Untersuchungen zur Wechselwirkung von Listeria monocytogenes mit Komponenten des Mikrofilamentsystems der Wirtszelle und funktionelle Charakterisierung des Aktin-Nukleationsfaktors ActA

Der intrazelluläre Krankheitserreger Listeria monocytogenes kann die lebensbedrohende Listeriose verursachen. Mit dem Aktin-Nukleationsfaktor ActA induzieren diese Bakterien die Polymerisierung von Aktin an ihrer Oberfläche, um sich intra- und interzellulär zu verbreiten. In der vorliegenden Arbeit wurden verschiedene Aspekte der Interaktion von ActA mit wichtigen Komponenten des Aktinsystems der Wirtszelle bearbeitet. 1) Nach Immunodepletion von VASP aus Thrombozytenextrakten waren die Listerien nicht mehr motil. Die EVH1-Domäne von VASP, Mena und Evl inhibierte die Fortbewegung der Bakterien in dem zellfreien System. Damit konnte erstmals die direkte Beteiligung von VASP an der Fortbewegung der Listerien nachgewiesen werden 2) Mit Hilfe polyklonaler und monoklonaler Antikörper gegen p21-Arc konnte der Arp2/3-Komplex in infizierten Gewebekulturzellen auf der Oberfläche stationärer und im Aktinschweif motiler Listerien lokalisiert werden. Die Rekrutierung des Arp2/3-Komplexes war unabhängig von existierenden Aktinfilamenten, da p21-Arc auch nach Zerstörung der Aktinfilamente durch Latrunculin B noch auf der Listerienoberfläche nachgewiesen werden konnte. 3) Durch gezielte Mutagenese des KKRRK-Motivs wurde ActA charakterisiert. Die Analyse im Mitochondrien-Targetingsystem und die Charakterisierung isogener Listeria-Mutanten konnte zeigen, daß die zwei Argininreste an Position 148 und 149 für die Aktinnukleation und die Rekrutierung des Arp2/3-Komplexes essentiell waren. Die erzielten Resultate lieferten neue Erkenntnisse über die Wechselwirkung des ActA-Proteins mit dem Mikrofilamentsystem der Wirtszelle, und es wurde ein verbessertes Dreistufenmodell für die Nukleation von Aktinfilamenten durch intrazelluläre Listerien vorgestellt.The intracellular pathogen Listeria monocytogenes is causing the life threatening disease Listeriosis. With the actin nucleating factor ActA Listeria induce the polymerization of actin at their surface for intra- and intercellular spreading. The work presented here is dealing with different aspects of the interaction of ActA with the microfilament system. 1) After immunodepletion of VASP from platelet extracts, Listeria were no longer able to move. The EVH1-domain of VASP, Mena and Evl inhibited listerial movement in a cell free system. This was the first evidence for a direct implication of VASP in listerial movement. 2) With polyclonal and monoclonal antibodies against p21-Arc was the Arp2/3-complex localized on the surface of stationery and in the actin tail of moving Listeria. Recruitment of the Arp2/3-complex was independent of preexisting actin filaments, since after destruction of filaments with Latrunculin B, p21-Arc was still detectable on the surface of Listeria. 3) ActA was characterized by site-directed mutagenesis of the KKRRK motiv. Analysis in the mitochondria targeting system and characterization of isogenic Listeria-mutants could show, that the two Arginin residues at positions 148 and 149 are essentiell for actin filament nucleation and recruitment of the Arp2/3-complex. These results revealed new insight into the interaction of the ActA protein with the microfilament system of the host cell and a refined three-step-model for the nucleation of actin filaments by intracellular Listeria is presented

Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans.

Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ~3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (ComX-GFP) reporter, and the transcriptome analysis demonstrated the successful combination of both methods, because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (mutacin V) confirmed this expression pattern on the single-cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity

Assessing the viability of bacterial species in drinking water by combined cellular and molecular analyses.

The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining ("live/dead staining" indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotypes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water

Proteome analysis of distinct developmental stages of human natural killer (NK) cells.

The recent Natural Killer (NK) cell maturation model postulates that CD34(+) hematopoietic stem cells (HSC) first develop into CD56(bright) NK cells, then into CD56(dim)CD57(-) and finally into terminally maturated CD56(dim)CD57(+). The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56(bright) NK cells versus CD56(dim) NK cells and CD56(dim)CD57(-) NK cells versus CD56(dim)CD57(+) NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa

Staphylococcus aureus binding to Seraph® 100 Microbind® Affinity Filter: Effects of surface protein expression and treatment duration

Introduction Extracorporeal blood purification systems represent a promising alternative for treatment of blood stream infections with multiresistant bacteria. Objectives The aim of this study was to analyse the binding activity of S. aureus to Seraph affinity filters based on heparin coated beads and to identify effectors influencing this binding activity. Results To test the binding activity, we used gfp-expressing S. aureus Newman strains inoculated either in 0.9% NaCl or in blood plasma and determined the number of unbound bacteria by FACS analyses after passing through Seraph affinity filters. The binding activity of S. aureus was clearly impaired in human plasma: while a percent removal of 42% was observed in 0.9% NaCl (p-value 0.0472) using Seraph mini columns, a percent removal of only 10% was achieved in human plasma (p-value 0.0934). The different composition of surface proteins in S. aureus caused by the loss of SarA, SigB, Lgt, and SaeS had no significant influence on its binding activity. In a clinically relevant approach using the Seraph® 100 Microbind® Affinity Filter and 1000 ml of human blood plasma from four different donors, the duration of treatment was shown to have a critical effect on the rate of bacterial reduction. Within the first four hours, the number of bacteria decreased continuously and the reduction in bacteria reached statistical significance after two hours of treatment (percentage reduction 64%, p-value 0.01165). The final reduction after four hours of treatment was close to 90% and is dependent on donor. The capacity of Seraph® 100 for S. aureus in human plasma was approximately 5 x 108 cells. Conclusions The Seraph affinity filter, based on heparin-coated beads, is a highly efficient method for reducing S. aureus in human blood plasma, with efficiency dependent on blood plasma composition and treatment duration

Serum response factor contributes selectively to lymphocyte development.

Serum response factor (SRF), is a crucial transcription factor for murine embryonic development and for the function of muscle cells and neurons. Gene expression data show that SRF and its transcriptional cofactors are also expressed in lymphocyte precursors and mature lymphocytes. However, the role of SRF in lymphocyte development has not been addressed in vivo so far, attributed in part to early embryonic lethality of conventional Srf-null mice. To determine the in vivo role of SRF in developing lymphocytes, we specifically inactivated the murine Srf gene during T or B cell development using lymphocyte-specific Cre transgenic mouse lines. T cell-specific Srf deletion led to a severe block in thymocyte development at the transition from CD4/CD8 double to single positive stage. The few residual T cells detectable in the periphery retained at least one functional Srf allele, thereby demonstrating the importance of SRF in T cell development. In contrast, deletion of Srf in developing B cells did not interfere with the growth and survival of B cells in general, yet led to a complete loss of marginal zone B cells and a marked reduction of the CD5+ B cell subset. Our study also revealed a contribution of SRF to the expression of the surface molecules IgM, CD19, and the chemokine receptor 4 in B lymphocytes. We conclude that SRF fulfills essential and distinct functions in the differentiation of different types of lymphocytes

Selective Bacterial Targeting and Infection-Triggered Release of Antibiotic Colistin Conjugates.

In order to render potent, but toxic antibiotics more selective, we have explored a novel conjugation strategy that includes drug accumulation followed by infection-triggered release of the drug. Bacterial targeting was achieved using a modified fragment of the human antimicrobial peptide ubiquicidin, as demonstrated by fluorophore-tagged variants. To limit the release of the effector colistin only to infection-related situations, we introduced a linker that was cleaved by neutrophil elastase (NE), an enzyme secreted by neutrophil granulocytes at infection sites. The linker carried an optimized sequence of amino acids that was required to assure sufficient cleavage efficiency. The antibacterial activity of five regioisomeric conjugates prepared by total synthesis was masked, but was released upon exposure to recombinant NE when the linker was attached to amino acids at the 1- or the 3-position of colistin. A proof-of-concept was achieved in co-cultures of primary human neutrophils and Escherichia coli that induced the secretion of NE, the release of free colistin, and an antibacterial efficacy that was equal to that of free colistin