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J Hacker

Publications and source records attributed to J Hacker.

At least 37 records · Page 2Linked to original sources
PubMed2000

Functional analysis of the minor subunits of S fimbrial adhesion (SfaI) in pathogenic Escherichia coli.

S fimbrial adhesins I and II (SfaI and II), produced by extraintestinal Escherichia coli pathogens that cause urinary tract infections (UTI) and newborn meningitis (NBM), respectively, mediate bacterial adherence to sialic acid-containing glycoprotein receptors present on host epithelial cells and extracellular matrix. The S fimbrial adhesin complexes consist of four proteins: SfaI-A, the major subunit protein and the minor subunit proteins SfaI-G, SfaI-S and SfaI-H. Sialic acid-specific binding is mediated by the minor subunit protein SfaI-S. In order to determine whether the minor subunit proteins SfaI-G, -S and -H play a role in the modulation of adherence and the degree of fimbriation, a trans-complementation system was developed. A non-adhesive E. coli K-12 derivative, harbouring the sfaI-A gene but lacking sfaI-G, -S and -H, was transformed with sfaI-G, -S or -H. Only SfaI-S was able to increase the degree of fimbriation and to confer adhesion properties on the recombinant E. coli K-12 strains. Amino acid residues in SfaI-S that are involved in modulation of fimbriation as well as in receptor recognition were localized by random and site-directed mutagenesis.

Adhesins, Escherichia coli↗
PubMed2000

Symbiosis and pathogenesis: evolution of the microbe-host interaction.

Symbiotic and pathogenic bacteria have in common that they live in or on host organisms or host cells. To make a successful living in eukaryotic hosts, bacteria must possess the traits to recognize a given host and establish adherence. When the bacterial location is internal or intracellular, they must further have the ability to invade, to establish a niche, and finally to multiply within a host. The underlying mechanisms which allow this form of existence show similarities between symbiotic and pathogenic bacteria. The final outcome, however, may result in a wide spectrum of consequences for the host ranging from the acquisition of novel metabolic pathways to damage or death. Despite the vastly different forms of interactions, symbiotic and pathogenic bacteria have in common that they are adapted to a particular environmental niche represented by the host organism or compartment thereof. This contribution reviews the evolutionary forces which have shaped the microbial-host interactions. Particular emphasis is placed on the genetic and molecular mechanisms that drive bacterial evolution in response to the selective pressures of the host environment.

Animals↗
PubMed2000

Cloning and characterization of a Legionella pneumophila-specific gene encoding a member of the LysR family of transcriptional regulators.

Flagellin gene regulation in Legionella pneumophila is modulated by various environmental factors. The expression of the virulent phenotype seems to be linked genetically to flagellum expression. To better understand the mechanisms of flagellin gene expression in L. pneumophila (Lp), we screened a pool of plasmids from a L. pneumophila Corby genomic library for the ability to prevent or reduce luciferase activity in the Escherichia coli strain YK410, which harbours a Lp-pflaA-luxAB fusion. We cloned a DNA fragment encoding the N-terminal part of a protein with significant similarity to members of the LysR family of transcriptional regulators (LTTRs). The entire gene, cloned by inverse PCR, was named flaR. It encodes a protein of 302 amino acids, and computer-assisted analysis of the amino acid sequence revealed a helix-turn-helix motif located near the N-terminus of the protein. The FlaR protein exhibits 21-31% identity to various LTTRs. Furthermore, gel retardation experiments indicate that the FlaR protein is able to bind to its own promoter region and, to a lesser extent, to the flaA promoter of L. pneumophila. The flaR promoter region contains putative LysR binding motifs and two putative Fur boxes. Taken together, these results indicate that FlaR is a DNA-binding protein which belongs to the LTTR family. Southern analysis with a L. pneumophila Corby-specific flaR probe revealed homologous genes in various L. pneumophila strains, but not in the 12 nonpneumophila strains tested so far.

Amino Acid Sequence↗
PubMed2000

Common molecular mechanisms of symbiosis and pathogenesis.

Traditionally, symbiotic and pathogenic interactions were considered different manifestations of the bacteria-host interaction. However, the molecular mechanisms that mediate communication between and cellular modulation of the involved partners are quite similar. With this review we aim to contribute to a reduction of the traditional gap between symbiosis and pathogenesis research.

Animals↗
PubMed2000

Toxin genes on pathogenicity islands: impact for microbial evolution.

Toxin-specific genes are often located on mobile genetic elements such as phages, plasmids and pathogenicity islands (PAIs). The uropathogenic E. coli strain 536 carries two alpha-hemolysin gene clusters, which are part of the pathogenicity islands I536 and II536, respectively. Using different genetic techniques, two additional PAIs were identified in the genome of the E. coli strain 536, and it is likely that further PAIs are located on the genome of this strain. Pathogenicity islands are often associated with tRNA genes. In the case of the E. coli strain 536, the PAI-associated tRNA gene leuX, which encodes a minor leucyl-tRNA, affects the expression of various virulence traits including alpha-hemolysin production. The exact mode of action of the tRNA5Leu-dependent gene expression has to be identified in the future.

Bacterial Toxins↗
PubMed2000

Influence of pathogenicity islands and the minor leuX-encoded tRNA5Leu on the proteome pattern of the uropathogenic Escherichia coli strain 536.

The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries four distinct DNA regions in its chromosome, termed pathogenicity islands (PAIs I536 to IV536). Each of these PAIs encodes at least one virulence factor. All four PAIs are associated with tRNA genes. PAI I536 and PAI II536 can be spontaneously deleted from the chromosome by homologous recombination between flanking direct repeats. The deletion of PAI II536 results in the truncation of the associated gene leuX encoding the tRNALeu. This tRNA influences the expression of various virulence traits. In order to get a deeper insight into the role of PAI I536/II536 and of the tRNA5LeU for the protein expression, the protein expression patterns of Escherichia coli 536 and different derivatives were studied. Differences in the protein expression patterns of the wild-type strain Escherichia coli 536, its mutants 536-21 (PAI I536-, PAI II536-, leuX-), 536delta102 (PAI I536+, PAI II536+, leuX-) as well as of the strain 536R3 (PAI I536-, PAI II536-, leuX+) were analyzed by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry. We identified about 39 different intracellular proteins whose expression is markedly altered in the different strain backgrounds. These differences can be linked either to the presence or absence of the PAI I536 and PAI II536 or to that of the tRNA gene leuX. The identities of 34 proteins have been determined by MALDI-TOF-MS. The identification of five proteins was not possible. The results suggest that proteome analysis is an efficient approach to study differences in global gene expression. The comparison of protein expression patterns of the uropathogenic E. coli strain 536 and different derivatives revealed that in this strain the expression of various proteins including those encoded by many housekeeping genes is affected by the presence of PAI I536 and Pai II536 or by that of the tRNA5Leu.

Bacterial Proteins↗
PubMed2000

Characterization of the Legionella pneumophila gene ligA.

Legionella pneumophila is a bacterial pathogen that resides and multiplies in macrophages as well as in its natural aquatic hosts, the protozoa. Different bacterial factors contribute to pathogenicity and accompanying eukaryotic intracellular events. Sequencing of mip flanking regions revealed a gene of 2610 bp, ligA, that has no significant similarity to any of the genes identified previously. Epidemiological studies indicate that this gene is present in Legionella pneumophila, the species most often associated with cases of the Legionnaires' disease, but not in Legionella species other than L. pneumophila. The isogenic ligA deletion mutant was resistant to NaCl, and showed decreased cytotoxicity to human monocytes and decreased hemolytic activity to red blood cells. However, the most prominent effect of the L. pneumophila ligA mutant strain LEPF1 was the nearly completely reduced replication within the natural host Acanthamoeba castellanii. Since this gene is L. pneumophila specific and regulates numerous bacterial properties we designated this gene ligA for Legionella pneumophila infectivity gene A.

Acanthamoeba↗
PubMed2000

Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella.

The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. In this study, we infected single-cell stages of D. discoideum with different Legionella species. Intracellular growth of Legionella in this new host system was compared with their growth in the natural host Acanthamoeba castellanii. Transmission electron microscopy of infected D. discoideum cells revealed that legionellae reside within the phagosome. Using confocal microscopy, it was observed that replicating, intracellular, green fluorescent protein (GFP)-tagged legionellae rarely co-localized with fluorescent antibodies directed against the lysosomal protein DdLIMP of D. discoideum. This indicates that the bacteria inhibit the fusion of phagosomes and lysosomes in this particular host system. In addition, Legionella infection of D. discoideum inhibited the differentiation of the host into the multicellular fruiting stage. Co-culture studies with profilin-minus D. discoideum mutants and Legionella resulted in higher rates of infection when compared with infections of wild-type amoebae. Because the amoebae are amenable to genetic manipulation as a result of their haploid genome and because a number of cellular markers are available, we show for the first time that D. discoideum is a valuable model system for studying intracellular pathogenesis of microbial pathogens.

Acanthamoeba↗
PubMed2000

Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus.

The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA.

Amino Acid Sequence↗
PubMed2000

Impact of flavophospholipol and vancomycin on conjugational transfer of vancomycin resistance plasmids.

The influence of vancomycin and flavophospholipol (FPL) on the transfer rate of conjugative plasmids harboring the vancomycin resistance operon vanA was determined in several clinical and animal isolates of Enterococcus faecium. FPL significantly inhibited the frequency of transfer of conjugative VanA plasmids up to 70-fold. Vancomycin had no significant effect on the transfer rate of VanA plasmids.

Anti-Bacterial Agents↗
PubMed2000

Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis.

Biofilm production is an important step in the pathogenesis of Staphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from Escherichia coli was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on ica expression in S. epidermidis. It was shown that S. epidermidis biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance ica expression 9- to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on ica expression. A weak (i.e., 2.5-fold) induction of ica expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of ica transcription and quantitative analyses of biofilm formation in a colorimetric assay.

Adhesins, Bacterial↗
PubMed2000

New fimbrial gene cluster of S-fimbrial adhesin family.

Fimbrial adhesins that mediate attachment to host cells are produced by most virulent Escherichia coli isolates. These virulence factors play an important role in the initial stages of bacterial colonization and also in determination of the host and tissue specificity. Isolates belonging to serotype O78 are known to cause a large variety of clinical syndromes in farm animals and humans and have been shown to produce several types of adherence fimbriae. We studied the fimbrial adhesin from an avian septicemic E. coli isolate of serotype O78. Analysis of the genetic organization of the fac (fimbria of avian E. coli) gene cluster indicates that it belongs to the S-fimbrial adhesin family. Seven open reading frames coding for major and minor structural subunits were identified, and most of them showed a high degree of homology to the corresponding Sfa and Foc determinants. The least-conserved open reading frame was facS, encoding a protein known to play an important role in determining adherence specificity in other S-fimbrial gene clusters.

Adhesins, Escherichia coli↗
PubMed2000

Receptor structure for F1C fimbriae of uropathogenic Escherichia coli.

F1C fimbriae are correlated with uropathogenic Escherichia coli strains. Although F1C fimbriae mediate binding to kidney tubular cells, their receptor is not known. In this paper, we demonstrate for the first time specific carbohydrate residues as receptor structure for F1C-fimbria-expressing E. coli. The binding of the F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) and purified F1C fimbriae to reference glycolipids of different carbohydrate compositions was evaluated by using thin-layer chromatography (TLC) overlay and solid-phase binding assays. TLC fimbrial overlay analysis revealed the binding ability of purified F1C fimbriae only to glucosylceramide (GlcCer), beta1-linked galactosylceramide 2 (GalCer2) with nonhydroxy fatty acids, lactosylceramide, globotriaosylceramide, paragloboside (nLc(4)Cer), lactotriaosylceramide, gangliotriaosylceramide (asialo-GM(2) [GgO(3)Cer]) and gangliotetraosylceramide (asialo-GM(1) [GgO(4)Cer]). The binding of purified F1C fimbriae as well as F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) was optimal to microtiter plates coated with asialo-GM(2) (GgO(3)Cer). The bacterial interaction with asialo-GM(1) (GgO(4)Cer) and asialo-GM(2) (GgO(3)Cer) was strongly inhibited only by disaccharide GalNAcbeta1-4Galbeta linked to bovine serum albumin. We observed no binding to globotetraosylceramide or Forssman antigen (Gb(5)Cer) glycosphingolipids or to sialic-acid-containing gangliosides. It was demonstrated that the presence of a GalCer or GlcCer residue alone is not sufficient for optimal binding, and additional carbohydrate residues are required for high-affinity adherence. Indeed, the binding efficiency of F1C fimbriated recombinant bacteria increased by 19-fold when disaccharide sequence GalNAcbeta1-4Galbeta is linked to glucosylceramide as in asialo-GM(2) (GgO(3)Cer). Thus, it is suggested that the disaccharide sequence GalNAcbeta1-4Galbeta of asialo-GM(2) (GgO(3)Cer) which is positioned internally in asialo-GM(1) (GgO(4)Cer) is the high-affinity binding epitope for the F1C fimbriae of uropathogenic E. coli.

Adhesins, Escherichia coli↗
PubMed2000

Alternative transcription factor sigma(B) is involved in regulation of biofilm expression in a Staphylococcus aureus mucosal isolate.

Osmotic stress was found to induce biofilm formation in a Staphylococcus aureus mucosal isolate. Inactivation of a global regulator of the bacterial stress response, the alternative transcription factor sigma(B), resulted in a biofilm-negative phenotype and loss of salt-induced biofilm production. Complementation of the mutant strain with an expression plasmid encoding sigma(B) completely restored the wild-type phenotype. The combined data suggest a critical role of sigma(B) in S. aureus biofilm regulation under environmental stress conditions.

Bacterial Proteins↗
PubMed2000

Characterization of the sigma(B) regulon in Staphylococcus aureus.

The sigma(B)-dependent stress regulon in gram-positive bacteria might fulfill a physiological role in stress response and virulence similar to that of the sigma(S) regulon in Escherichia coli and other gram-negative bacteria. In order to obtain evidence for the function of the sigma(B) regulon of Staphylococcus aureus, especially in virulence control, sigma(B)-dependent stress genes were identified. The two-dimensional protein pattern of wild-type cells of S. aureus COL was compared with that of an isogenic sigB mutant. By this approach, we found that the synthesis of about 27 cytoplasmic proteins seemed to be under the positive control of sigma(B). N-terminal sequencing of 18 proteins allowed the identification of their genes on the almost finished genome sequence of S. aureus COL and the analysis of the promoter structure. Transcriptional analyses of 11 of these genes confirmed their sigma(B) dependency, and moreover, about 7 additional sigma(B)-dependent genes were found which are cotranscribed with the newly detected genes, forming operons. Altogether, we identified 23 sigma(B)-dependent genes and their corresponding proteins. Among them are proteins probably involved in the generation of NADH or in membrane transport mechanisms. Furthermore, at least one clpC-homologous gene was localized on the S. aureus sequence solely transcribed by sigma(B). In contrast, a second clpC-homologous gene in S. aureus forming an operon with ctsR, yacH, and yacI was sigma(B) independently expressed.

Bacterial Proteins↗
PubMed2000

Pathogenicity islands and the evolution of microbes.

Virulence factors of pathogenic bacteria (adhesins, toxins, invasins, protein secretion systems, iron uptake systems, and others) may be encoded by particular regions of the prokaryotic genome termed pathogenicity islands. Pathogenicity islands were first described in human pathogens of the species Escherichia coli, but have recently been found in the genomes of various pathogens of humans, animals, and plants. Pathogenicity islands comprise large genomic regions [10-200 kilobases (kb) in size] that are present on the genomes of pathogenic strains but absent from the genomes of nonpathogenic members of the same or related species. The finding that the G+C content of pathogenicity islands often differs from that of the rest of the genome, the presence of direct repeats at their ends, the association of pathogenicity islands with transfer RNA genes, the presence of integrase determinants and other mobility loci, and their genetic instability argue for the generation of pathogenicity islands by horizontal gene transfer, a process that is well known to contribute to microbial evolution. In this article we review these and other aspects of pathogenicity islands and discuss the concept that they represent a subclass of genomic islands. Genomic islands are present in the majority of genomes of pathogenic as well as nonpathogenic bacteria and may encode accessory functions which have been previously spread among bacterial populations.

Bacteria↗
PubMed1999

Evolution of bacterial pathogenesis.

The evolution of bacteria is associated with continuous generation of novel genetic variants. The major driving forces in this process are point mutations, genetic rearrangements, and horizontal gene transfer. A large number of human and animal bacterial pathogens have evolved the capacity to produce virulence factors that are directly involved in infection and disease. Additionally, many bacteria express resistance traits against antibiotics. Both virulence factors and resistance determinants are subject to intrastrain genetic and phenotypic variation. They are often encoded on unstable DNA regions. Thus, they can be readily transferred to bacteria of the same species or even to non-related prokaryotes. This review article focuses on the main mechanisms of bacterial microevolution responsible for the rapid emergence of variants with novel virulence and resistance properties. In addition, processes of macroevolution are described with special emphasis on gene transfer and fixation of adaptive mutations in the genome of pathogens.

Animals↗