A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration

Abstract

Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration.

Pathogen-Mediated Inhibition of Anorexia Promotes Host Survival and Transmission

“…Thus SlrP does not inhibit anorexia by alleviating tissue damage in target organs of this pathogen. SlrP has been shown in in vitro assays to inhibit dendritic cell (DC) migration (McLaughlin et al, 2014). We found no difference in the levels of DCs or other immune cells associated with ST infection or intestinal inflammation in the LP, MLNs, liver or spleen between 0–24 and 24–48hrs post-infection, indicating that SlrP does not inhibit anorexia by limiting immune cell infiltration (Figure 3D, E and S2B–H).…”

Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis

“…The CCC is flanked by two arrays of micro-capillaries, which increase fluidic resistance and prevent fluid flow and the generation of shear forces within the CCC 28 . The absence of fluid flow within the CCC makes it easy to adapt the device to observe the behavior of loosely adherent cells 29,35 or cells in 3D matrices 36,37 . Channels on either side of the arrays are perfused with medium, such that the direction of fluid flow runs perpendicular to the micro-capillaries.…”

Salmonellae interactions with host processes

“…Another SPI-1/2 effector, SteA elicits changes in the expression of several genes in HeLa cells, including the activation of genes that regulate extracellular matrix organization, cell proliferation and serine/threonine kinase signaling pathways; and the repression of genes involved in regulating immune processes, cell death, adhesion and migration 72 , which suggests that this effector can also modulate inflammation in the host. The effectors SseF, SifA, SspH2, SlrP, PipB2 and SseI can inhibit the migration of dendritic cells 73 , and several of these effectors (SspH2, SlrP, PipB2, SifA, SopD2 and SseFG) also inhibit antigen presentation by dendritic cells and inhibit T-cell proliferation 74 . Collectively, the results of these studies suggest that both the innate and adaptive immune response of the host is activated and inhibited during salmonellae infection.…”