Vasodilator-stimulated phosphoprotein restricts cell-to-cell spread of Shigella flexneri at the cell periphery

Abstract

Shigella spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. Shigella utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to Shigella flexneri spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting S. flexneri spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in Shigella pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells.

Actin-based motility and cell-to-cell spread of bacterial pathogens

“…For example, S. flexneri spread is enhanced by host Diaphanous (Dia) related formins [49], and inhibited by Ena/VASP family members VASP and EVL [50]. These factors do not influence bacterial motility in the cytosol [49,51], suggesting they specifically influence actin networks at the cortex and intercellular junctions.…”

Transfer of HTLV-1 p8 and Gag to target T-cells depends on VASP, a novel interaction partner of p8

“…The bacterial protein ActA interacts with VASP to foster bacterial spreading [ 39 , 58 , 62 ]. Contrary, during infections with the intracellular bacterial pathogens Shigella spp ., which cause diarrheal disease in humans, VASP and the related protein Ena/VASP-like (EVL) limit bacterial spread [ 61 ]. Interestingly, VASP activity is also required for Coxiella burnetii growth in human macrophages [ 64 ].…”

“…Interestingly, we could show that intracellular cAMP levels are upregulated in chronically HTLV-1 infected T-cells [60], and it is conceivable that phosphorylated VASP-species are present in these cells. Since not only the EVH1 domain, but also phosphorylation of Ser153 are required for VASP localization to focal adhesions and for the affinity of VASP to F-Actin [61,62], it cannot be excluded yet that phosphorylation of VASP is important for interacting with p8, or even for p8 and HTLV-1 transfer.…”