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Biomedical subjects

Martin Aepfelbacher

Publications and source records attributed to Martin Aepfelbacher.

At least 19 recordsLinked to original sources
PubMed2007

Lactobacillus fermentum attenuates the proinflammatory effect of Yersinia enterocolitica on human epithelial cells.

BACKGROUND: Lactobacilli represent a major component of the human microbiota. In this study we investigated whether and how Lactobacillus fermentum inhibits the proinflammatory responses of human epithelial cells on Yersinia enterocolitica infection. METHODS: Human epithelial cells were exposed to Y. enterocolitica pYV(-) or L. fermentum or to both strains, combinations of heat-killed L. fermentum or supernatant of L. fermentum cultures and Y. enterocolitica. The modulation of Y. enterocolitica induced IL-8 levels in the culture supernatants was determined and activation of Rac, p38, and NF-kappaB was investigated. RESULTS: Exposure of human epithelial cells to L. fermentum does not induce NF-kappaB activation and subsequent IL-8 secretion in HeLa cells, whereas Y. enterocolitica induces NF-kappaB activation and high levels of IL-8. Viable L. fermentum, supernatant of L. fermentum cultures, but not heat-killed L. fermentum, inhibited IL-8 secretion of HeLa cells triggered by Y. enterocolitica. Lactobacillus fermentum-exposed HeLa cells showed decreased Rac, p38, and NF-kappaB activation after Y. enterocolitica infection. Treatment of L. fermentum supernatants with phospholipase C abolished the inhibitory effect, indicating that a secreted phospholipid mediates the antiinflammatory properties of L. fermentum. Adhesion to or invasion of Y. enterocolitica into epithelial cells was not altered by coincubation with L. fermentum. CONCLUSION: Our results lead to the conclusion that L. fermentum inhibits the Y. enterocolitica-induced IL-8 production by a possibly secreted phospholipid of <10 kDa molecular weight. These data suggest that L. fermentum may have probiotic properties modulating intestinal inflammatory responses and might offer new therapeutic strategies in the treatment of intestinal inflammatory diseases.

Bacterial Adhesion↗
PubMed2006

Yersinia protein kinase YopO is activated by a novel G-actin binding process.

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.

Actins↗
PubMed2006

Staphylococcus aureus fibronectin binding protein-A induces motile attachment sites and complex actin remodeling in living endothelial cells.

Staphylococcus aureus fibronectin binding protein-A (FnBPA) stimulates alpha5beta1-integrin signaling and actin rearrangements in host cells. This eventually leads to invasion of the staphylococci and their targeting to lysosomes. Using live cell imaging, we found that FnBPA-expressing staphylococci induce formation of fibrillar adhesion-like attachment sites and translocate together with them on the surface of human endothelial cells (velocity approximately 50 microm/h). The translocating bacteria recruited cellular actin and Rab5 in a cyclic and alternating manner, suggesting unsuccessful attempts of phagocytosis by the endothelial cells. Translocation, actin recruitment, and eventual invasion of the staphylococci was regulated by the fibrillar adhesion protein tensin. The staphylococci also regularly produced Neural Wiskott-Aldrich syndrome protein-controlled actin comet tails that further propelled them on the cell surface (velocity up to 1000 microm/h). Thus, S. aureus FnBPA produces attachment sites that promote bacterial movements but subvert actin- and Rab5 reorganization during invasion. This may constitute a novel strategy of S. aureus to postpone invasion until its toxins become effective.

Actins↗
PubMed2006

The proteasome pathway destabilizes Yersinia outer protein E and represses its antihost cell activities.

Pathogenic Yersinia spp. neutralize host defense mechanisms by engaging a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell. Although the modulation of the cellular responses by individual Yops has been intensively studied, little is known about the fate of the translocated Yops inside the cell. In this study, we investigated involvement of the proteasome, the major nonlysosomal proteolytic system in eukaryotic cells, in Yop destabilization and repression. Our data show that inhibition of the proteasome in Yersinia enterocolitica-infected cells selectively stabilized the level of YopE, but not of YopH or YopP. In addition, YopE was found to be modified by ubiquitination. This suggests that the cytotoxin YopE is physiologically subjected to degradation via the ubiquitin-proteasome pathway inside the host cell. Importantly, the increased levels of YopE upon proteasome inhibition were associated with decreased activity of its cellular target Rac. Thus, the GTPase-down-regulating function of YopE is enhanced when the proteasome is inhibited. The stabilization of YopE by proteasome inhibitor treatment furthermore led to aggravation of the cytotoxic YopE effects on the actin cytoskeleton and on host cell morphology. Together, these data show that the host cell proteasome functions to destabilize and inactivate the Yersinia effector protein YopE. This implies the proteasome as integral part of the cellular host immune response against the immunomodulatory activities of a translocated bacterial virulence protein.

Bacterial Outer Membrane Proteins↗
PubMed2006

Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes.

Small colony variants (SCVs) of Staphylococcus aureus have been proposed to persist within vascular endothelium, thereby sustaining chronic infections. To identify the intracellular SCV location we infected primary human endothelial cells with various wild type and SCV strains of S. aureus and visualised maturation of phagosomes using live cell imaging. Staphylococci-containing phagosomes were matured by sequential and dynamic interactions with Rab5- and Rab7-positive vesicles. Within 45-60 min all internalised staphylococci accumulated in LAMP-1- and LysoTracker-enriched lysosomal organelles and remained there for up to 5 days. Recovery of most staphyloccocal strains was below 1% after a 24 h intracellular stay, indicating a high bactericidal activity of the endothelial cell lysosomes. However, the menadione auxotroph SCV strain JB1 displayed a recovery rate of 4% and, furthermore, through multiple intracellular passaging a subtype (JB1-P4) with a recovery rate of 25-30% could be generated. Interestingly, both JB1 and JB1-P4 also resided exclusively in lysosomes. Thus, on one hand we document effective bactericidal activity of human endothelial cell lysosomes towards staphylococci, and on the other hand we provide evidence that certain SCVs are capable to withstand this activity.

Endothelial Cells↗
PubMed2006

The kinesin KIF1C and microtubule plus ends regulate podosome dynamics in macrophages.

Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end-enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells.

Cell Differentiation↗
PubMed2006

Detection and genotyping of SHV beta-lactamase variants by mass spectrometry after base-specific cleavage of in vitro-generated RNA transcripts.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR-amplified and in vitro-transcribed bla(SHV) genes was used for the identification and genotyping of SHV beta-lactamases. For evaluation, bla(SHV) stretches of 21 clinical Enterobacteriaceae isolates were PCR amplified using T7 promoter-tagged forward and reverse primers, respectively. In vitro transcripts were generated with T7 RNA and DNA polymerase in the presence of modified analogues replacing either CTP or UTP. Using RNase A, the in vitro transcripts were base-specifically cleaved at every "T" or "C" position. Resulting cleavage products were analyzed by MALDI-TOF MS, generating a characteristic signal pattern based on the fragment masses. All 21 individual SHV genes were identified unambiguously using reference sequences, and the results were in perfect concordance with those obtained by fluorescent dideoxy sequencing, which represents the current standard method. As multiple point mutations can be detected in a single assay and newly emerged mutations which are not yet described in public databases can be identified too, MALDI-TOF MS appears to be an ideal tool for analysis of sequence polymorphisms in resistance-associated gene loci.

Bacteriological Techniques↗
PubMed2004

Microviscoelasticity of the apical cell surface of human umbilical vein endothelial cells (HUVEC) within confluent monolayers.

We studied the local viscoelasticity of the apical membrane of human umbilical vein endothelial cells within confluent layers by magnetic tweezers microrheometry. Magnetic beads are coupled to various integrins by coating with fibronectin or invasin. By analyzing the deflection of beads evoked by various force scenarios we demonstrate that the cell envelope behaves as a linear viscoelastic body if forces up to 2 nN are applied for short times (<20 s) but can respond in an adaptive way if stress pulses are applied longer (>30 s). The time-dependent shear relaxation modulus G(t) exhibits three time regimes: a fast response (t < 0.05 s) where the relaxation modulus G(t) obeys a power law G(t) approximately t(-0.82+/-0.02); a plateau-like behavior (at 0.05 s < t < 0.15 s); and a slow flow-like response which is, however, partially reversible. Strain field mapping experiments with colloidal probes show that local forces induce a strain field exhibiting a range of zeta = 10 +/- 1 microm, but which could only be observed if nonmagnetic beads were coupled to the cell surface by invasin. By application of the theory of elasticity of planar bodies we estimated a surface shear modulus of 2.5 x10(-4) N/m. By assuming a thickness of the actin cortex of approximately 0.5 microm we estimate a Young modulus micro approximately 400 Pa for the apical membrane. The value agrees with a plateau modulus of an entangled or weakly cross-linked actin network of an actin concentration of 100 microM (mesh size 0.2 microm). This result together with our observation of a strong reduction of the shear modulus by the actin destabilizing agent latrunculin A suggests that the shear modulus measured by our technique is determined by the actin cortex. The effect of two ligands inducing actin stress fiber formation and centripetal contraction of cells (associated with the formation of gaps in the confluent cell monolayer) on the viscoelastic responses were studied: histamine and lysophosphatidic acid (LPA). Histamine evoked a dramatic increase of the cell stiffness by >1 order of magnitude within <30 s, which is attributed to a transient rise of the intracellular Ca(2+) level, since DMSO exerted a similar effect. The stiffening is accompanied by a concomitant rounding of the cells as observed by microinterferometry and relaxes partially in the timescale of 5 min, whereas gaps between cells close after approximately 30 min. LPA did not exert a remarkable and reproducible effect other than an occasional very weak transient increase of the shear stiffness, which shows that the gap formation activated by LPA is mediated by a different mechanism than that induced by histamine.

Cell Membrane↗
PubMed2003

Characterization of YopT effects on Rho GTPases in Yersinia enterocolitica-infected cells.

Pathogenic yersiniae employ a type III secretion system for translocating up to six effector proteins (Yersinia outer proteins (Yops)) into eukaryotic target cells. YopT is a cysteine protease that was shown to remove the C-terminal isoprenoid group of RhoA, Rac, and CDC42Hs. Here we characterized the cell biological and biochemical activities of YopT in cells infected with pathogenic Yersinia enterocolitica. Bacterially injected YopT located to cell membranes from which it released RhoA but not Rac or CDC42Hs. In the infected cells RhoA was dissociated from guanine nucleotide dissociation inhibitor-1 (GDI-1) and accumulated as a monomeric protein in the cytosol, whereas Rac and CDC42Hs remained GDI-bound. Direct transfer of isoprenylated RhoA to YopT and RhoA modification could be reconstituted in vitro by guanosine 5'-3-O-(thio)triphosphate loading of a recombinant RhoA.GDI-1 complex. Finally, in macrophages infected with a Yersinia strain selectively translocating YopT podosomal adhesion structures required for chemotaxis as well as phagocytic cups mediating uptake of yersiniae were disrupted. These findings indicate that bacterially translocated YopT acts on membrane-bound and GDI-complexed RhoA but not Rac or CDC42, and this is sufficient for disruption of macrophage immune functions.

Animals↗
PubMed2003

Local force induced conical protrusions of phagocytic cells.

Magnetic tweezers were used to study the passive and active response of macrophages to local centripetal nanonewton forces on beta1 integrins. Superparamagnetic beads coated with the beta1-integrin-binding protein invasin were attached to J774 murine macrophages to mimic phagocytosis of bacterial pathogens. Forces exceeding approximately 0.5 nN induce the active formation of trumpet-like protrusions resembling pseudopodia after an initial elastic deflection and a response time of approximately 30 seconds. The speed of advancement of the protrusion is =0.065+/-0.020 micro m second(-1) and is force independent. After saturation (after about 100 seconds) the protrusion stops abruptly and is completely retracted again against forces exceeding 5 nN with an effective relaxation time of approximately 30 seconds. The active protrusion is tentatively attributed to the growth of the actin cortex in the direction of the force, and evidence for the involvement of actin is provided by the finding that Latrunculin A abolishes the activated cone growth. The growth is assumed to be activated by cell signaling mediated by the invasin-specific integrins (exhibiting beta1 chains) and could play a role in phagocytic and protrusive events during immune response by macrophages.

Adhesins, Bacterial↗
PubMed2003

Podosomes: adhesion hot-spots of invasive cells.

Podosomes are highly dynamic, actin-rich adhesion structures of monocyte-derived cells, certain transformed fibroblasts and carcinoma cells and have recently also been discovered in an increasing number of other cell types. Because they are found mainly in motile cells and control the activity of matrix metalloproteases, podosomes are thought to contribute to tissue invasion and matrix remodeling. Importantly, podosomes are physiologically relevant organelles because they can be found in ex vivo models of invasive cells. Regulators of podosome turnover include tyrosine kinases, RhoGTPases, actin regulators and the microtubule system. Podosomes might also serve as an attractive model to study how integration of various signaling pathways controls actin dynamics. Here, we summarize and discuss the known structural, regulatory and functional features of podosomes, our aim being to stimulate further research into these unique structures.

Actin Cytoskeleton↗
PubMed2003

Macrophages of patients with X-linked thrombocytopenia display an attenuated Wiskott-Aldrich syndrome phenotype.

The immunodeficiency disorder Wiskott-Aldrich syndrome and its milder form X-linked thrombo-cytopenia are caused by mutations in the WASp gene. Wiskott-Aldrich syndrome is characterized by a plethora of clinical symptoms which are due to functional defects of haematopoietic cells, including the inability of macrophages to form actin-rich adhesion structures called podosomes. In contrast, X-linked thrombocytopenia patients show reduced platelet size and counts but no cytoskeletal white blood cell defects have been detected so far. Here we use immunofluorescence technique to evaluate podosome formation in macrophages from X-linked thrombocyto-penia and Wiskott-Aldrich syndrome patients and from healthy donors. We find that X-linked thrombocytopenia macrophages, cells previously thought to be unaffected in this disorder, are compromised in the formation of podosomes. Western blot analysis shows that this phenotype is not due to lower levels of WASp expression. Interestingly, the bacterial chemoattractant formyl-methionyl-leucyl-phenylalanine can rescue podosome formation in X-linked thrombocytopenia cells. Our findings indicate that: 1. The spectrum of WASp-dependent disorders contains defects more subtle than originally recognized and 2. in X-linked thrombocytopenia, some of these defects may not be evident under conditions of bacterial stimulation. Further evaluation of this and other, as yet unrecognized, cellular defects may provide a more complete picture of the continuum of Wiskott-Aldrich syndrome and X-linked thrombocytopenia defects.

Case-Control Studies↗
PubMed2003

Activation of NF-kappaB and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascades.

Yersinia enterocolitica triggers activation of the nuclear factor (NF)-kappaB and production of the proinflammatory chemokine interleukin (IL)-8 in intestinal epithelial cells. This activation is due to adhesion of the bacteria via their outer membrane protein invasin to the host cells. Using Clostridium difficile toxins that specifically inactivate small GTPases, and transfection of inhibitory proteins of the Rho-GTPases, we demonstrate that Rac1, but not Cdc42 or Rho, is required for activation of NF-kappaB by invasin. Invasin activated the mitogen activated protein kinases (MAPK) p38 and c-Jun N-terminal protein kinase (JNK) but not extracellular signal regulated kinase (ERK). The functional relevance of these pathways for invasin-mediated IL-8 expression was assessed by protein kinase inhibitors and dominant-negative kinase mutants. While NF-kappaB and JNK contribute to IL-8 transcription, p38 MAPK also acts through stabilization of IL-8 mRNA, as confirmed by quantitative RT-PCR and electrophoretic mobility shift assays. Transfection experiments with I-kappaB kinase (IKK)1 and IKK2 mutants indicate that the release of NF-kappaB from its cytoplasmic inhibitor I-kappaB and its translocation into the nucleus is mediated by these kinases. Our data identify Rac1 as a key intermediate in invasin-triggered IL-8 synthesis and demonstrate that maximum IL-8 induction involves several MAP kinase cascades.

Adhesins, Bacterial↗
PubMed2003

Analysis of chaperone-dependent Yop secretion/translocation and effector function using a mini-virulence plasmid of Yersinia enterocolitica.

We have constructed a mini-pYV plasmid (pTTSS) harboring the Yersinia type three secretion system (TTSS) and the adhesin yadA on a low-copy vector. Using this system we could demonstrate for the first time that YopO, YopP, YopM, and YopQ do not require any of the known or orphan chaperones for efficient secretion/translocation. Y. enterocolitica harboring pTTSS, (WA-C(pTTSS)) was able to secrete and translocate single Yop effector proteins in trans. WA-C(pTTSS) proved to be stable and secretion of Yops was Ca2+ and temperature dependent as is the case for the parental strain. This shows that all genes necessary for translocation and expression of the Ca(2+)-dependent phenotype are contained within the cloned region. In contrast to previously published multiple yop mutants which were constructed by sequential deletion of yops, our system which harbors only the TTSS region without yops, chaperones, and unknown ORFs can be sequentially complemented with yops and sycs of choice. WA-C(pTTSS) was able to translocate YopE, YopM and YopT into HeLa cells as demonstrated by Western blotting. Translocation of YopE and YopT was strictly dependent on the presence of their respective chaperones, whereas YopM did not require a chaperone for translocation. WA-C(pTTSS) harboring yopT and sycT was shown to translocate active YopT by demonstrating modification of the small GTP-binding protein RhoA. This shows for the first time that RhoA modification is strictly dependent on YopT and does not require additional effector Yops. WA-C(pTTSS) harboring YopP was shown to induce apoptosis. This system is ideal to study chaperone-dependent Yop secretion/ translocation without the background of other effector Yops (YopE, YopM, YopO, YopP, YopT, YopH), chaperones (SycE, SycH, SycT) and unknown ORFs. In addition this system can secrete heterologous proteins fused to the N-terminal secretion/translocation domain of YopE.

Bacterial Outer Membrane Proteins↗
PubMed2003

Cytotoxic necrotizing factor 1 of Escherichia coli stimulates Rho/Rho-kinase-dependent myosin light-chain phosphorylation without inactivating myosin light-chain phosphatase in endothelial cells.

Cytotoxic necrotizing factor 1 (CNF-1) is an exotoxin of Escherichia coli that constitutively activates the GTPases Rho, Rac, and CDC42. Stimulation of Rho was shown to enhance myosin light-chain (MLC) phosphorylation via Rho kinase-mediated inhibition of MLC phosphatase in endothelial cells. Here we report that 3 h after CNF stimulation of endothelial cells, RhoA was activated and MLC phosphorylation was increased in a Rho/Rho-kinase-dependent manner, but no decrease in MLC phosphatase activity could be detected. Despite continuous RhoA activation, MLC phosphatase activity was doubled after 24 h of CNF stimulation, and this coincided with decreased MLC phosphorylation and cell spreading. Rac was also activated at 3 to 24 h but did not contribute to MLC phosphorylation, and its amount gradually decreased in the CNF-stimulated cells. CDC42Hs was not activated above control values by CNF. These results suggest that CNF can induce specific decoupling (Rho kinase from MLC phosphatase) and deactivation events in Rho GTPase signaling, potentially reflecting cellular protection mechanisms against permanently active Rho GTPases.

Actomyosin↗
PubMed2002

Yersinia V-antigen exploits toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression.

A characteristic of the three human-pathogenic Yersinia spp. (the plague agent Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica) is the expression of the virulence (V)-antigen (LcrV). LcrV is a released protein which is involved in contact-induced secretion of yersinia antihost proteins and in evasion of the host's innate immune response. Here we report that recombinant LcrV signals in a CD14- and toll-like receptor 2 (TLR2)-dependent fashion leading to immunosuppression by interleukin 10 induction. The impact of this immunosuppressive effect for yersinia pathogenesis is underlined by the observation that TLR2-deficient mice are less susceptible to oral Y. enterocolitica infection than isogenic wild-type animals. In summary, these data demonstrate a new ligand specificity of TLR2, as LcrV is the first known secreted and nonlipidated virulence-associated protein of a Gram-negative bacterium using TLR2 for cell activation. We conclude that yersiniae might exploit host innate pattern recognition molecules and defense mechanisms to evade the host immune response.

Amino Acid Sequence↗