Distribution and genetic structure of the Colibactin gene cluster in Enterobacteriaceae

Horizontaler Gentransfer zwischen Bakterien – sogar zwischen verschiedenen Spezies – ist ein wichtiger Mechanismus für den Austausch genetischer Information. Dies kann dem Rezipienten einen selektiven Vorteil verleihen, z. B. durch die schnelle Aneignung von Genclustern, die für Pathogenitäts- oder Fitnessfaktoren kodieren. Die Variabilität bakterieller Genome durch Aneignung und Inkorporation genetischen Materials in das Genom trägt somit erheblich zur Evolution von Bakterien bei. Bakterielle Genome neigen allerdings dazu, nutzlose genetische Information zu verlieren und daher kann horizontal erworbener DNA häufig eine distinkte biologische Funktion zugeordnet werden. Das Colibactin-Gencluster, welches zuerst in Escherichia coli gefunden wurde, weist mehrere Charakteristika einer horizontal erworbenen genomischen Insel auf. Die Größe dieser genomischen Insel beträgt 54 kb und sie umfasst 20 offene Leseraster (ORFs), von denen acht für putative Polyketidsynthasen (PKS), nichtribosomale Peptidsynthasen (NRPS) und Hybride dieser kodieren. Colibactin übt einen zytopathischen Effekt (CPE) auf eukaryotische Zellen in vitro aus. Nach Kokultivierung Colibactin-Gencluster-positiven Bakterien mit eukaryotischen Zellen kommt es zu DNA Doppelstrang Brüchen, Zellzyklus-Arrest in der G2-Phase, Megalozytose und schließlich zum Zelltod. Diese Effekte sind mit denen des Zyklomodulins „Cytolethal Distending Toxin“ (CDT) vergleichbar, allerdings konnte die biologische Funktion des Colibactins in vivo bisher nicht aufgeklärt werden. Das Colibactin-Gencluster wurde bisher nur in Escherichia coli Stämmen der phylogenetischen Gruppe B2 als individuelle genomische Insel, integriert im tRNA-asnW-Gen, vorgefunden. Im Rahmen dieser Arbeit konnte das Colibactin-Gencluster auch in E. coli der phylogenetischen Gruppe B1 und in Citrobacter koseri, Enterobacter aerogenes und Klebsiella pneumoniae subsp. pneumoniae nachgewiesen werden. In diesen Bakterienstämmen ist das Colibactin-Gencluster Teil eines genetischen Elements, das Ähnlichkeit zu integrativen und konjugativen Elementen (ICE) aus E. coli und K. pneumoniae aufweist. Im Gegensatz zur hochkonservierten Integrationsstelle des Colibactin-Genclusters in tRNA-asnW in E. coli der phylogenetischen Gruppe B2 konnte die Integrationsstelle dieses ICE in E. coli der Gruppe B1 in tRNA-asnU bestimmt werden. In Bakterienstämmen der Spezies K. pneumoniae subsp. pneumoniae wurden vier verschiedene Integrationsstellen in fünf analysierten Stämmen identifiziert. Neben der Studien zur Verbreitung und chromosomalen Integration des Colibactin-Genclusters wurden Kolonisierungsstudien im murinen streptomycinbehandelten Intestinaltrakt mit E. coli Stamm Nissle 1917 durchgeführt, um eine mögliche Funktion des Colibactins im Darmtrakt näher zu untersuchen. Weder in nicht-kompetitiven noch in kompetitiven Versuchsdurchführungen konnte dabei ein Kolonisierungsvorteil durch Colibactin nachgewiesen werden. Die Ergebnisse dieser Arbeit haben gezeigt, dass das Colibactin-Gencluster in verschiedenen Spezies der Enterobacteriaceae vorhanden und funktional ist. Das Auftreten dieses sowohl als individuelle genomische Insel als auch als Teil eines ICE veranschaulicht die genetische Plastizität dieses Elements und die Bedeutung des horizontalen Transfers genetischen Materials. Die biologische Funktion des Colibactins in vivo bleibt weiterhin unklar und könnte sowohl die bakterielle Fitness als auch die Virulenz beeinflussen.Horizontal gene transfer between bacteria – even between different species – has been shown to be an important mechanism for exchange of genetic material. This may confer a selective advantage to the recipient, e. g. the rapid acquisition of gene clusters coding for pathogenicity or fitness factors. The variability of bacterial genomes enabled by acquisition and incorporation of genetic material into their genome contributes considerably to bacterial evolution. Bacterial genomes tend to lose useless genetic information and therefore horizontally acquired DNA can most frequently be connected to a distinct biological function. The colibactin gene cluster initially discovered in Escherichia coli displays several features of a horizontally acquired genomic island. This genomic island is approximately 54 kb in size and consists of 20 open reading frames (ORFs), of which eight code for putative polyketide synthases (PKS), non-ribosomal peptide synthases (NRPS) and hybrids thereof. The synthesized hybrid non-ribosomal peptide-polyketide colibactin exerts a cytopathic effect (CPE) on eukaryotic cells, DNA double strand breaks are induced, the cells are arrested in the G2-phase of the cell cycle and exhibit megalocytosis and cell death. These effects are comparable to the effects of the cyclomodulin cytolethal distending toxin (CDT), but the biological function of colibactin in vivo is still unknown. So far the colibactin gene cluster has only been found in Escherichia coli strains of the phylogenetic lineage B2 as an individual genomic island integrated at the tRNA-asnW gene. In context of this thesis the colibactin gene cluster could be identified in E. coli strains of the phylogenetic group B1 as well as in Citrobacter koseri, Enterobacter aerogenes and Klebsiella pneumoniae subsp. pneumoniae. In those bacterial strains the colibactin gene cluster is part of a genetic element, which exhibits similarities to integrative and conjugative elements (ICE) previously described in E. coli and K. pneumoniae. In contrast to the highly conserved integration site of the colibactin gene cluster at tRNA-asnW in E. coli of the phylogenetic lineage B2, integration at tRNA-asnU was determined in E. coli of group B1. In bacterial strains of the species K. pneumoniae subsp. pneumoniae four different integration sites in a total of five strains were identified. Besides the surveys concerning the distribution and chromosomal integration of the colibactin gene cluster colonization studies of the murine streptomycin-treated intestinal tract were conducted using E. coli strain Nissle 1917 to examine a possible effect of colibactin in this context. However, there was no evidence providing a colibactin-related advantage during colonization neither in non-competitive nor in competitive experimental setups. In this thesis the existence and functionality of the colibactin gene cluster within different species of the Enterobacteriaceae was shown. Its occurrence as an individual genomic island as well as a part of an ICE demonstrates the genetic plasticity of this element and the impact of horizontally transferred genetic material. The biological function of colibactin in vivo remains to be elucidated and may affect both bacterial fitness and virulence

Cache-, Hash- and Space-Efficient Bloom Filters

A Bloom filter is a very compact data structure that supports approximate membership queries on a set, allowing false positives. We propose several new variants of Bloom filters and replacements with similar functionality. All of them have a better cache-efficiency and need less hash bits than regular Bloom filters. Some use SIMD functionality, while the others provide an even better space efficiency. As a consequence, we get a more flexible trade-off between false positive rate, space-efficiency, cache-efficiency, hash-efficiency, and computational effort. We analyze the efficiency of Bloom filters and the proposed replacements in detail, in terms of the false positive rate, the number of expected cachemisses, and the number of required hash bits. We also describe and experimentally evaluate the performance of highly-tuned implementations. For many settings, our alternatives perform better than the methods proposed so far

Mental workload during n-back task—quantified in the prefrontal cortex using fNIRS

When interacting with technical systems, users experience mental workload. Particularly in multitasking scenarios (e.g., interacting with the car navigation system while driving) it is desired to not distract the users from their primary task. For such purposes, human-machine interfaces (HCIs) are desirable which continuously monitor the users' workload and dynamically adapt the behavior of the interface to the measured workload. While memory tasks have been shown to elicit hemodynamic responses in the brain when averaging over multiple trials, a robust single trial classification is a crucial prerequisite for the purpose of dynamically adapting HCIs to the workload of its user. The prefrontal cortex (PFC) plays an important role in the processing of memory and the associated workload. In this study of 10 subjects, we used functional Near-Infrared Spectroscopy (fNIRS), a non-invasive imaging modality, to sample workload activity in the PFC. The results show up to 78% accuracy for single-trial discrimination of three levels of workload from each other. We use an n-back task (n ∈ {1, 2, 3}) to induce different levels of workload, forcing subjects to continuously remember the last one, two, or three of rapidly changing items. Our experimental results show that measuring hemodynamic responses in the PFC with fNIRS, can be used to robustly quantify and classify mental workload. Single trial analysis is still a young field that suffers from a general lack of standards. To increase comparability of fNIRS methods and results, the data corpus for this study is made available online

Uropathogenic E. coli Exploit CEA to Promote Colonization of the Urogenital Tract Mucosa

Attachment to the host mucosa is a key step in bacterial pathogenesis. On the apical surface of epithelial cells, members of the human carcinoembryonic antigen (CEA) family are abundant glycoproteins involved in cell-cell adhesion and modulation of cell signaling. Interestingly, several gram-negative bacterial pathogens target these receptors by specialized adhesins. The prototype of a CEACAM-binding pathogen, Neisseria gonorrhoeae, utilizes colony opacity associated (Opa) proteins to engage CEA, as well as the CEA-related cell adhesion molecules CEACAM1 and CEACAM6 on human epithelial cells. By heterologous expression of neisserial Opa proteins in non-pathogenic E. coli we find that the Opa protein-CEA interaction is sufficient to alter gene expression, to increase integrin activity and to promote matrix adhesion of infected cervical carcinoma cells and immortalized vaginal epithelial cells in vitro. These CEA-triggered events translate in suppression of exfoliation and improved colonization of the urogenital tract by Opa protein-expressing E. coli in CEA-transgenic compared to wildtype mice. Interestingly, uropathogenic E. coli expressing an unrelated CEACAM-binding protein of the Afa/Dr adhesin family recapitulate the in vitro and in vivo phenotype. In contrast, an isogenic strain lacking the CEACAM-binding adhesin shows reduced colonization and does not suppress epithelial exfoliation. These results demonstrate that engagement of human CEACAMs by distinct bacterial adhesins is sufficient to blunt exfoliation and to promote host infection. Our findings provide novel insight into mucosal colonization by a common UPEC pathotype and help to explain why human CEACAMs are a preferred epithelial target structure for diverse gram-negative bacteria to establish a foothold on the human mucosa.publishe

<i>E</i>. <i>coli</i> AfaE-III shows improved mucosal colonization and induces CD105 expression.

<p><i>(A)</i> Wild-type or CEAtg female mice were infected with <i>E</i>. <i>coli</i> AfaE-III or <i>E</i>. <i>coli</i> ΔAfaE-III. 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10). Data were compiled from two independent experiments. The median for each experimental group of animals is indicated by a line; numbers of recovered bacteria were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. <i>(B</i>, <i>C)</i> Animals were infected as in (A) and genital tracts were excised, fixed and frozen. <i>(B)</i> Cryosections of genital tracts were co-stained with rabbit antibodies against <i>E</i>. <i>coli</i> (green) and a mouse monoclonal antibody against CEA. Cell nuclei were visualized by Hoechst (blue). Numerous <i>E</i>. <i>coli</i> AfaE-III can be detected in close association with the CEA-positive epithelium (arrowhead), whereas non-CEACAM binding <i>E</i>. <i>coli</i> are rarely observed. (C) Cryosections were co-stained with rabbit antibodies against <i>E</i>. <i>coli</i> (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). Strong local expression of CD105 can be observed on the mucosal surface of CEAtg mice upon association with <i>E</i>. <i>coli</i> AfaE-III (arrowhead). Pictures in B) and C) are representative for three independent biological replicates.</p

The interaction of <i>E</i>. <i>coli</i> Opa_CEA with CEA facilitates mucosal colonization and leads to CD105 expression in epithelial cells.

<p><i>(A)</i> Wild-type (●) or CEAtg (▼) female mice were infected with the indicated bacterial strains and 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10; except for CEAtg animals infected with <i>E</i>. <i>coli</i> Opa_CEA, where n = 15). Data were compiled from four independent experiments. The median for each experimental group of animals is indicated by a line; groups were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. <i>(B)</i> Individual re-isolated bacterial colonies from the genital tract of CEAtg mice infected with either <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA were plated on LB-ampicillin agar plates (post infection). Five isolates for each strain were analysed by Western blotting with an antibody against Opa protein. As a control, lysates of the <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA used for infection (input) were also analysed. <i>(C)</i> Genital tracts from CEAtg mice infected for 24 hours with <i>E</i>. <i>coli</i> or <i>E</i>. <i>coli</i> Opa_CEA were excised, and cryosections were co-stained with antibodies against <i>E</i>. <i>coli</i> (green) and against CEA (red). Cell nuclei were stained with Hoechst dye (blue). Arrowheads indicate host-associated <i>E</i>. <i>coli</i>. Pictures are representative for three independent biological replicates. <i>(D)</i> Cryosections as in (C) were co-stained with antibodies against <i>E</i>. <i>coli</i> (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). CD105 expression on the mucosal surface of CEAtg mice infected with <i>E</i>. <i>coli</i> Opa_CEA is highlighted by small arrows. Pictures are representative for three independent biological replicates.</p

<i>E</i>. <i>coli</i> AfaE-III suppresses epithelial exfoliation in CEAtg mice.

<p><i>(A)</i> Whole mount urogenital tracts of uninfected wild-type or CEAtg female mice were fixed and processed for scanning electron microscopy (SEM). Pictures show the luminal surface of the upper vaginal and cervical regions and are representative for five animals each treatment group. <i>(B)</i> Wild-type or CEAtg female mice were infected with <i>E</i>. <i>coli</i> AfaE-III or <i>E</i>. <i>coli</i> ΔAfaE-III for 24 h and the genital tracts processed as in (A). SEM pictures (at two different magnifications, as indicated by the scale bars) show the luminal surface of the upper vaginal and cervical regions. Whereas massive epithelial exfoliation is evident in infected wildtype mice and in CEAtg mice infected with <i>E</i>. <i>coli</i> ΔAfaE-III, a strongly reduced detachment of epithelial cells is observed in CEAtg mice infected with CEACAM-binding <i>E</i>. <i>coli</i> AfaE-III. Adherent bacteria can be observed at higher magnifications (arrows). Pictures are representative for five animals each treatment group. <i>(C)</i> Quantification of exfoliating epithelial cells from samples in (B). Bars represent mean ± S.D. of exfoliating cells from at least n = 26 areas (~0.075 mm²) derived from at least five animals each treatment group. Results were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***.</p

CEA engagement by <i>E</i>. <i>coli</i> AfaE-III triggers enhanced cell-matrix adhesion via integrin activation.

<p><i>(A)</i> 293 cells were transfected with a control vector (GFP), or plasmids encoding CEA or CD105. Cells were left uninfected or infected for 8 h with the indicated bacteria. After infection, cells were used in adhesion assays on collagen. Bars represent means ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.–not significant. Shown is one representative experiment out of three independent biological replicates <i>(B)</i> 293 cells were transfected with CEA and seeded on 25 μg/ml collagen. Confluent layers were left uninfected or infected for 14 h with <i>E</i>. <i>coli</i>, <i>E</i>. <i>coli</i> AfaE-III, or <i>E</i>. <i>coli</i> ΔAfaE-III. Following infection, cells were washed and remaining cells were stained with crystal violet. Representative areas with remaining cells were photographed. Pictures are representative for three independent biological replicates. <i>(C)</i> 293 cells were infected and stained as in (B). Staining intensity was determined after dye elution in a spectrophotometer at 550 nm. Bars represent mean ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates. <i>(D)</i> 293 cells were transfected with plasmids encoding CEA or CD105. Cells were left uninfected or infected for 8 h with the indicated bacteria. After infection, cells were used in adhesion assays on collagen in the absence or presence of 1 mM Mn²⁺. Bars represent means ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates. <i>(E)</i> CEA-transfected 293 cells were infected for 14 h with the indicated bacteria and analyzed by flow cytometry for CD105 expression. Gray area indicates staining of uninfected cells with an isotype matched control antibody. Shown is one representative experiment out of three independent biological replicates. <i>(F</i>, <i>G)</i> 293 cells were transfected with plasmids encoding either CEA or CD105. As indicated, transfected cells were infected with bacteria for 14 h or left uninfected. After infection, cells were replated onto collagen-coated culture dishes for 90 min and stimulated or not for 5 min with 1 mM Mn²⁺ before fixation. Fixed samples were either stained with a rat monoclonal integrin β1 antibody (clone AIIB2), which recognizes the integrin β1 extracellular domain irrespective of its conformation (total integrin) (F) or samples were stained with an activation-epitope specific rat monoclonal integrin β1 antibody (clone 9EG7), which recognizes the extended, ligand-bound conformation of integrin β1 (active integrin)(G). Bars represent the mean ± S.D. of 5 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates.</p