Birgit Manncke

Gene expression patterns of epithelial cells modulated by pathogenicity factors of Yersinia enterocolitica

Epithelial cells express genes whose products signal the presence of pathogenic microorganisms to the immune system. Pathogenicity factors of enteric bacteria modulate host cell gene expression. Using microarray  technology  we  have  profiled  epithelial cell gene expression upon interaction with Yersinia enterocolitica. Yersinia enterocolitica wild‐type and isogenic mutant strains were used to identify host genes modulated by invasin protein (Inv), which is involved in enteroinvasion, and Yersinia outer protein P (YopP) which inhibits innate immune responses. Among 22 283 probesets (14 239 unique genes), we found 193 probesets (165 genes) to be regulated by Yersinia infection. The majority of these genes were induced by Inv, whose recognition leads to expression of NF‐κB‐regulated factors such as cytokines and adhesion molecules. Yersinia virulence plasmid (pYV)‐encoded factors counter regulated Inv‐induced gene expression. Thus, YopP repressed Inv‐induced NF‐κB regulated genes at 2 h post infection whereas other pYV‐encoded factors repressed host cell genes at 4 and 8 h post infection. Chromosomally encoded factors of Yersinia, other than Inv, induced expression of genes known to be induced by TGF‐β receptor signalling. These genes were also repressed by pYV‐encoded factors. Only a few host genes were exclusively induced by pYV‐encoded factors. We hypothesize that some of these genes may contribute to pYV‐mediated silencing of host cells. In conclusion, the data demonstrates that epithelial cells express a limited number of genes upon interaction with enteric Yersinia. Both Inv and YopP appear to modulate gene expression in order to subvert epithelial cell functions involved in innate immunity.

IFIT2 Is an Effector Protein of Type I IFN–Mediated Amplification of Lipopolysaccharide (LPS)-Induced TNF-α Secretion and LPS-Induced Endotoxin Shock

Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor– and IFN regulatory factor (Irf)9–dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow–derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-β mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.

Bacteroides fragilis adheres to laminin significantly stronger than bacteroides thetaiotaomicron and other species of the genus

The laminin binding properties of eight species of the genus Bacteroides were examined using latex particle agglutination assay. B. fragilis was found to bind strongly to laminin, whereas all other species tested showed no or only weak laminin adherence. The pronounced differences in laminin binding activity between B. fragilis on the one side and B. thetaiotaomicron and B. ovatus on the other were determined to be statistically significant (p < 0.001 and p < 0.01, respectively). With regard to the relevance of laminin adherence for bacterial pathogenicity and invasiveness, our results give a possible explanation for the well-known finding that B. fragilis is the most frequently isolated pathogen in anaerobic bacteremia.

Fibronectin and vitronectin binding of Bacteroides fragilis and eight other species of the genus

The fibronectin and vitronectin bindings of 152 strains belonging in 9 Bacteroides species of different origins were tested by means of latex agglutination. 23% of the strains isolated from faeces exhibited fibronectin binding, as did 46% of the strains obtained from severe infections. Most of the strains displaying fibronectin binding belonged to the species Bacteroides fragilis or Bacteroides vulgatus. The binding could be inhibited by preincubation of the cells with an excess amount of fibronectin. Vitronectin binding was less common, but was always observed in parallel with fibronectin binding.

Yersinia enterocolitica YopT and Clostridium difficile Toxin B Induce Expression of GILZ in Epithelial Cells

Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.

Fibronectin binding and cell surface hydrophobicity contribute to adherence properties of group B streptococci

The matrix protein, fibronectin, which is detectable in various tissues, when present in the vaginal fluid of women in labour, indicates the rupture of membranes. It is known that many bacteria adhere to fibronectin, thus establishing a first step of infection. In women in labour, group B streptococci are common agents of chorioamnionitis. For group B streptococci, unspecific adherence mechanisms like negative net charge and hydrophobic interactions have already been discussed in literature. In the present study, group B streptococci isolates from 57 patients with premature rupture of membranes were studied for fibronectin binding activities, using a particle agglutination assay and for cell surface hydrophobicity, by testing adhesion to hydrocarbons. Particle agglutination assays and adhesion assays were done with strains grown on blood-containing media and media without blood. Fibronectin binding was shown to be present in 14 and 11 out of 57 isolates grown on Mueller-Hinton and Tryptic Soy agar, respectively. When the strains were grown on blood-containing media, fibronectin-binding was found to be concomitant with decreased hydrophobicity. According to the results obtained in a total of 57 strains, cell surface hydrophobicity is an unspecific adhesion factor in group B streptococci. Fibronectin binding seems to be an additional adherence factor in some of the strains and may be assumed to play a major role in establishing infectious processes.

Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection.

Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors.

Erreger mit Weltraumverwandtschaft

Ein 64-jahriger bisher gesunder Patient erkrankt im Januar 2001 an einer starken Erkaltung. Trotz der Erkaltung geht er in der Folgezeit seiner schweren korperlichen Arbeit weiterhin nach. In den folgenden Monaten hat er aber mehrere Rezidive zunehmenden Schweregrades. Schlieslich muss er im Juli deswegen sogar seinen Baubetrieb aufgeben. Die progrediente korperliche Schwache ist vergesellschaftet mit zunehmenden Gedachtnisstorungen, Fieber von bis zu 39°C, Orientierungsstorungen sowie Hyperasthesien. Zur Abklarung und Behandlung dieser Symptome erfolgt schlieslich die stationare Krankenhausaufnahme. Die dort durchgefuhrte Untersuchung des Liquors ergibt eine Zellzahl von ca. 300 Zellen/μl. Bei Verdacht auf eine virale Enzephalitis erfolgt eine 7-tagige Aciclovirtherapie. Bei der anschliesenden Kontrolluntersuchung ist die Zellzahl im Liquor zwar auf ca. 15Zellen/μl vermindert, das Fieber persistiert dennoch, wahrend sich die neurologische Symptomatik sogar verstarkt.