Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

Koshi Imami,Amit P Bhavsar,Hongbing Yu,Nat F Brown,Lindsay D Rogers,B Brett Finlay,Leonard J Foster

doi:10.1074/mcp.m112.026161

Abstract

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.

Highlights

Type III secretion systems (T3SSs)1 are specialized virulence factors in Gram-negative pathogens that play an impor
Quantitative Phosphoproteome and Proteome Profiling of the Host Cells Modulated by Salmonella pathogenicity island 2 (SPI2) Effectors—Intracellular replication of the SPI2 mutant (⌬ssaR [26]) strain was seen to decrease in macrophages (RAW264.7) more than 90% compared with the wild-type (WT) SL1344 strain [5]
The majority of the molecular interactions between SPI2 effectors and host targets has yet to be defined and will be the subject of future investigations, in this study we have revealed a landscape of key host players and their phosphorylation sites involving particular cellular processes/pathways altered by SPI2 effectors (Fig. 2 and supplemental Fig. S3)

Summary

The abbreviations used are

T3SS, type III secretion system; SPI, Salmonella pathogenicity island; SCV, Salmonella-containing vacutant role in delivering effector proteins to host cells. SteC is capable of promoting assembly of an F-actin meshwork around the SCV; this is dependent on its kinase activity but does not require activation of signaling pathways through Rho-associated protein kinase [8], Cdc, Rac, N-WASP, Scar/WAVE, and Arp2/3 [10]. These host signaling proteins are the main targets of T3SS-secreted effectors from many pathogens, including the SPI1 system in Salmonella [11] and Shigella [12]. We explore a molecular target of SPI2 effector kinase SteC by integrating the phosphoproteomics data and other quantitative proteomics screens

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION