Phosphorylated vimentin-triggered fibronectin matrix disaggregation enhances the dissemination of Treponema pallidum subsp. pallidum across the microvascular endothelial barrier
Fibronectin (FN) is an essential component of the extracellular matrix (ECM) that protects the integrity of the microvascular endothelial barrier (MEB). However, Treponema pallidum subsp . pallidum ( Tp ) breaches this barrier through elusive mechanisms and rapidly disseminates throughout the host. We aimed to understand the impact of Tp on the surrounding FN matrix of MEB and the underlying mechanisms of this effect. In this study, immunofluorescence assays (IF) were conducted to assess the integrity of the FN matrix surrounding human microvascular endothelial cell-1 (HMEC-1) with/without Tp co-culture, revealing that only live Tp exhibited the capability to mediate FN matrix disaggregation in HMEC-1. Western blotting and IF were employed to determine the protein levels associated with the FN matrix during Tp infection, which showed the unaltered protein levels of total FN and its receptor integrin α5β1, along with reduced insoluble FN and increased soluble FN. Simultaneously, the integrin α5β1-binding protein–intracellular vimentin maintained a stable total protein level while exhibiting an increase in the soluble form, specifically mediated by the phosphorylation of its 39 th residue (pSer39-vimentin). Besides, this process of vimentin phosphorylation, which could be hindered by a serine-to-alanine mutation or inhibition of phosphorylated-AKT1 (pAKT1), promoted intracellular vimentin rearrangement and FN matrix disaggregation. Moreover, within the introduction of additional cellular FN rather than other Tp -adhered ECM protein, in vitro endothelial barrier traversal experiment and in vivo syphilitic infectivity test demonstrated that viable Tp was effectively prevented from penetrating the in vitro MEB or disseminating in Tp -challenged rabbits. This investigation revealed the active pAKT1/pSer39-vimentin signal triggered by live Tp to expedite the disaggregation of the FN matrix and highlighted the importance of FN matrix stability in syphilis, thereby providing a novel perspective on ECM disruption mechanisms that facilitate Tp dissemination across the MEB. Treponema pallidum subsp . pallidum ( Tp ), dominating the global concerned disease—syphilis, can mediate various pathogenic processes through its dissemination, during which the vascular endothelial barrier plays a vital role in the pathogen-host interaction. Tp alters the characteristics of the barrier components, including vascular endothelial cell and the surrounding extracelluar matrix (ECM), while the initial step of Tp dissemination involves breaching the ECM and moving toward the surface within unknown mechanisms. Here, we demostrated that live Tp triggered the pAKT1/pSer39-vimentin signals in microvascular endothelial cells, which promoted the redistribution of vimentin around the perinucleus. And pSer39-vimentin facilitated the detachment of intergrein α5β1 from fibronectin (FN), which is an essential component of ECM for Tp adhesion. Furthermore, we discovered that pAKT1 inhibition or addition of cellular FN could delay the FN matrix disaggregation, ultimately preventing live Tp from traversing the microvascular endothelial barrier. These results provide insights into ECM disruption employed by Tp and identify potential therapeutic targets against syphilis.
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