Search

The intermediate filament vimentin is present in immune cells and is implicated in proinflammatory immune responses. Whether and how it supports antimicrobial activities of neutrophils are not well established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts the production of proinflammatory cytokines and reactive oxygen species (ROS), but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and inflammasome activation. Moreover, gene expression analysis demonstrated that the presence of vimentin was associated with changes in expression of multiple genes required for mitochondrial function and ROS overproduction. Treatment of wild-type cells with rotenone, an inhibitor for complex I of the electron transport chain, increases the ROS levels. Likewise, treatment with mitoTEMPO, a SOD mimetic, rescues the ROS production in cells lacking vimentin. Together, these data show vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity. [ABSTRACT FROM AUTHOR]

Type 3 secretion systems (T3SSs) enable bacterial virulence by translocating virulence proteins (effectors) into host cells. Shigella flexneri require T3SS to invade and to spread between cells in the colon. In order to spread, S. flexneri forms membrane protrusions that push into the adjacent host cell. These protrusions are resolved into double membrane vacuoles (DMVs) that the bacteria quickly escape. The mechanisms required for escape from the DMV are poorly understood, but the T3SS translocon pore protein IpaC is essential. Here, we show IpaC forms a pore that is competent for translocation of T3SS effectors as bacteria spread between cells. To do so, we used a genetic approach to test mutations of IpaC that disrupt its ability to translocate and to form pores. We show that during spread, IpaC is efficiently inserted into the plasma membrane, the membrane-embedded IpaC forms pore complexes, and the IpaC-dependent pores translocate effectors that are necessary for S. flexneri to escape the DMV. We further show that T3SS activation is regulated through a distinct mechanism at spread compared to at invasion; activation of T3SS secretion does not require pore formation during spread. Thus, we show that a distinct regulation of the T3SS during S. flexneri intercellular spread enables the placement of effectors both around S. flexneri and across membranes of the DMV. Altogether, this study provides new insights into how S. flexneri escapes the DMV., Importance: The type 3 secretion system (T3SS) is required for virulence in many bacterial pathogens that infect humans. The T3SS forms a pore through which virulence proteins are delivered into host cells to enable bacterial infection. Our work investigates the Shigella translocon pore protein IpaC, which is essential not only for bacteria to invade cells, but also for bacteria to spread between cells. An ability to spread between cells is essential for pathogenesis, thus understanding the mechanisms that enable spread is important for understanding how S. flexneri infection causes illness. We show that IpaC delivers virulence factors across the host membrane for S. flexneri to efficiently spread. This study furthers our understanding of the mechanisms involved in T3SS secretion and of translocon pore function during S. flexneri intercellular spread.

The intermediate filament vimentin is present in immune cells and is implicated in proinflammatory immune responses. Whether and how it supports antimicrobial activities of neutrophils are not well established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts the production of proinflammatory cytokines and reactive oxygen species (ROS), but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and inflammasome activation. Moreover, gene expression analysis demonstrated that the presence of vimentin was associated with changes in expression of multiple genes required for mitochondrial function and ROS overproduction. Treatment of wild-type cells with rotenone, an inhibitor for complex I of the electron transport chain, increases the ROS levels. Likewise, treatment with mitoTEMPO, a SOD mimetic, rescues the ROS production in cells lacking vimentin. Together, these data show vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity., Competing Interests: MM consults for NED Biosystems, Vericel. He received grants from Karius, Danaher, Genentech, and Thermo-Fisher Scientific. He is a medical writer for UpToDate. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Huynh, Toperzer, Scherer, Gumina, Brunetti, Mansour, Markovitz and Russo.)