Pertussis Toxin Exploits Host Cell Signaling Pathways Induced by Meningitis-Causing E. coli K1-RS218 and Enhances Adherence of Monocytic THP-1 Cells to Human Cerebral Endothelial Cells.

Pertussis toxin (PTx), the major virulence factor of the whooping cough-causing bacterial pathogen Bordetella pertussis, permeabilizes the blood-brain barrier (BBB) in vitro and in vivo. Breaking barriers might promote translocation of meningitis-causing bacteria across the BBB, thereby facilitating infection. PTx activates several host cell signaling pathways exploited by the neonatal meningitis-causing Escherichia coli K1-RS218 for invasion and translocation across the BBB. Here, we investigated whether PTx and E. coli K1-RS218 exert similar effects on MAPK p38, NF-κB activation and transcription of downstream targets in human cerebral endothelial TY10 cells using qRT-PCR, Western blotting, and ELISA in combination with specific inhibitors. PTx and E. coli K1-RS218 activate MAPK p38, but only E. coli K1-RS218 activates the NF-κB pathway. mRNA and protein levels of p38 and NF-κB downstream targets including IL-6, IL-8, CxCL-1, CxCL-2 and ICAM-1 were increased. The p38 specific inhibitor SB203590 blocked PTx-enhanced activity, whereas E. coli K1-RS218's effects were inhibited by the NF-κB inhibitor Bay 11-7082. Further, we found that PTx enhances the adherence of human monocytic THP-1 cells to human cerebral endothelial TY10 cells, thereby contributing to enhanced translocation. These modulations of host cell signaling pathways by PTx and meningitis-causing E. coli support their contributions to pathogen and monocytic THP-1 cells translocation across the BBB.

Identification of specific miRNAs targeting proteins of the apical junctional complex that simulate the probiotic effect of E. coli Nissle 1917 on T84 epithelial cells.

In the intestine, dysregulation of miRNA is associated with inflammation, disruption of the gastrointestinal barrier, and the onset of gastrointestinal disorders. This study identifies miRNAs involved in the maintenance of intercellular junctions and barrier integrity. For the functional identification of barrier affecting miRNAs, we took advantage of the barrier-enforcing effects of the probiotic bacterium Escherichia coli Nissle 1917 (EcN) which can be monitored by enhanced transepithelial resistance (TER). miRNA-profiling of T84 monolayers prior and after co-incubation with EcN revealed for the first time differentially regulated miRNAs (miR-203, miR-483-3p, miR-595) targeting tight junction (TJ) proteins. Using real-time PCR, Western blotting and specific miRNA mimics, we showed that these miRNAs are involved in the regulation of barrier function by modulating the expression of regulatory and structural components of tight junctional complexes. Furthermore, specific inhibitors directed at these miRNA abrogated the disturbance of tight junctions induced by enteropathogenic E. coli (EPEC). The half-maximal inhibitory concentration (IC(50)) was determined to 340 nM by monitoring inhibitor kinetics. In summary, we conclude that specific miRNAs effect regulatory as well as structural proteins of the junctional complex which in turn are involved in the barrier enhancing effect of EcN. Hence, we suggest that the application of miRNAs might be refined and further developed as a novel supportive strategy for the treatment of gastrointestinal disorders.

Prion protein 90-231 contains a streptavidin-binding motif.

The biological function of prion protein (PrP) and the physiological relevance of its truncated subtypes and glycoforms is still enigmatic. In this paper, we adduce evidence that recombinant murine PrP fragment 90-231 (mPrP90-231) contains a biotin-mimicking sequence motif that causes binding of the bacterial protein streptavidin to mPrP90-231. As indicated by epitope mapping and proven by analysis of a deletion mutant (mPrP101-231), streptavidin binding is primarily mediated by the amino-terminus of mPrP90-231 with the core-binding sequence represented by residues 94-100. Competition with biotin significantly reduces the interaction pointing to an involvement of streptavidin's biotin-binding site (BBS). Since the BBS of streptavidin shares similarities with the active sites of proteins involved in biotin metabolism we speculate that biotin mimicry by truncated PrP-species may have an impact in vivo.

Interferon-gamma mediates antigen trafficking to MHC class II-positive late endosomes of enterocytes.

MHC class II-positive late endosomes of enterocytes are thought to be involved in antigen presentation to CD4(+) T cells. In contrast to enterocytes of BALB/c mice, severe combined immunodeficiency (SCID) enterocytes lack MHC class II expression and fail to transport internalized ovalbumin (OVA) into late endosomes. IFN-gamma is known to induce MHC class II in enterocytes and antigen targeting to late endosomes in macrophages. In this study, we investigated the influence of IFN-gamma and MHC class II on the processes of antigen traffic in enterocytes. Subcellular targeting of OVA and MHC class II expression within enterocytes were examined in SCID, IFN-gamma-treated SCID, BALB/c and C57BL/6 MHC class II knockout (KO) mice after a single feed with OVA. Sorting of OVA into late endosomes was found in enterocytes from BALB/c, C57BL/6 KO and IFN-gamma-stimulated SCID mice, but not from untreated SCID mice. MHC class II expression was restricted to enterocytes of IFN-gamma-treated SCID and BALB/c mice, present at basolateral membranes and within endosomal compartments. These enterocytes further revealed colocalization of class II antigens and OVA in endosomes. We suggest that antigen trafficking into late endosomes of enterocytes is mediated by IFN-gamma and occurs in the absence of MHC class II.