EscO, a Functional and Structural Analog of the Flagellar FliJ Protein, Is a Positive Regulator of EscN ATPase Activity of the Enteropathogenic Escherichia coli Injectisome

Abstract

Type III secretion systems (T3SSs) are multiprotein molecular devices used by many Gram-negative bacterial pathogens to translocate effector proteins into eukaryotic cells. A T3SS is also used for protein export in flagellar assembly, which promotes bacterial motility. The two systems are evolutionarily related, possessing highly conserved components in their export apparatuses. Enteropathogenic Escherichia coli (EPEC) employs a T3SS, encoded by genes in the locus of enterocyte effacement (LEE) pathogenicity island, to colonize the human intestine and cause diarrheal disease. In the present work, we investigated the role of the LEE-encoded EscO protein (previously Orf15 or EscA) in T3SS biogenesis. We show that EscO shares similar properties with the flagellar FliJ and the Yersinia YscO protein families. Our findings demonstrate that EscO is essential for secretion of all categories of T3SS substrates. Consistent with its central role in protein secretion, it was found to interact with the ATPase EscN and its negative regulator, EscL, of the export apparatus. Moreover, we show that EscO stimulates EscN enzymatic activity; however, it is unable to upregulate ATP hydrolysis in the presence of EscL. Remarkably, EscO partially restored the swimming defect of a Salmonella flagellar fliJ mutant and was able to stimulate the ATPase activity of FliI. Overall, our data indicate that EscO is the virulence counterpart of the flagellar FliJ protein.

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome

“…It has been reported that functionally related T3SS-associated proteins, despite not having significant sequence similarity, are encoded by genes with similar genetic contexts (32,57,66,67). Our BLAST search identified multiple uncharacterized EscK homologs encoded by genes sharing the same context as that for genes coding for members of the SctK family (YscK, MxiK, and OrgA) (68).…”

“…The escK gene is located downstream of the cesAB gene, encoding a chaperone for the EspA and EspB translocators (50), and upstream of escL, encoding a member of the SctL protein family that acts as a negative regulator of EscN ATPase activity (32). Likewise, the genes located downstream of the identified escK homologs (Fig.…”

“…The export apparatus is composed of a set of integral membrane proteins (EscR, EscS, EscT, EscU, and EscV) essential for protein secretion, which are embedded in the cytoplasmic membrane and surrounded by the inner membrane ring (17,29). The cytoplasmic components include an ATPase complex formed by the ATPase EscN and negative (EscL) and positive (EscO) regulators of EscN ATPase activity, which act in concert to ensure optimal coupling of energy derived from ATP hydrolysis to protein secretion (30)(31)(32). Finally, a ring-shaped structure termed the C-ring, composed of multiple copies of the EscQ protein, is docked at the cytoplasmic interface of the basal body ( Fig.…”

The Salmonella Type III Secretion System Virulence Effector Forms a New Hexameric Chaperone Assembly for Export of Effector/Chaperone Complexes

“…A key component of the export apparatus is a cytoplasmic nonameric ring structure called the export gate, which localizes to the base of the basal body and is proposed to sequester effectors prior to secretion (3). The ATPase (called EscN in EPEC) forms complexes with its positive and negative regulators, EscO and EscL, respectively (4). EscN is one of the best-characterized T3SS ATPases and is suggested to form a hexameric ring structure, analogous to that of the F o F 1 -ATPase, with ATP bound between adjacent monomers (5).…”

“…The peripheral stalk, EscL, inhibits the EscN ATPase activity and may form a trimeric heterocomplex with EscN and EscO, resembling the homologous flagellar FliH-FliI-FliJ complex (19). Based on the structural and functional similarities of the T3SS and flagellar ATPase complexes, EscL is predicted to bind the N-terminal region of EscN, while EscO binds the C-terminal region of EscN (4,20). EscL and EscO are linked to the export gate while holding the EscN ATPase far from the export gate in an export-off state (6) (Fig.…”