An mRNA degrading complex in Rhodobacter capsulatus.

An RNA degrading, high molecular weight complex was purified from Rhodobacter capsulatus. N-terminal sequencing, glycerol-gradient centrifugation, and immunoaffinity purification as well as functional assays were used to determine the physical and biochemical characteristics of the complex. The complex comprises RNase E and two DEAD-box RNA helicases of 74 and 65 kDa, respectively. Most surprisingly, the transcription termination factor Rho is a major, firmly associated component of the degradosome.

Bartonella henselae induces NF-kappaB-dependent upregulation of adhesion molecules in cultured human endothelial cells: possible role of outer membrane proteins as pathogenic factors.

The endothelium is a specific target for Bartonella henselae, and endothelial cell infection represents an important step in the pathogenesis of cat scratch disease and bacillary angiomatosis. Mechanisms of Bartonella-endothelial cell interaction as well as signaling pathways involved in target cell activation were analyzed. B. henselae strain Berlin-1, isolated from bacillary angiomatosis lesions of a human immunodeficiency virus-infected patient, potently stimulated human umbilical cord vein endothelial cells (HUVEC), as determined by NF-kappaB activation and enhanced adhesion molecule expression. These effects were accompanied by increased PMN rolling on and adhesion to infected endothelial cell monolayers, as measured in a parallel-plate flow chamber assay. Monoclonal antibodies against E-selectin significantly reduced PMN rolling and adhesion. In our hands, B. henselae Berlin-1 was substantially more active than the typing strain B. henselae ATCC 49882. E-selectin and ICAM-1 upregulation occurred for up to 9 days, as verified by Northern blotting and cell surface enzyme-linked immunosorbent assay. Induction of adhesion molecules was mediated via NF-kappaB activation and could be blocked by a specific NF-kappaB inhibitor. Additional studies indicated that B. henselae-induced effects did not require living bacteria or Bartonella lipopolysaccharides. Exposure of HUVEC to purified B. henselae outer membrane proteins (OMPs), however, reproduced all aspects of endothelial cell activation. In conclusion, B. henselae, the causative agent of cat scratch disease and bacillary angiomatosis, infects and activates endothelial cells. B. henselae OMPs are sufficient to induce NF-kappaB activation and adhesion molecule expression followed by enhanced rolling and adhesion of leukocytes. These observations identify important new properties of B. henselae, demonstrating its capacity to initiate a cascade of events culminating in a proinflammatory phenotype of infected endothelial cells.

Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells.

Bacterial endotoxin (lipopolysaccharide, or LPS) has potent proinflammatory properties by acting on many cell types, including endothelial cells. Secretion of the CXC-chemokine interleukin-8 (IL-8) by LPS-activated endothelial cells contributes substantially to the inflammatory response. Using human umbilical vein endothelial cells (HUVECs), we analyzed the role of small GTP-binding Rho proteins and p38 mitogen-activated protein kinase (MAPK) for LPS-dependent IL-8 expression in endothelial cells. Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced tyrosine phosphorylation, nuclear factor (NF)-kappaB-dependent gene expression, IL-8 messenger RNA, and IL-8 protein accumulation but showed no effect on LPS-dependent p38 MAPK activation. Inhibition of p38 MAPK by SB 202190 also blocked LPS-induced NF-kappaB activation and IL-8 synthesis. Furthermore, selective activation of the p38 MAPK pathway by transient expression of a constitutively active form of MAPK kinase (MKK)6, the upstream activator of p38, was as effective as LPS with respect to IL-8 expression in HUVECs. In summary, our data suggest that LPS-induced NF-kappaB activation and IL-8 synthesis in HUVECs are regulated by both a Rho-dependent signaling pathway and the MKK6/p38 kinase cascade.

Initial events in the degradation of the polycistronic puf mRNA in Rhodobacter capsulatus and consequences for further processing steps.

Individual segments of the polycistronic puf mRNA of Rhodobacter capsulatus exhibit extremely different half-lives contributing to the stoichiometry of light-harvesting and reaction centre complexes of this facultative phototrophic bacterium. While earlier investigations shed light on the processes leading to the degradation of the 2.7 kb pufBALMX mRNA and, consequently, to the formation of the highly stable 0.5 kb pufBA mRNA processing product, we have now investigated the initial events in the degradation of the highly unstable 3.2 kb pufQBALMX primary transcript. Sequence modifications of two putative RNase E recognition sites within the pufQ coding region provide strong evidence that RNase E-mediated cleavage of a sequence at the 3' end of pufQ is involved in rate-limiting cleavage of the primary pufQBALMX transcript in vivo. The putative RNase E recognition sequence at the 5' end of pufQ is cleaved in vitro but does not contribute to rate-limiting cleavage in vivo. Analysis of the decay of puf mRNA segments transcribed from wild-type and mutated puf DNA sequences in R. capsulatus and Escherichia coli reveal that RNase E-mediated cleavage within the pufQ mRNA sequence also affects the stability of the 0.5 kb pufBA processing product. These findings demonstrate that the stability of a certain mRNA segment depends on the pathway of processing of its precursor molecule.

Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae.

Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (beta1 and beta2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-kappaB activation/translocation occurred within 10-15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis.

Two distinct phospholipases C of Listeria monocytogenes induce ceramide generation, nuclear factor-kappa B activation, and E-selectin expression in human endothelial cells.

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGD delta plcA and EGD delta plcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGD delta plcA or EGD delta plcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-kappa B activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-kappa B activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.