SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

Abstract

The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE-and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE-and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL-and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCEMany bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into the host cells to cause disease. The secreted proteins perform different functions at various stages during infection and are classified into three substrate categories (T3SS components, translocators, and effectors). They all contain secretion signals at their N termini, but how their secretion hierarchy is determined is poorly understood. Here, we show that the N-terminal secretion signals from different substrate categories all behave the same and do not confer substrate specificity. We further characterize the secretion signals of the translocators and identify a translocator-specific signal, demonstrating that substrate-specific secretion signals are required in regulating T3SS substrate hierarchy. T he type III secretion system (T3SS) is one of the best-studied bacterial protein secretion systems and plays a central role in the virulence and pathogenesis of a large number of medically and agriculturally important bacterial pathogens, including Yersinia, Salmonella, Pseudomonas, Shigella, and Escherichia spp. (1-3). The T3SS consists of a series of mu...

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

“…To identify them, we generated whole and partial (i.e., encoding the large cytoplasmic domains of escV and escU) chromosomal gene deletions. None of the derivative strains secreted EspA in vivo ( Fig 3B; Appendix Fig S1A), in agreement with previous results (Dziva et al, 2004;O'Connell et al, 2004;Nadler et al, 2006;Deng et al, 2015), nor formed actin pedestals on HeLa cells (Appendix Fig S1D-L), an assay reporting effector injection (e.g., Tir) via the EspA filaments and the EspB/D translocon pore (Creasey et al, 2003).…”

“…We anticipate that the novel receptor role revealed here and analyzed in detail for CesAB might be universal for translocator chaperones, even those structurally distinct from CesAB [e.g., the CesD and CesD2 class III chaperones of EspB and D (Wainwright & Kaper, ; Neves et al , )]. This would be in agreement with, structures of gatekeepers co‐complexed with chaperone homologues (Archuleta & Spiller, ), pull‐down assays (Kubori & Galan, ; Silva‐Herzog et al , ), and the abolished secretion of all translocators in SepL, SepD, and CesL mutants (Fig EV5H; Wang et al , ; Younis et al , ; Deng et al , ). In this capacity, SepL may act indeed as an early pseudo‐effector (Younis et al , ), that occupies EscV, switches it to preferentially accepting translocators and blocks binding of all other effectors.…”

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

“…The chimeric proteins were expressed in WT EPEC (normal secretion), the Δ escN mutant (no secretion), and the Δ sepD and Δ sepL mutants (hypersecretion of effectors). Because many T3SS proteins containing a secretion signal exhibit significantly higher secretion in the Δ sepD and Δ sepL mutant strains (37, 49), we used these strains to enable the detection of low levels of secreted substrates.…”

“…SepL is a known member of the gatekeeper family of proteins, which includes YopN in Yersinia , InvE and SsaL in Salmonella , and MxiC in Shigella (7). SepD, on the other hand, is not well conserved in other pathogens, and as such, it has a single, functional homolog, SpiC, which was identified in Salmonella pathogenicity island 2 (36–38). In addition, the YscB protein was suggested to belong to the SepD family of proteins because of its analogous position within the Yersinia pathogenicity island (39).…”

Type III-Dependent Translocation of HrpB2 by a Nonpathogenic hpaABC Mutant of the Plant-Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria

“…Similarly, the secretion and translocation signal in the N-terminal 20 amino acids of translocon proteins from enteropathogenic E. coli is not sufficient to mediate translocon-specific secretion in mutant strains which lack the control proteins SepD and SepL (74)(75)(76)(77)(78). Therefore, it was suggested that T3S is controlled by composite signals, which are involved in a multistep recognition process (68,74,75). Notably, however, this model does not appear to apply to HrpB2, suggesting that the presence of additive export signals in T3S substrates is not a general rule.…”