoneal (i.p.) injection. In the present study, a wild-type M3 isolate and an M3 speB mutant derivative were used to investigate the mechanism responsible for altered virulence. Following i.p. injection, the mutant and wild-type strains induced virtually identical cellular inflammatory responses, characterized largely by an influx of polymorphonuclear leukocytes (PMNs). In addition, the mutant and wild-type strains rapidly entered the blood and were recovered from all organs examined. However, significantly fewer (P < 0.05) CFUs of the isogenic mutant derivative than of the wild-type parent strain were recovered from blood and organs. PMNs effectively cleared the M3 speB mutant from the peritoneum by 22 h, thereby sparing the host. In contrast, the wild-type M3 strain continued to replicate intraperitoneally and had the ability to kill phagocytes. This process allowed the wild-type strain to continuously disseminate, resulting in host death. Our results indicate that genetic inactivation of the cysteine protease decreased the resistance of the mutant to phagocytosis and impaired its subsequent dissemination to organs. These results provide insight into the detrimental effect of SpeB inactivation on virulence.

Animals↗
PubMed1998

Production of stabilized virulence factor-negative variants by group A streptococci during stationary phase.

Many of the virulence factors associated with fulminant group A streptococci (GAS) infection are expressed under in vitro exponential growth conditions. However, the survival of GAS in tissue and intracellularly, as well as colonization of asymptomatic carriers, has been reported for GAS. The bacteria associated with these niches may encounter high-density, low-nutrient-flowthrough conditions that may more closely mimic in vitro stationary-phase conditions than exponential growth. Therefore, the behavior of GAS in stationary-phase culture was examined. We observed that after 24 h in stationary phase, GAS serotypes M49 and M2 developed a unstable colony dimorphism of typical large and atypical small colonies. Between days 4 and 5, we isolated stabilized atypical small colonies which remained stable for up to nine passages (approximately 200 generations) on fresh medium before fully reverting to the large-colony phenotype. Upon analysis, the small colonies showed no difference in cell number per colony, growth rate, survival in prolonged stationary-phase culture, or antibiotic sensitivity. However, the small colonies showed decreased transcription of hyaluronic acid capsule, the global positive virulence factor regulator gene mga, the mga-regulated emm mRNA (M-protein structural gene), and speB (cysteine protease). Accordingly, the small colonies were completely sensitive in a traditional phagocytosis assay. The production of virulence factors and phagocytosis resistance of the small-colony isolates was recovered when, after several passages on fresh medium, the colony morphology began to revert.

Antigens, Bacterial↗
PubMed1997

Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains.

Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions.

Animals↗
PubMed1997

Inactivation of single genes within the virulence regulon of an M2 group A streptococcal isolate result in differences in virulence for chicken embryos and for mice.

An M2 streptococcal isolate and isogenic mutants in which either the emm or mrp gene was insertionally inactivated were tested for virulence using either a mouse model or a chicken embryo model. The results of the studies using the mouse model demonstrated that neither the emm nor mrp gene products had a significant effect on virulence when mice were challenged via the i.p. route. However, when the bacteria were injected into the skin the emm gene product was identified as a virulence factor. In parallel studies in the chicken embryo model the mrp gene product was found to be a major virulence factor, while a minor contribution to virulence could also be attributed to the emm gene product. The importance of these gene products to virulence was noted when the chicken embryo were injected either i.v or when the bacteria were placed on top of the chorioallantoic membrane. The direct comparison of a single wild type group A organism and its paired isogenic mutants in two animal models suggests that different combinations of bacterial factors are required to overcome host defense strategies associated with different animal species.

Animals↗
PubMed1997

Identification of key gene products required for acquisition of plasmin-like enzymatic activity by group A streptococci.

Group A streptococci incubated in human plasma can acquire a plasmin-like enzymatic activity. This process involves at least two bacterial proteins and two human protein cofactors. In this study, the key bacterial proteins were identified by using a series of isogenic mutants of group A isolate, CS101. These studies confirm a key role for the secreted plasminogen activator, streptokinase, and identify the major surface fibrinogen-binding protein as the product of the mrp gene. The requirement for human fibrinogen and plasminogen as key cofactors was also confirmed.

Bacterial Proteins↗
PubMed1996

Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS).

Ten novel streptococcal shuttle vectors for genomic integration and allelic replacements have been constructed based on plasmid pSF152. These vectors can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The basic vector pFW5 (2.8 kb, aad9 spectinomycin-resistance marker) carries two multiple cloning sites MCS-I and MCS-II (10 and 15 restrictions sites, respectively) to either side of the aad9 resistance gene. Each MCS is flanked by transcription termination sites for stabilization of recombinant plasmids. In vector pFW6 the transcription terminator between aad9 and MCS-II was deleted. Plasmids pFW7 through pFW10 carry resistance genes for kanamycin, chloramphenicol, erythromycin, and tetracyclin instead of aad9. Vectors pFW11 and pFW12 are pFW5/6 derivatives harboring an improved synthetic aad9 promoter. In pFW-phoA and pFW-gfp, promoterless alkaline phosphatase and green fluorescent protein boxes were integrated into MCS-I. If streptococcal DNA fragments are cloned into MCS-I and MSC-II, these vectors can be used for specific allelic replacements in streptococci via double-crossover recombinations. Depending on the vector used, this event will not lead to polar effects, facilitating mutagenesis within operons. The vectors containing reporter boxes allow in vivo studies of gene expression and promoter activity in pathogenic streptococci and potentially, also in other Gram-positive bacteria.

Alkaline Phosphatase↗