Pertussis Toxin Exploits Specific Host Cell Signaling Pathways for Promoting Invasion and Translocation of Escherichia coli K1 RS218 in Human Brain-derived Microvascular Endothelial Cells

Sascha Karassek,Laura Starost,Johanna Solbach,Lilo Greune,Yasuteru Sano,Takashi Kanda,Kwangsik Kim,M Alexander Schmidt

doi:10.1074/jbc.m115.650101

Abstract

Pertussis toxin (PTx), an AB5 toxin and major virulence factor of the whooping cough-causing pathogen Bordetella pertussis, has been shown to affect the blood-brain barrier. Dysfunction of the blood-brain barrier may facilitate penetration of bacterial pathogens into the brain, such as Escherichia coli K1 (RS218). In this study, we investigated the influence of PTx on blood-brain barrier permissiveness to E. coli infection using human brain-derived endothelial HBMEC and TY10 cells as in vitro models. Our results indicate that PTx acts at several key points of host cell intracellular signaling pathways, which are also affected by E. coli K1 RS218 infection. Application of PTx increased the expression of the pathogen binding receptor gp96. Further, we found an activation of STAT3 and of the small GTPase Rac1, which have been described as being essential for bacterial invasion involving host cell actin cytoskeleton rearrangements at the bacterial entry site. In addition, we showed that PTx induces a remarkable relocation of VE-cadherin and β-catenin from intercellular junctions. The observed changes in host cell signaling molecules were accompanied by differences in intracellular calcium levels, which might act as a second messenger system for PTx. In summary, PTx not only facilitates invasion of E. coli K1 RS218 by activating essential signaling cascades; it also affects intercellular barriers to increase paracellular translocation.

Highlights

Pertussis toxin (PTx) affects the blood-brain barrier in different models
HBMEC as well as TY10 cells were grown to confluence on filter membranes and stimulated with PTx 6 h before infection with the pathogenic E. coli K1 RS218 or the non-pathogenic E. coli HB101 strain (Fig. 1, A and B)
Quantification of the translocation rates showed that PTx increases the translocation of E. coli K1 RS218 as well as of E. coli HB101 in HBMEC cells

Summary

Background

Pertussis toxin (PTx) affects the blood-brain barrier in different models. Results: PTx activates host cell signaling pathways identical to those exploited by E. coli K1 RS218 in brain-derived microvascular endothelial cells. The observed changes in host cell signaling molecules were accompanied by differences in intracellular calcium levels, which might act as a second messenger system for PTx. In summary, PTx facilitates invasion of E. coli K1 RS218 by activating essential signaling cascades; it affects intercellular barriers to increase paracellular translocation. Bordetella pertussis is the causative agent of the respiratory disease whooping cough, which in young infants may occasionally be associated with neurological disorders [1,2,3] It has been shown in several studies that pertussis toxin (PTx), a decisive and secreted virulence factor of B. pertussis, affects cerebral vascular barriers and transiently increases the permeability of the blood-brain barrier [1, 3,4,5,6,7,8]. To gain further insight into the mode of action of PTx, we investigated which host cell signaling cascades might be affected and whether the toxin alters the same signaling pathways as E. coli K1 RS218 in brainderived endothelial cells

Experimental Procedures

Results

Discussion