Live Cell Imaging Reveals Novel Functions of Salmonella enterica SPI2-T3SS Effector Proteins in Remodeling of the Host Cell Endosomal System

Abstract

Intracellular Salmonella enterica induce a massive remodeling of the endosomal system in infected host cells. One dramatic consequence of this interference is the induction of various extensive tubular aggregations of membrane vesicles, and tubules positive for late endosomal/lysosomal markers are referred to as Salmonella-induced filaments or SIF. SIF are highly dynamic in nature with extension and collapse velocities of 0.4-0.5 mm x sec(-1). The induction of SIF depends on the function of the Salmonella Pathogenicity Island 2 (SPI2) encoded type III secretion system (T3SS) and a subset of effector proteins. In this study, we applied live cell imaging and electron microscopy to analyze the role of individual effector proteins in SIF morphology and dynamic properties of SIF. SIF in cells infected with sifB, sseJ, sseK1, sseK2, sseI, sseL, sspH1, sspH2, slrP, steC, gogB or pipB mutant strains showed a morphology and dynamics comparable to SIF induced by WT Salmonella. SIF were absent in cells infected with the sifA-deficient strain and live cell analyses allowed tracking of the loss of the SCV membrane of intracellular sifA Salmonella. In contrast to analyses in fixed cells, in living host cells SIF induced by sseF- or sseG-deficient strains were not discontinuous, but rather continuous and thinner in diameter. A very dramatic phenotype was observed for the pipB2-deficient strain that induced very bulky, non-dynamic aggregations of membrane vesicles. Our study underlines the requirement of the study of Salmonella-host interaction in living systems and reveals new phenotypes due to the intracellular activities of Salmonella.