Functional Characterization of the Type III Secretion ATPase SsaN Encoded by Salmonella Pathogenicity Island 2

Yukie Yoshida,Nobuhiko Okada,Takeshi Haneda,Masahiro Ito,Sayaka Ono,Tsuyoshi Miki,Eric Cascales

doi:10.1371/journal.pone.0094347

Yukie Yoshida, Nobuhiko Okada + Show 5 more

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https://doi.org/10.1371/journal.pone.0094347

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Journal: PloS one	Publication Date: Apr 10, 2014
Citations: 82	License type: CC BY 4.0

Affiliation: Kitasato University

Abstract

A type III secretion system (T3SS) is utilized by a large number of gram-negative bacteria to deliver effectors directly into the cytosol of eukaryotic host cells. One essential component of a T3SS is an ATPase that catalyzes the unfolding of proteins, which is followed by the translocation of effectors through an injectisome. Here we demonstrate a functional role of the ATPase SsaN, a component of Salmonella pathogenicity island 2 T3SS (T3SS-2) in Salmonella enterica serovar Typhimurium. SsaN hydrolyzed ATP in vitro and was essential for T3SS function and Salmonella virulence in vivo. Protein-protein interaction analyses revealed that SsaN interacted with SsaK and SsaQ to form the C ring complex. SsaN and its complex co-localized to the membrane fraction under T3SS-2 inducing conditions. In addition, SsaN bound to Salmonella pathogenicity island 2 (SPI-2) specific chaperones, including SsaE, SseA, SscA, and SscB that facilitated translocator/effector secretion. Using an in vitro chaperone release assay, we demonstrated that SsaN dissociated a chaperone-effector complex, SsaE and SseB, in an ATP-dependent manner. Effector release was dependent on a conserved arginine residue at position 192 of SsaN, and this was essential for its enzymatic activity. These results strongly suggest that the T3SS-2-associated ATPase SsaN contributes to T3SS-2 effector translocation efficiency.

Highlights

A number of gram-negative pathogenic bacteria utilize a type III secretion system (T3SS) for their interactions with eukaryotic host cells
Typhimurium is predominantly attributed to horizontally acquired genomic islands, termed as Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2), which encode for different T3SSs
SsaN is crucial for SPI-2 effector protein translocation In the T3SS-2 locus of S

Summary

Introduction

A number of gram-negative pathogenic bacteria utilize a type III secretion system (T3SS) for their interactions with eukaryotic host cells. T3SS delivers bacterial effectors through a needle-like structure that extends across the inner and outer membranes of a bacterium and into the cytosol of eukaryotic cells [1,2]. Typhimurium) is an enteropathogenic bacterium that causes gastroenteritis in humans and typhoid-like fever in mice. Typhimurium is predominantly attributed to horizontally acquired genomic islands, termed as Salmonella pathogenicity island 1 (SPI-1) and SPI-2, which encode for different T3SSs. SPI-1 T3SS (T3SS-1) facilitates host cell invasion and inflammation [3,4], whereas SPI-2 T3SS (T3SS-2) mediates intracellular survival and immune evasion [5,6]

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