Phenotypic heterogeneity: a bacterial virulence strategy.

Growing knowledge of the complexity of the host-pathogen interactions during the course of an infection revealed an amazing variability of bacterial pathogens within the same host tissue site. This heterogeneity in bacterial populations is either the result of a different bacterial response to a slightly divergent tissue microenvironment or is caused by a genetic circuit in which small endogenous fluctuations in a small number of transcription factors drive gene expression in combination with a positive feedback loop. As a result host-pathogen encounters can have different outcomes in individual cells, which enables bet-hedging and/or a co-operative behavior that enhance bacterial fitness and virulence, drive different host responses and promote resistance of small subpopulations to antibiotic treatment. This has a strong impact on the progression and control of the infection, which must be considered for the development of successful antimicrobial therapies.

[Individualized treatment strategies for Clostridium difficile infections]. / Individuelle Therapiestrategien bei Clostridium-difficile-Infektionen.

Upon hospitalization, up to 15.5% of patients are already colonized with a toxigenic Clostridium difficile strain (TCD). The rate of asymptomatic colonization is 0-3% in healthy adults and up to 20-40% in hospitalized patients. The incidence and mortality of C. difficile infection (CDI) has significantly increased during recent years. Mortality lies between 3 and 14%. CDI is generally caused by intestinal dysbiosis, which can be triggered by various factors, including antibiotics or immune suppressants. If CDI occurs, ongoing antibiotic therapy should be discontinued. The choice of treatment is guided by the clinical situation: Mild courses of CDI should be treated with metronidazole. Oral vancomycin is suitable as a first-line therapy of mild CDI occurring during pregnancy and lactation, as well as in cases of intolerance or allergy to metronidazole. Severe courses should be treated with vancomycin. Recurrence should be treated with vancomycin or fidaxomicin. Multiple recurrences should be treated with vancomycin or fidaxomicin; if necessary, a vancomycin taper regimen may also be used. An alternative is fecal microbiota transplant (FMT), with healing rates of more than 80%. Bezlotoxumab is the first available monoclonal antibody which neutralizes the C. difficile toxin B, and in combination with an antibiotic significantly reduces the rate of a new C. difficile infection compared to placebo. A better definition of clinical and microbiota-associated risk factors and the ongoing implementation of molecular diagnostics are likely to lead to optimized identification of patients at risk, and an increasing individualization of prophylactic and therapeutic approaches.

Studies of the efficacy of Enterocoliticin, a phage-tail like bacteriocin, as antimicrobial agent against Yersinia enterocolitica serotype O3 in a cell culture system and in mice.

The efficacy of enterocoliticin, a phage tail-like bacteriocin, as antimicrobial compound against infections with pathogenic Yersinia enterocolitica serotype O3 strains was assessed. In cell cultures, which were infected with the Y. enterocolitica strains 13 169 or 6471/76, bactericidal activity of enterocoliticin was found for bacteria adhering to the surface of eukaryotic cells, whereas bacteria, which had invaded the eukaryotic cells, were not accessible to the bacteriocin. The interaction of enterocoliticin with Y. enterocolitica was further examined in animals. Female BALB/c mice were experimentally infected with the two Y. enterocolitica strains and enterocoliticin was applied as antimicrobial compound by the oral route. Experimental variations concerning the infectious doses of the Y. enterocolitica strains and the time points of application of the bacteriocin were investigated. The increase of the Yersinia CFU titre in animals was retarded at time points shortly after the application of enterocoliticin indicating that the particles were effective on recently introduced Yersinia. The repeated application of enterocoliticin, however, did not prevent the colonization of the gastrointestinal tract by Yersinia.

[Not Available]. / Zelluläre Invasion Eine bakterielle Überlebensstrategie : Eine bakterielle Überlebensstrategie.

The entry of pathogenic bacteria into host cells is an important step in the induction and progression of many infectious diseases. It confers several advantages to the microorganism during pathogenesis. Intracellular bacteria are protected from the host immune response and the action of antibiotics. Furthermore, pathogens can use the invasion process to transmigrate through epithelial layers and colonize deeper host tissue and organs. To promote cell entry, the bacteria synthesize various surface molecules, called invasins, with which they tightly adhere to eukaryotic cell receptors. This interaction induces signal transduction pathways in the host cell cytoplasm that can lead to dramatic rearrangements of the cytoskeleton, i.e. actin filament nucleation. This modulation results in the formation of pseudopods or membrane ruffles which enclose the bacteria inside the cell.

Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains.

Yersinia enterocolitica 29930 (biogroup 1A; serogroup O:7,8) produces a bacteriocin, designated enterocoliticin, that shows inhibitory activity against enteropathogenic strains of Y. enterocolitica belonging to serogroups O:3, O:5,27 and O:9. Enterocoliticin was purified, and electron micrographs of enterocoliticin preparations revealed the presence of phage tail-like particles. The particles did not contain nucleic acids and showed contraction upon contact with susceptible bacteria. Enterocoliticin addition to logarithmic-phase cultures of susceptible bacterial strains led to a rapid dose-dependent reduction in CFU. Calorimetric measurements of the heat output of cultures of sensitive bacteria showed a complete loss of cellular metabolic activity immediately upon addition of enterocoliticin. Furthermore, a dose-dependent efflux of K(+) ions into the medium was determined, indicating that enterocoliticin has channel-forming activity.

Environmental control of invasin expression in Yersinia pseudotuberculosis is mediated by regulation of RovA, a transcriptional activator of the SlyA/Hor family.

Invasin is the primary invasive factor of Yersinia pseudotuberculosis that allows efficient internalization into eukaryotic cells. We investigated invasin expression and found that the inv gene is regulated in response to a variety of environmental signals, such as temperature, growth phase, nutrients, osmolarity and pH, and requires the product of rovA, a member of the SlyA/Hor transcriptional activator family. The rovA gene was found by a genetic complementation strategy that restores temperature regulation of an unexpressed inv-phoA fusion in Escherichia coli K-12. RovA plays a role in the invasion of Y. pseudotuberculosis into mammalian cells and mediates the regulation of invasin in response to all environmental signals analysed. Deletion analysis of the inv promoter region revealed a DNA segment extending 207 bp upstream of the transcriptional start site, which is required for maximal RovA-induced inv transcription. Gel retardation assays showed that RovA interacts preferentially with this promoter fragment and suggested two potential RovA binding sites. Studies with chromosomal gene fusions also demonstrated that rovA follows the same pattern of regulation as invasin, indicating that environmental control of inv expression is mainly mediated by the control of RovA synthesis. Furthermore, we showed that a rovA-lacZ fusion is only slightly expressed in a rovA mutant strain, indicating that a positive autoregulatory mechanism is also involved in rovA expression.

Intimin from Shiga toxin-producing Escherichia coli and its isolated C-terminal domain exhibit different binding properties for Tir and a eukaryotic surface receptor.

The outer membrane protein intimin plays a crucial role in the attaching and effacing process employed by different enteropathogens to colonize the epithelial surface of their hosts. In this study we have characterized the C-terminal binding domain of intimin from the Shiga toxin-producing Escherichia coli strain 413/89-1, that belongs to the beta-subtype of intimins. We found that a fusion of this domain to the maltose-binding protein binds efficiently to both the translocated intimin receptor (Tir) and the surface of uninfected eukaryotic host cells. In contrast, no such binding was observed with the full-length protein localized on the bacterial surface. As the C-terminal domain of intimin and the full-length protein differ in their binding activity, we suggest that the intimin-binding domain might be controlled by the N-terminal portion of the molecule to prevent unproductive interactions with molecules in the lumen of the gut.

Signaling and invasin-promoted uptake via integrin receptors.

The invasin protein encoded by enteropathogenic Yersinia allows entry of bacteria into intestinal M cells by binding to integrin receptors. In cultured cells, invasin-mediated uptake requires proteins involved in endocytosis and signaling to the cell cytoskeleton. At least four different factors have been demonstrated to play a role in regulating the efficiency of invasin-promoted uptake. These include receptor-ligand affinity, receptor clustering, signaling through focal adhesion kinase, and stimulation of cytoskeletal rearrangements by small GTP binding proteins.

An immunoglobulin superfamily-like domain unique to the Yersinia pseudotuberculosis invasin protein is required for stimulation of bacterial uptake via integrin receptors.

The binding of the Yersinia pseudotuberculosis and Yersinia enterocolitica invasin proteins to beta(1) integrin receptors allows internalization of these organisms by cultured cells. The C-terminal 192-residue superdomain of the Y. pseudotuberculosis invasin is necessary and sufficient for integrin recognition, while a region located outside, and N-terminal to, this superdomain strongly enhances the efficiency of bacterial uptake. Within the enhancer region is a domain called D2 that allows invasin-invasin interaction. To investigate the role of the enhancer region, bacterial cell binding and entry mediated by the Y. pseudotuberculosis invasin protein (invasin(pstb)) was compared to that of Y. enterocolitica invasin (invasin(ent)), which lacks the D2 self-association domain. Invasin(ent) was shown to be unable to promote self-interaction, using the DNA binding domain of lambda repressor as a reporter. Furthermore, two genetically engineered in-frame deletion mutations that removed D2 from invasin(pstb) were significantly less proficient than wild-type invasin(pstb) at promoting uptake, although the amount of surface-exposed invasin as well as the cell binding capacity of the recombinant Escherichia coli strains remained similar. Competitive uptake assays showed that E. coli cells expressing invasin(pstb) had a significant advantage in the internalization process versus either E. coli cells expressing invasin(ent) or the invasin(pstb) derivatives deleted for D2, further demonstrating the importance of invasin self-interaction for the efficiency of invasin-mediated uptake.

A region of the Yersinia pseudotuberculosis invasin protein enhances integrin-mediated uptake into mammalian cells and promotes self-association.

Invasin allows efficient entry into mammalian cells by Yersinia pseudotuberculosis. It has been shown that the C-terminal 192 amino acids of invasin are essential for binding of beta1 integrin receptors and subsequent uptake. By analyzing the internalization of latex beads coated with invasin derivatives, an additional domain of invasin was shown to be required for efficient bacterial internalization. A monomeric derivative encompassing the C-terminal 197 amino acids was inefficient at promoting entry of latex beads, whereas dimerization of this derivative by antibody significantly increased uptake. By using the DNA-binding domain of lambda repressor as a reporter for invasin self-interaction, we have demonstrated that a region of the invasin protein located N-terminal to the cell adhesion domain of invasin is able to self-associate. Chemical cross-linking studies of purified and surface-exposed invasin proteins, and the dominant-interfering effect of a non-functional invasin derivative are consistent with the presence of a self-association domain that is located within the region of invasin that enhances bacterial uptake. We conclude that interaction of homomultimeric invasin with multiple integrins establishes tight adherence and receptor clustering, thus providing a signal for internalization.

Differential effects of integrin alpha chain mutations on invasin and natural ligand interaction.

To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human alpha3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the alpha3 chain, alpha3-W220A, failed to promote adhesion to the natural alpha3 beta1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, alpha3-Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, alpha3-D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-alpha3 antibody, indicating that the second and third repeats of the alpha3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin alpha chain mutations, as alpha4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.

Recombinant soluble human alpha 3 beta 1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner.

Using insect cells, we expressed large quantities of soluble human integrin alpha 3 beta 1 ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble alpha 3 beta 1 specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble alpha 3 beta1 integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the alpha 3 beta 1 integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg2+, alpha 3 beta 1's binding affinity for invasin (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha 3 beta 1 ectodomain heterodimer resembles that of wild-type membrane-anchored beta 1 integrins. The integrin alpha 3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the alpha 3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant alpha 3 beta 1 integrin have provided new insights into alpha 3 beta1 structure, ligand binding function, specificity, and regulation.

The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment.

The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures. We show here that the ability to cope efficiently with a cold environment (12 degrees C and 25 degrees C) is strongly impaired in E. coli strains carrying hns mutations. Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93). A protein fragment (H-NS*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process. Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns' genes that result in the production of C-terminally truncated H-NS proteins supports this proposal. H-NS* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant. In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.

Low-copy-number T7 vectors for selective gene expression and efficient protein overproduction in Escherichia coli.

A set of low-copy-number vectors (pPD) has been constructed that permit selective gene expression and high-level protein overproduction in Escherichia coli, based on the bacteriophage T7 RNA polymerase/T7 promoter system. These plasmids carry a chloramphenicol resistance gene (cat) as a selective marker and an extended multiple cloning site for convenient gene cloning. Their replication is mediated by ori sequences derived from the low-copy-number vector pSC101. The efficient T7 gene 10 promoter present on these vectors allows selective and high-level transcription of cloned genes carrying their own translational initiation signals. In addition, low-copy-number T7 vectors were constructed that permit expression of genes lacking their own transcription and translation initiation elements by providing a ribosome binding site, an ATG start codon and a multiple cloning site devised for the cloning in all three reading frames. The pPD expression vectors were used to achieve high-level overproduction of the E. coli integral outer membrane protein Tsx, and the cytoplasmic enzymes beta-galactosidase (beta Gal) and UTP:alpha-D-glucose-1-phosphate uridylyltransferase (GalU). The characteristics of these low-copy-number T7 expression vectors should prove very useful for the cloning and high-level overexpression of genes whose gene products are deleterious to the E. coli host.

Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli.

The Escherichia coli hns gene encodes the abundant nucleoid-associated DNA-binding protein H-NS. Mutations in hns alter the expression of many genes with unrelated functions and result in a derepression of the proU operon (proVWX) without abolishing the osmotic control of its transcription. We have investigated the interactions of H-NS with the proU regulatory region by deletion analysis of cis-acting sequences, competitive gel retardation assays, and DNase I footprinting. The negative effect of H-NS on proU transcription was mediated by cis-acting sequences within proV but did not depend on the presence of a curved DNA segment upstream of the proU-35 region previously characterized as a target for H-NS binding in vitro. We detected a 46-base pair high affinity H-NS binding region downstream of the proU promoter at the 5' end of the proV gene and a complex array of additional H-NS binding sites which suggest the presence of an extended H-NS nucleoprotein complex. Most of the H-NS binding sites were highly A+T-rich and carried stretches of 5 or more consecutive A-T base pairs. The implications of our results for the osmotic regulation of proU transcription are discussed.

Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation.

Mutations in the structural gene (hns) for the Escherichia coli nucleoid-associated DNA-binding protein H-NS cause highly pleiotropic effects on gene expression, site-specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA. We have investigated the regulation of hns expression and found that hns transcription is subjected to stationary phase induction and negative autoregulation. A set of hns-lacZ protein and operon fusions was constructed in vitro and integrated in single copy into the attB site of the bacterial genome. Quantification of beta-galactosidase activity along the bacterial growth curve showed that hns expression increases approximately 10-fold in stationary phase compared with exponentially growing cells. Immunological detection of the H-NS protein in growing and stationary phase cells supported the genetic data and showed that H-NS synthesis varies with growth phase. In addition, primer extension experiments demonstrated that the amount of hns mRNA is elevated in stationary phase cultures and that hns transcription is directed by a unique promoter functioning in both log and stationary phase. Disruption of the hns gene by an insertion mutation led to the derepression (approximately fourfold) of the expression of an hns-lacZ operon fusion integrated at the attB site, showing that hns transcription is subjected to negative regulation by its own gene product. Autoregulation of hns expression is particularly pronounced in log phase. Both stationary phase control and autoregulation of hns transcription are associated with a 130 bp fragment that contains the hns promoter. In order to study the interaction of H-NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H-NS. DNA gel retardation assays showed that the H-NS protein can preferentially interact with a restriction fragment carrying the hns promoter. This restriction fragment showed features of curved DNA as judged by two-dimensional polyacrylamide gel electrophoresis performed at 4 degrees C and 60 degrees C.

The osmZ (bglY) gene encodes the DNA-binding protein H-NS (H1a), a component of the Escherichia coli K12 nucleoid.

A class of trans-acting mutations, which alter the osmoregulated expression of the Escherichia coli proU operon, maps at 27 min on the chromosome in a locus we have called osmZ. Mutations in osmZ are allelic to bglY, pilG and virR, affect gene expression, increase the frequency of the site-specific DNA inversion mediating fimbrial phase variation, stimulate the formation of deletions, and influence in vivo supercoiling of reporter plasmids. We have cloned the osmZ+ gene, mapped it at 1307 kb of the E. coli restriction map, identified its gene product as a 16 kDa protein, and determined the nucleotide sequence of the osmZ+ gene. The deduced amino acid sequence for OsmZ predicts a protein of 137 amino acid residues with a calculated molecular weight of 15,530. The primary sequence of OsmZ is identical to that of H-NS (H1a), a DNA-binding protein that affects DNA topology and is known to be associated with the bacterial nucleoid. Thus, osmZ is the structural gene for the H-NS (H1a) protein. The nucleotide sequence of osmZ is almost identical to that of hns; however, hns was incorrectly located at 6.1 min on the E. coli linkage map. Increased osmZ gene dosage leads to cell filament formation, altered gene expression, and reduced frequency of fimbrial phase variation. Our results suggest that the nucleoid-associated DNA-binding protein H-NS (H1a) plays a critical role in gene expression and in determining the structure of the genetic material.