Role of EscP (Orf16) in Injectisome Biogenesis and Regulation of Type III Protein Secretion in Enteropathogenic Escherichia coli

Abstract

bEnteropathogenic Escherichia coli employs a type III secretion system (T3SS) to translocate virulence effector proteins directly into enterocyte host cells, leading to diarrheal disease. The T3SS is encoded within the chromosomal locus of enterocyte effacement (LEE). The function of some of the LEE-encoded proteins remains unknown. Here we investigated the role of the Orf16 protein in T3SS biogenesis and function. An orf16 deletion mutant showed translocator and effector protein secretion profiles different from those of wild-type cells. The orf16 null strain produced T3S structures with abnormally long needles and filaments that caused weak hemolysis of red blood cells. Furthermore, the number of fully assembled T3SSs was also reduced in the orf16 mutant, indicating that Orf16, though not essential, is required for efficient T3SS assembly. Analysis of protein secretion revealed that Orf16 is a T3SS-secreted substrate and regulates the secretion of the inner rod component EscI. Both pulldown and yeast two-hybrid assays showed that Orf16 interacts with the C-terminal domain of an inner membrane component of the secretion apparatus, EscU; the inner rod protein EscI; the needle protein EscF; and the multieffector chaperone CesT. These results suggest that Orf16 regulates needle length and, along with EscU, participates in a substrate specificity switch from early substrates to translocators. Taken together, our results suggest that Orf16 acts as a molecular measuring device in a way similar to that of members of the Yersinia YscP and flagellar FliK protein family. Therefore, we propose that this protein be renamed EscP.

The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL

“…We observed that deletion of the escP gene results in the unregulated secretion of translocator and effector proteins, but transformation of the mutant with the pEscP-Flag vector fully restored T3SS secretion regulation. Similar results were obtained for an EscP protein labeled with a double HA tag (2HA; data not shown) and are consistent with previously reported data (42). Download .…”

“…Similar to the results obtained by Monjarás Feria et al, the Δ escP mutant EPEC strain grown in regular DMEM (1.8 mM CaCl 2 ) showed lower translocator and higher effector secretion levels than WT EPEC (Fig. 2A) (42). Interestingly, the secretion profile of the escP null mutant in regular DMEM with 1.8 mM CaCl 2 was strikingly similar to that of WT EPEC grown in calcium-free DMEM.…”

“…In this study, we found that the EPEC protein EscP, which was previously suggested to function as the ruler protein and is involved in the first substrate specificity switch (42), is also involved in regulation of the second substrate specificity switch. We showed that the EPEC Δ escP mutant is calcium blind and its type III secretion (T3S) protein profile exactly resembles that of wild-type (WT) EPEC grown in Ca 2+ -free medium.…”

The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing

“…Structure of the P. aeruginosa Autoprotease PscU-The inner membrane-spanning T3SS autoprotease has also been implicated in substrate switching, and a direct interaction with the ruler protein has been reported in the flagellum and in the enteropathogenic E. coli and Shigella T3SS systems (14,58,59). We therefore purified the cytoplasmic domain from the P. aeruginosa autoprotease PscP.…”

“…The molecular mechanism of a substrate specificity switch on the cytosolic side remains to be elucidated. The T3SS rod protein (YscI, MxiI, PrgJ, and EscI in the Yersinia T3SS, Shigella T3SS, Salmonella SPI-1 T3SS, and enteropathogenic E. coli T3SS, respectively) has been implicated in this process, and direct interaction between the autoprotease and rod has been reported in a number of systems (14,(71)(72)(73)(74). However, no obvious rod homologue is found in the flagellum, where four proteins (FlgB, -C, -F, and -G) have been proposed to form a rodlike structure but have not been linked to substrate switching.…”