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

Abstract

The type III secretion system (T3SS) is a supramolecular machine used by many bacterial pathogens to translocate effector proteins directly into the eukaryotic host cell cytoplasm. Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrheal disease in underdeveloped countries. EPEC virulence relies on a T3SS encoded within a chromosomal pathogenicity island known as the locus of enterocyte effacement (LEE). In this work, we pursued the functional characterization of the LEE-encoded protein EscK (previously known as Orf4). We provide evidence indicating that EscK is crucial for efficient T3S and belongs to the SctK (OrgA/YscK/MxiK) protein family, whose members have been implicated in the formation of a sorting platform for secretion of T3S substrates. Bacterial fractionation studies showed that EscK localizes to the inner membrane independently of the presence of any other T3SS component. Combining yeast two-hybrid screening and pulldown assays, we identified an interaction between EscK and the C-ring/sorting platform component EscQ. Site-directed mutagenesis of conserved residues revealed amino acids that are critical for EscK function and for its interaction with EscQ. In addition, we found that T3S substrate overproduction is capable of compensating for the absence of EscK. Overall, our data suggest that EscK is a structural component of the EPEC T3SS sorting platform, playing a central role in the recruitment of T3S substrates for boosting the efficiency of the protein translocation process.IMPORTANCE The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

Novel insights into the mechanism of SepL‐mediated control of effector secretion in enteropathogenic Escherichia coli

“…SepL binding to the Ni‐NTA resin was not detected (Figure a lower panel). As an additional control, we showed that under the conditions tested, His‐EscVc does not bind to MBP‐EscK (data not shown), as previously observed in a yeast two hybrid screen (Soto et al., ), corroborating the specificity of the EscV‐SepL interaction.…”

“…The export apparatus resides within the inner membrane ring and is formed by five highly conserved proteins, named EscR, EscS, EscT, EscU, and EscV (Moraes, Spreter, & Strynadka, ). The cytoplasmic side of the basal body is associated to a ring‐shaped oligomeric structure (EscQ and EscK), which functions as a substrate recognition platform, and provides a docking site for the ATPase complex (EscN, EscO, and EscL) (Andrade, Pardo, Espinosa, Perez‐Hernandez, & Gonzalez‐Pedrajo, ; Biemans‐Oldehinkel, Sal‐Man, Deng, Foster, & Finlay, ; Romo‐Castillo et al., ; Soto et al., ; Zarivach et al., ). Moreover, the extracellular part of the injectisome comprises a 23 nm in length needle‐like structure (EscF), which is extended by a long filament (EspA) (Knutton et al., ; Monjaras Feria et al., ; Ogino et al., ; Sekiya et al., ; Wilson, Shaw, Daniell, Knutton, & Frankel, ).…”

A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome

“…SctK / YscK is the least studied of the four soluble T3SS components, and is the only protein with no clear flagellar homologue. SctK/YscK localizes to the membrane in E. coli , independent of the presence of other T3SS components, and is required for the efficient export of effectors 21 , but its exact role in secretion remains unclear. In vitro experiments have established a chain of interactions Sct/YscK-Q-L-N 19 22 23 , and we will refer to these four core proteins as ‘soluble T3SS components’.…”

Two Type VI Secretion Systems of Enterobacter cloacae Are Required for Bacterial Competition, Cell Adherence, and Intestinal Colonization

“…The quantification of bacterial hemolysis was performed as described ( Soto et al, 2017 ). E. cloacae strains were grown in TSB until the OD 600 nm = 1.0, and 0.5 ml culture was added to 0.5 ml of a 4% ( v / v ) red blood cells (RBC)/saline solution (0.9% NaCl), and centrifuged at 2500 × g for 1 min.…”