Abstract
Intestinal epithelial tight junctions (TJ) are a major barrier restricting the entry of various harmful factors including pathogens; however, they also represent an important entry portal for pathogens. Although the rotavirus-induced early disruption of TJ integrity and targeting of TJ proteins as coreceptors are well-defined, the precise molecular mechanisms involved remain unknown. In the present study, infection of polarized MDCK cells with the species A rotavirus (RVA) strains human DS-1 and bovine NCDV induced a redistribution of TJ proteins into the cytoplasm, a reversible decrease in transepithelial resistance, and an increase in paracellular permeability. RhoA/ROCK/MLC signaling was identified as activated at an early stage of infection, while inhibition of this pathway prevented the rotavirus-induced early disruption of TJ integrity and alteration of TJ protein distribution. Activation of pMYPT, PKC, or MLCK, which are known to participate in TJ dissociation, was not observed in MDCK cells infected with either rotavirus strain. Our data demonstrated that binding of RVA virions or cogent VP8* proteins to cellular receptors activates RhoA/ROCK/MLC signaling, which alters TJ protein distribution and disrupts TJ integrity via contraction of the perijunctional actomyosin ring, facilitating virion access to coreceptors and entry into cells.
Highlights
The gastrointestinal epithelium consists of a multitude of cell types and acts as a selective barrier that prevents potentially harmful luminal agents, such as microorganisms and their products, food antigens, or toxins from penetrating underlying tissues, while allowing for the exchange of ions and small molecules[1]
RhoA and its downstream effector Rho kinase (ROCK) as well as protein kinase C (PKC) and its downstream effector myosin light chain kinase (MLCK) are crucial in mediating tight junctions (TJ) dissociation; this can be mediated through direct phosphorylation of the myosin II regulatory light chain (MLC) or indirectly through inhibition of dephosphorylation of MLC via activation of the regulatory subunit of myosin light chain phosphatase (MYPT), providing the force for disruption of TJs upon contraction of the perijunctional actomyosin ring[4,13]
In contrast to mock-infected Madin-Darby canine kidney (MDCK) cells, in which pMLC remained unchanged during the entire experiment, infection with either RVA strain resulted in a remarkable increase in pMLC that was detected as early as 5 min post-infection, peaked at 30 mpi, and was sustained at a high level until 60 mpi, declining afterwards (Fig. 1a,b)
Summary
The gastrointestinal epithelium consists of a multitude of cell types and acts as a selective barrier that prevents potentially harmful luminal agents, such as microorganisms and their products, food antigens, or toxins from penetrating underlying tissues, while allowing for the exchange of ions and small molecules[1]. Of TJs include activation of host cell signaling pathways via binding of virus particles to their primary receptors, reorganization or degradation of specific TJ proteins, and/or contraction of the perijunctional actomyosin ring (formed from stress fibers)[1,2,6,11]. In polarized epithelial cells (e.g., MDCK cells), RVA infection induces a drop in TER and an increase in paracellular flux via dissociation of TJs, which is induced by specific binding of the VP8* protein to its cellular receptors[7]. RVA infection induces the activation of RhoA GTPase and the formation of stress fibers, resulting in disruption of the cell cytoskeleton[33]; the specific involvement of cellular signaling pathways, in particular RhoA/ROCK and its associated PKC signaling pathway, in RVA-induced TJ disruption, has remained elusive
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