Salmonella Effectors SseF and SseG Interact with Mammalian Protein ACBD3 (GCP60) To Anchor Salmonella-Containing Vacuoles at the Golgi Network.

Xiu-Jun Yu,Mei Liu,David W Holden,Scott J Hultgren

doi:10.1128/mbio.00474-16

Abstract

ABSTRACTFollowing infection of mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) replicates within membrane-bound compartments known as Salmonella-containing vacuoles (SCVs). The Salmonella pathogenicity island 2 type III secretion system (SPI-2 T3SS) translocates approximately 30 different effectors across the vacuolar membrane. SseF and SseG are two such effectors that are required for SCVs to localize close to the Golgi network in infected epithelial cells. In a yeast two-hybrid assay, SseG and an N-terminal variant of SseF interacted directly with mammalian ACBD3, a multifunctional cytosolic Golgi network-associated protein. Knockdown of ACBD3 by small interfering RNA (siRNA) reduced epithelial cell Golgi network association of wild-type bacteria, phenocopying the effect of null mutations of sseG or sseF. Binding of SseF to ACBD3 in infected cells required the presence of SseG. A single-amino-acid mutant of SseG and a double-amino-acid mutant of SseF were obtained that did not interact with ACBD3 in Saccharomyces cerevisiae. When either of these was produced together with the corresponding wild-type effector by Salmonella in infected cells, they enabled SCV-Golgi network association and interacted with ACBD3. However, these properties were lost and bacteria displayed an intracellular replication defect when cells were infected with Salmonella carrying both mutant genes. Knockdown of ACBD3 resulted in a replication defect of wild-type bacteria but did not further attenuate the growth defect of a ΔsseFG mutant strain. We propose a model in which interaction between SseF and SseG enables both proteins to bind ACBD3, thereby anchoring SCVs at the Golgi network and facilitating bacterial replication.

Highlights

Following infection of mammalian cells, Salmonella enterica serovar Typhimurium
Upon invasion of epithelial cells, the majority of Salmonella-containing vacuoles (SCVs) migrate to the perinuclear region-located microtubule-organizing center (MTOC) and Golgi network and remain in this region of the cell during the first few rounds of bac
Their SCVs move erratically throughout the cytoplasm of infected cells, resulting in a scattered phenotype [9, 12, 13]. This phenotype suggests that SseF and SseG function by tethering SCVs to a Golgi network-associated compartment and/or by modulating the activities of microtubule motors, which are known to interact with SCVs [10, 11, 14, 19, 20], in a way that favors minus-end-directed dynein activity on SCVs

Summary

Introduction

Following infection of mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) replicates within membrane-bound compartments known as Salmonella-containing vacuoles (SCVs). IMPORTANCE Upon invasion of epithelial cells, the majority of vacuoles containing Salmonella enterica migrate to the perinuclear region-located Golgi network and remain in this region of the cell during the first few rounds of bacterial replication, forming a clustered microcolony of vacuoles This process requires the action of SseF and SseG, two effector proteins that are translocated by the Salmonella SPI-2 type III secretion system. The second hypothesis proposes that SCV positioning is controlled by physical tethering of SCV membranes to Golgi networkassociated molecules [9, 13] Whatever their precise mechanism(s), SseF and SseG have key functions in regulating SCV localization: imaging of SCVs in live epithelial cells at approximately 8 h postinvasion revealed that Golgi network-associated vacuoles containing wild-type bacteria are relatively immobile, whereas those containing sseG mutant bacteria are highly motile and display erratic large-scale movements throughout host cells [13]. Close apposition of vacuoles with Golgi membranes could facilitate fusion of Golgi network-derived material with SCVs and thereby provide a source of SCV membrane or proteins, lipids, or other molecules that could be exploited by bacteria for nutrition

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