Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins

YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU_CC. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca²⁺ conditions) and calcium-depleted medium (−Ca²⁺ conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca²⁺-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca²⁺ conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca²⁺ conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.The type III secretion system (T3SS) occurs in many gram-negative pathogenic or symbiotic bacteria (6, 16, 19). The T3SS is evolutionarily related to the bacterial flagellum (19, 24), but while the flagellar apparatus is dedicated to bacterial motion, the T3SS specifically allows bacterial targeting of effector proteins across eukaryotic cell membranes into the lumen of the target cell (19). The main function of the effectors is to reprogram the cell to the benefit of the bacterium (28). The two organelles are superficially similar in form and can be divided into two physical substructures; a basal body is connected to a multimeric filamentous protein structure protruding from the bacterial surface. The basal body is embedded in the cell wall and spans from the cytosol to the surface of the bacterium with a cytosolic extension called the C-ring. The proximal center of the basal body is likely involved in the actual export of nonfolded substrates, which are thought to pass through the cell wall through this hollow structure (6, 16, 41). Early and elegant work by Macnab''s group showed that morphogenesis of the flagella is ordered such that first the cell-proximal hook structure is polymerized and then the flagellar filament is assembled on top of the hook structure (43). Thus, there is ordered switching from secretion of hook proteins to flagellin, which was called substrate specificity switching by Macnab et al. (15, 27). Mutants expressing extraordinarily long hooks have been isolated and connected to regulation and determination of hook buildup and subsequent substrate specificity switching (18, 29, 43). A central factor in this process is the integral 42-kDa cytoplasmic membrane protein FlhB, which has four putative transmembrane helices in its N-terminal domain, which is designated FlhB_TM. The hydrophilic C-terminal domain (FlhB_C) is predicted to protrude into the cytosol. In addition, FlhB_C can be further divided into two subdomains, FlhB_CN (amino acids 211 to 269) and FlhB_CC (amino acis 270 to 383), that are connected via a proposed flexible hinge region (27). The hinge region contains a highly conserved NPTH motif, which is found in all T3SSs. Interestingly, FlhB_C is specifically cleaved within this NPTH sequence (N269↓P270) (27). Site-specific mutagenesis of the NPTH site has a significant effect on the substrate switching, and the ability of flhB(N269A) and flhB(P270A) mutants to cleave FlhB is impaired, indicating that autoproteolysis is important (13, 15). Interestingly, the proteolysis is most likely the outcome of an autochemical process rather than an effect of external proteolytic enzymes (13). The FlhB homolog in the Yersinia pseudotuberculosis plasmid-encoded T3SS is the YscU protein, which has been shown to be essential for proper function of the T3SS since a yscU-null mutant is unable to secrete Yop proteins (Yops) into the culture supernatant (1, 21). YscU has been coupled to needle and Yop secretion regulation, as second-site suppressor mutations introduced into YscU_CC restore the yscP-null mutant phenotype. A yscP mutant is unable to exhibit substrate specificity switching and carries excess amounts of the needle protein YscF on the bacterial surface compared to the wild type. (11) Furthermore, YscP has been implicated in regulation of the T3SS needle length as a molecular ruler, where the size and helical content of YscP determine the length of the needle (20, 42). Together, these findings suggest that YscP and YscU interact and that this interaction is important for regulation of needle length, as well as for Yop secretion. As in FlhB, four predicted transmembrane helices followed by a cytoplasmic tail can be identified in YscU (1). In addition, the cytoplasmic part (YscU_C) can be divided into the YscU_CN and YscU_CC subdomains (Fig. ​(Fig.1A).1A). Variants of YscU with a single substitution in the conserved NPTH sequence (N263A) have been found to be unable to generate YscU_CC, suggesting that YscU of Yersinia also is autoproteolysed (21, 33, 38). The T3SS of Y. pseudotuberculosis secretes about 11 proteins, which collectively are called Yops (Yersinia outer proteins). These Yops have different functions during infection. Some are directly involved as effector proteins, attacking host cells to prevent phagocytosis and inflammation, while others have regulatory functions. Although the pathogen is extracellularly located, the Yop effectors are found solely in the cytosol of the target cell, and secretion of Yops occurs only at the zone of contact between the pathogen and the eukaryotic target cell (7, 36). Close contact between the pathogen and the eukaryotic cell also results in elevated expression and secretion of Yops (12, 30). Hence, cell contact induces the substrate switching; therefore, here we studied the connection between YscU autoproteolysis and expression, as well as secretion and translocation of Yops. Previous studies of YscU function were conducted mainly with in trans constructs instead of introduced YscU mutations in cis. Such studies reported loss of T3SS regulation (21). To avoid potential in trans problems, we introduced all mutations in cis with the aim of elucidating the function of YscU in type III secretion (T3S). Our results suggest that YscU autoproteolysis is not an absolute requirement either for Yop/LcrV secretion or for Yop translocation but is important for accurate regulation of Yop expression and secretion.Open in a separate windowFIG. 1.Autoproteolysis of YscU. (A) Schematic diagram of YscU in the bacterial inner membrane. The diagram shows the NPTH motif and the different parts of YscU after autoproteolysis and is the result of a prediction of transmembrane helices in proteins performed at the site http://www.cbs.dtu.dk/services/TMHMM. IM, inner membrane. (B) E. coli expressing C-terminally His-tagged YscU_C was induced with IPTG, which was followed by sonication and solubilization and denaturation of the protein in binding buffer (8 M urea and 10 mM imidazole). The lysate (lane L) was flushed over the Ni column, and the flowthrough (lane FT) was collected. The column was washed five times with binding buffer, and the wash fractions (lanes W1 to W5) were collected. Elution buffer (8 M urea and 300 mM imidazole) was flushed over the column to release proteins bound to the column, resulting in the eluate (lane E). The eluate was diluted 1:30 in 10 mM Tris (pH 7.4) to obtain a urea concentration of 0.2 M and incubated at 21°C overnight. The resulting overnight eluate fraction (lane E/ON) was TCA precipitated and taken up in binding buffer. Samples were analyzed by 15% Tris-Tricine SDS-PAGE. The cleavage of YscU_C-His₆ to YscU_CC-His₆ and YscU_CN was verified by N-terminal sequencing. All fractions were volume corrected. Lane ST contained a protein standard.