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

RESEARCH ARTICLE

crossm Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome Eduardo Soto,a Norma Espinosa,a Miguel Díaz-Guerrero,a Meztlli O. Gaytán,a José L. Puente,b Bertha González-Pedrajoa Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexicoa; Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexicob

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.

Received 19 July 2016 Accepted 3 October 2016 Accepted manuscript posted online 17 October 2016 Citation Soto E, Espinosa N, Díaz-Guerrero M, Gaytán MO, Puente JL, González-Pedrajo B. 2017. Functional characterization of EscK (Orf4), a sorting platform component of the enteropathogenic Escherichia coli injectisome. J Bacteriol 199:e00538-16. https://doi.org/ 10.1128/JB.00538-16. Editor Ann M. Stock, Rutgers University-Robert Wood Johnson Medical School Copyright © 2016 American Society for Microbiology. All Rights Reserved. Address correspondence to Bertha GonzálezPedrajo, [email protected].

IMPORTANCE The type III secretion system (T3SS) is an essential virulence determi-

nant 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. KEYWORDS enteropathogenic Escherichia coli, injectisome, sorting platform, type III

secretion system

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rotein secretion is particularly important for bacterial manipulation and colonization of certain ecological niches. Many diderm bacteria employ a sophisticated molecular device, known as a type III secretion system (T3SS) or injectisome, to translocate virulence proteins, called effectors, directly from the bacterial cytoplasm January 2017 Volume 199 Issue 1 e00538-16

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into eukaryotic cells (1, 2). Injected effectors exhibit a wide arsenal of biochemical activities in order to modulate diverse cellular functions of the eukaryotic host to the benefit of the bacterium (3). Hence, the T3SS is a key element in the virulence strategy adopted by multiple pathogenic bacteria. Enteropathogenic Escherichia coli (EPEC) is one of the most common etiological agents of infantile diarrheal disease in developing countries, where it remains a significant health threat due to poor sanitation services (4, 5). Once ingested, EPEC colonizes the human small intestine, producing a distinctive histological injury known as an attaching and effacing (A/E) lesion (6). The A/E phenotype is characterized by intimate adherence of the bacterium to the intestinal epithelium, large rearrangements of the enterocyte cytoskeleton leading to the destruction of surrounding microvilli, and the subsequent formation of a protruding pedestal structure underneath the bacterial attachment site (7, 8). EPEC virulence relies on a T3SS to deliver a repertoire of effector proteins into host cells (9). All the components needed to assemble the T3SS, as well as the effectors essential for A/E lesion formation, are encoded in a chromosomal pathogenicity island named the locus of enterocyte effacement (LEE) (10–14). Several other T3SS-translocated effectors encoded outside this island, termed non-LEEencoded effectors (Nle), also contribute to EPEC pathogenicity (15–17). The LEE island is also present in a family of related enteric pathogens that cause the same type of lesion (A/E pathogens), such as enterohemorrhagic Escherichia coli (EHEC) and the murine pathogen Citrobacter rodentium (18, 19). The EPEC injectisome can be divided into four major structural parts: an extracellular hollow needle-filament structure, a multiring basal body, an export apparatus, and cytoplasmic protein complexes. The needle is formed by the helical polymerization of the EscF protein and is further extended by a filament consisting of subunits of the hydrophilic translocator protein EspA (20, 21). The EspA filament serves as a scaffold for the assembly of the hydrophobic translocator proteins, EspB and EspD, which form the translocation pore in the host cell membrane (22). These components provide a continuous channel for protein translocation. The basal body spans the space between the outer and inner bacterial membranes and is formed by the annular oligomerization of the EscC protein in the outer membrane (OM) (23, 24) and the EscD and EscJ proteins in the inner membrane (IM) (Fig. 1) (25–27). The OM and IM rings are connected through a periplasmic inner rod formed by the EscI protein (28). 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 (30–32). 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. 1) (33–35). Throughout this work, we also use the T3SS unified nomenclature Sct (prefix standing for secretion and cellular translocation) (36) to facilitate direct comparison of proteins between species. The virulence T3SS is evolutionarily related to the bacterial flagellar system (37). A recent phylogenetic analysis revealed that the injectisome originated from an ancient flagellar system by an exaptation process, i.e., recruiting core components from a rotary engine used for bacterial propulsion into a machine employed for interkingdom protein transport (38). Among the components of these machineries that preserve an evolutionary footprint in their sequences are the export apparatus proteins EscR, EscS, EscT, EscU, and EscV (FliP, FliQ, FliR, FlhB, and FlhA, respectively, in the flagellum), the inner membrane ring protein EscJ (FliF), the cytosolic ATPase complex components EscL and EscN (FliH and FliI), and the C-ring major component EscQ (FliM and FliN) (39). The flagellar C-ring is composed of three proteins, FliG, FliM, and FliN, and has been involved in protein export, torque generation, and motor rotation switching (40–42). In the virulence T3SSs, members of the SctQ family share sequence similarity with both FliM and FliN proteins, although, to date, it is not clear whether a counterpart of FliG January 2017 Volume 199 Issue 1 e00538-16

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Enteropathogenic Escherichia coli EscK


FIG 1 Schematic representation of basal components of the EPEC injectisome. The EscK, EscQ, and EscL proteins forming the cytoplasmic sorting platform are indicated in bold. OM, outer membrane; IM, inner membrane.

exists in the injectisome. The flagellar C-ring is a stable substructure tightly bound to the basal body MS ring (FliF) through a direct interaction with FliG (43–45). In contrast, the injectisome C-ring is a less robust structure with a highly dynamic behavior (46, 47). Recently, this cytoplasmic complex was visualized by cryo-electron tomography of Shigella minicells, revealing that it is formed by six pods of the Spa33 (SctQ) protein radially arranged at the base of the injectisome. The C-ring appears to be connected, through a series of linkers formed by the MxiN (SctL) protein, to a central hub proposed to be the Spa47 ATPase (SctN) (48). For EPEC, the EscQ protein has been proposed to be the major component of the C-ring, and this protein was shown to interact with EscN and EscL (34). The molecular mechanisms that enable the injectisome to discern which specific proteins are recognized among the myriad T3S substrates in the bacterial cytoplasm, to allow their orderly secretion, are not fully understood. In Salmonella enterica, a highmolecular-weight complex made up of the SpaO, OrgA, and OrgB (SctQ, SctK and SctL) proteins is involved in this process (49). Lara-Tejero et al. provided evidence that this cytoplasmic complex serves as a sorting platform that is loaded with different categories of T3S substrates in a sequential manner, i.e., translocators are first loaded onto the sorting platform, and only once they have been secreted are effectors recruited to be secreted by the T3SS machinery (49). In the present work, we provide evidence showing that the product of the orf4 gene in EPEC is a distant homolog of the SctK (OrgA/YscK/MxiK) protein family; thereby, we renamed the protein EscK and carried out its functional characterization. We propose that EscK, together with EscQ and EscL, forms a sorting platform that besides serving as a structure for substrate secretion orchestration also functions as an affinity platform, increasing the local concentration of T3S substrates around the export apparatus. The results provide insight into the roles of members of the poorly characterized SctK protein family in T3S. RESULTS EscK is a distant member of the SctK protein family. The uncharacterized fourth gene of the LEE1 operon in EPEC, herein named escK (formerly known as orf4), encodes a 199-amino-acid protein of unknown function with a deduced molecular mass of 23.4 kDa and a theoretical pI of 7.64. A DELTA-BLAST search using the predicted EscK protein sequence as the query sequence retrieved homologous proteins from T3SSs of other A/E pathogens and several different bacterial species. A multiple-sequence alignment of EscK and a subset of the homologous proteins disclosed many conserved residues (see Fig. S1 in the supplemental material). However, as is the case for EscK, none of these homologs has been characterized functionally. January 2017 Volume 199 Issue 1 e00538-16

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FIG 2 Conserved genomic context among genes encoding EscK homologs and members of the OrgA/MxiK/YscK (SctK) protein family. (A) Schematic representation of the genomic neighborhood of genes encoding EscKorthologous proteins retrieved by a DELTA-BLAST search. (B) Schematic representation of the genomic context of genes encoding representative members of the SctK protein family. Genes are depicted as arrows and are represented to scale. Genes encoding homologous proteins are displayed in the same color. Similar characteristics (protein size and isoelectric point) among the proteins encoded by syntenic escK genes are shown.

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. S1) also encode members of the SctL protein family (Fig. 2A). Interestingly, the upstream region of genes encoding members of the SctL protein family from well-studied archetypical T3SSs corresponds to genes encoding members of the SctK protein family (OrgA, MxiK, and YscK) (49, 51, 52) that were not retrieved by the DELTA-BLAST search with EscK. These genes share the same genomic context as those encoding EscK homologs (Fig. 2B), and their encoded proteins possess characteristics similar to those of the SctK protein family, such as a comparable size and a nearly neutral to basic pI (Fig. 2). These data and functional evidence described below suggest that EscK is a remote member of the SctK protein family. EscK is a key component for type III protein secretion and translocon assembly. To examine EscK’s contribution to protein secretion via the T3SS, we generated an EPEC escK null mutant (ΔescK) and assessed its secretion phenotype. Secreted translocator and effector proteins were recovered from the culture supernatant and analyzed by Coomassie blue-stained SDS-PAGE and immunodetection. Wild-type EPEC displayed a typical protein secretion profile, secreting translocators (EspA, EspB, and EspD) and effectors (Tir and EspF), while the ΔescK null strain did not secrete any of these substrates, with a phenotype resembling that of the ΔescN T3S-defective mutant (Fig. 3A). The inability of the ΔescK strain to secrete type III substrates is not attributable to a defect in protein production, since comparable amounts of intracellular substrates were detected in all strains (Fig. 3A, “P” panels). Expression of His-EscK in trans fully January 2017 Volume 199 Issue 1 e00538-16

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FIG 3 EscK is a central component of the EPEC T3SS. (A) EPEC wild-type (WT), ΔescN, and ΔescK strains and the ΔescK strain complemented with a plasmid expressing His-EscK were grown in DMEM to induce type III secretion. Secreted proteins were analyzed by 15% SDS-PAGE and stained with Coomassie brilliant blue (CBB) (upper panel). The presence of the effectors Tir and EspF in the supernatant (S) and of Tir, EspF, and the translocator EspB in whole-cell lysates (P) was examined by immunodetection using anti-Tir, anti-EspF, and anti-EspB polyclonal antibodies. (B) T3SS-dependent hemolysis activity was analyzed by incubating EPEC strains with red blood cells (RBCs). Hemoglobin released into the supernatant was measured by spectrophotometry to determine the OD450. Percent hemolysis was calculated relative to the hemolytic activity caused by the EPEC WT strain. Results represent the means for three independent experiments. Error bars show standard deviations.

restored type III protein secretion of the null mutant, showing that the deletion of escK was nonpolar and that the N-terminally His-tagged version of EscK was functional (Fig. 3A). Additionally, we investigated whether an EscK homolog could complement the ΔescK mutant. We performed a heterologous complementation experiment with the EscK homolog STM1410 (Fig. S1), given that the S. enterica Salmonella pathogenicity island 2 (SPI-2) T3SS is closely related to the LEE-encoded T3SS in EPEC (53). The result showed that His-STM1410 was not able to restore protein secretion to the ΔescK strain (Fig. S2). In EPEC and other pathogenic bacteria, the assembly of functional translocation pores has been correlated with the ability to cause red blood cell (RBC) hemolysis (54–56). Thus, hemolysis assays have been used as a sensitive tool to assess translocon assembly (57). To further confirm the role of EscK in the biogenesis and function of injectisomes, we tested the hemolytic capacity of the ΔescK mutant strain. RBCs were incubated with EPEC wild-type, ΔescN, and ΔescK strains, and T3SS-dependent release of hemoglobin was measured as described in Materials and Methods. In comparison to that of the wild-type strain, the hemolytic activity of the ΔescK mutant was severely impaired, as this strain showed the same basal hemolytic levels as the ΔescN strain (Fig. 3B). Complementation of the mutant strain with a plasmid expressing His-EscK restored the hemolysis capability of the ΔescK strain to wild-type levels (Fig. 3B). Overall, these results demonstrate that the EscK protein plays a crucial role in the biogenesis and function of the EPEC T3SS and suggest that it may be a component of the secretion apparatus. EscK interacts with the C-ring-forming protein EscQ. To gain insight into the function of the EscK protein, we performed a screening for potential protein-protein January 2017 Volume 199 Issue 1 e00538-16

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FIG 4 EscK interacts with EscQ. (A) Yeast two-hybrid (Y2H) assay testing for interaction between the EscK and EscQ proteins. Diploid yeast strains carrying both pGADT7 and pGBKT7 or its derivatives were 10-fold serially diluted and spotted onto SD⫺Leu⫺Trp plates (lacking leucine and tryptophan) to confirm the presence of the vectors and onto SD⫺Leu⫺Trp⫺His⫺Ade plates (lacking leucine, tryptophan, histidine, and adenine) to test for protein-protein interactions. The plasmid pairs pGBKT7-53/pGADT7-T and pGBKT7-LAM/pGADT7-T served as positive and negative controls, respectively, and were provided by the manufacturer (Clontech). Absence of self-activation of fusion proteins was verified by testing the growth of a diploid yeast carrying a combination of the empty vector (⫺) and the fusion protein. (B) In vitro pulldown assay corroborating the interaction of EscK with EscQ. GST-EscQ, from a cleared lysate (L), was immobilized on glutathione-Sepharose beads. Subsequently, a cleared lysate of BDP cells expressing MBP or MBP-EscK (input [I]) was loaded onto the column. After extensive washing (W), bound proteins were eluted (E). All fractions were analyzed by CBB-stained 12% SDS-PAGE and immunoblotted with anti-MBP antibodies.

interactions with other structural components of the T3SS (e.g., EscD, EscF, EscJ, EscL, EscN, EscO, EscQ, EscU, and EscV) by using a yeast two-hybrid (Y2H) assay. The Y2H experiment revealed only one EscK-interacting partner among those tested, namely, the C-ring protein EscQ (Fig. 4A). The EscK-EscQ interaction was detected under high-stringency conditions (i.e., expression of two auxotrophic reporters), suggesting a strong interaction. In order to confirm this result, pulldown assays were carried out using an N-terminally glutathione S-transferase (GST)-tagged version of EscQ (GSTEscQ), expressed in E. coli BL21(DE3)/pLysS (BDP) cells and immobilized on glutathioneSepharose 4B beads. After washing of unbound proteins, a clarified cell lysate from BDP cells expressing maltose-binding protein (MBP) alone (negative control) or an N-terminally MBP-tagged version of EscK (MBP-EscK) was passed through a column containing immobilized GST-EscQ, and after extensive washing, proteins were eluted and analyzed by SDS-PAGE plus immunoblotting. MBP-EscK but not MBP alone coeluted with GST-EscQ, corroborating the interaction between these proteins (Fig. 4B). As an additional control, we showed that MBP-EscK did not coelute with GST alone, confirming the specificity of the EscK-EscQ interaction (Fig. S3). It is worth mentioning that the MBP-EscK and GST-EscQ recombinant proteins were functional for type III protein secretion in complementation assays (data not shown). January 2017 Volume 199 Issue 1 e00538-16

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FIG 5 EscK is located in both the cytoplasmic and inner membrane fractions. EPEC strains expressing His-EscK were fractionated into periplasmic (Per), cytoplasmic (C), and membrane (M) fractions. Equal amounts of total protein from each fraction, together with the whole-cell lysate (P), were subjected to 15% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with an anti-His antibody. The presence of cytoplasmic (DnaK), periplasmic (MBP), and membrane (EscJ) markers in each fraction was verified by immunoblotting to confirm proper fractionation. (A) Bacterial fractionation of EPEC ΔescK strain complemented with a plasmid encoding His-EscK. (B) Membrane subfractionation into inner and outer membranes by solubilization with N-lauroylsarcosine (see Materials and Methods). EscJ and EscC served as inner and outer membrane markers, respectively. (C) The bacterial fractionation experiment was performed in the ΔescK ΔescQ double mutant and Δler mutant backgrounds to determine whether EscK localization depends on EscQ or another T3SS component.

In this regard, it was previously shown that members of the SctK and SctQ protein families form part of the so-called sorting platform for classification of substrates (48, 49) and that the SctK-SctQ protein interaction is conserved among other T3SSs, as a direct physical binding between YscK and YscQ has been reported for Yersinia pestis (51), as well as binding between MxiK and Spa33 in Shigella flexneri (33, 52). Therefore, our result suggests that EscK, together with EscQ, is a component of the EPEC sorting platform. Subcellular localization of EscK. The direct protein-protein interaction between EscK and EscQ suggests that these two proteins colocalize in the cell. EscQ was previously found in the soluble and membrane fractions of EPEC (34). EscK has no predicted transmembrane helices or Sec-dependent signal sequence. Hence, to elucidate the subcellular localization of EscK, the EPEC ΔescK mutant strain carrying a plasmid expressing His-EscK was fractionated into periplasmic, cytoplasmic, and membrane fractions. The purity of each fraction was assessed by immunodetection of the periplasmic protein MBP, the cytoplasmic chaperone DnaK, and the T3SS inner membrane-associated ring protein EscJ. As shown in Fig. 5A, His-EscK was detected in both the cytoplasmic and membrane fractions. Furthermore, the inner and outer membranes were separated as described in Materials and Methods, and proper membrane fractionation was confirmed by immunodetection of EscJ and the T3SS outer membrane secretin EscC. The result showed that His-EscK was enriched in the inner membrane fraction (Fig. 5B). Additionally, to test the possibility that EscK is also a type III secreted substrate, we generated a C-terminally double-hemagglutinin (HA)-tagged version of EscK (EscK-2HA) and analyzed its secretion into the culture supernatant from January 2017 Volume 199 Issue 1 e00538-16

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EPEC wild-type, ΔescN, and ΔescK strains by using an anti-HA antibody. Even though the EscK-2HA recombinant protein was fully functional and produced in all strains, it was not detected in the supernatant (Fig. S4), confirming that it is an intracellular component of the type III secretion machinery. Taken together, these results are consistent with the role of EscK as part of a large cytoplasmic platform which is associated with the membrane-embedded base of the injectisome. Next, we addressed whether the subcellular localization of EscK depends on the presence of its interacting partner, EscQ. We performed bacterial fractionation of an EPEC ΔescK ΔescQ double mutant carrying a plasmid expressing His-EscK. As shown in Fig. 5C, His-EscK localized to the membrane fraction even in the absence of EscQ. Moreover, we found that the EscK membrane location did not require any other T3SS component, since the localization of His-EscK was not affected in the LEE master regulator ler deletion mutant (Fig. 5C), or even in the nonpathogenic E. coli BL21 strain (data not shown). Immunodetection of the sensor kinase ArcB in BDP cells was used as a membrane fraction control. These data demonstrate that, as observed for other components of virulence and flagellar T3SSs without predicted transmembrane domains (34, 58), the EscK protein is intrinsically targeted to the inner membrane. Likewise, to discern if membrane localization of EscQ is dependent on the presence of EscK, we generated wild-type and ΔescK EPEC strains expressing a chromosomally triple-FLAG-tagged version of EscQ and performed cell fractionation. The EscQ-3FLAG recombinant protein was detected in the membrane and cytoplasmic fractions of both the wild-type and ΔescK backgrounds (Fig. S5), indicating that membrane targeting of EscQ does not require EscK. Functional analysis of EscK. To investigate the function of EscK in more detail, we performed site-directed mutagenesis. Based on a multiple-sequence alignment of EscK and its homologs, we replaced highly conserved residues and some surrounding amino acids (W26, L93, D100, Y101, F103, S104, Y107, R108, Q122, and P171 [Fig. S1]) with alanine and tested the effects of these substitutions in an escK null strain complementation assay (Fig. 6). Each of the point mutants generated in the escK gene cloned into the pET19b vector were introduced into the ΔescK mutant strain, and the ability of the protein variants to restore T3 protein secretion was determined. The complementation analysis showed that the His-EscK D100A, Y101A, Y107A, and R108A replacements abolished the capacity of EscK to restore the secretion defect of the escK deletion strain, while substitutions at other positions had a minor or no effect on the function of the T3SS (Fig. 6). All His-EscK variants produced stable proteins at levels comparable to that of His-EscK when expressed from a pTrc99A-based plasmid, as judged by immunoblotting using an anti-His antibody (data not shown). Thus, it appears that a conserved domain in the central region of the EscK protein is essential for function. We then asked whether the loss-of-function EscK D100A, Y101A, Y107A, and R108A protein variants were able to interact with EscQ. To address this question, the escK variant alleles were subcloned into the pGBKT7 plasmid vector, and their ability to interact with pGADT7-produced EscQ was evaluated in the Y2H assay. The results showed that the EscK D100A and R108A protein variants retained the ability to interact with EscQ, whereas the Y101A and Y107A variants lost this interaction capability (Fig. 7A). To validate these results, the four point mutations were individually introduced into the plasmid encoding MBP-EscK by a new round of site-directed mutagenesis, and the interaction of each MBP-EscK mutant version with GST-EscQ was tested by pulldown assays as described above. All MBP-EscK protein variants were soluble and were produced at similar levels. The pulldown screening confirmed that the EscK Y101 and Y107 residues are directly involved in the interaction with EscQ (Fig. 7B) and suggested that the EscK-EscQ protein-protein interaction is necessary for T3SS function. Dominant negative effect of EscK protein variants on wild-type EPEC type III secretion. To test whether the loss-of-function variants of EscK exert a dominance effect on protein secretion, each escK variant allele was inserted into the expression vector pTrc99A and transformed into wild-type EPEC. Overproduction of His-EscK January 2017 Volume 199 Issue 1 e00538-16

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FIG 6 Site-directed mutagenesis defined critical residues for EscK function. (A) Protein secretion profiles of the EPEC WT strain and ΔescK mutant strains carrying the empty vector pET19b (⫺) or a pET19b plasmid encoding His-EscK (his-escK) or His-EscK single-amino-acid mutant derivatives. Secreted proteins from each strain were recovered from the supernatant and analyzed by CBB-stained 15% SDS-PAGE. (B) The presence of the Tir and EspA T3S substrates in both the supernatant (S) and whole-cell lysate (P) was examined by immunodetection using anti-Tir and anti-EspA polyclonal antibodies.

D100A completely abolished T3S, whereas His-EscK R108A severely diminished the secretion of both translocators and effectors. In contrast, overproduction of the HisEscK Y101A or Y107A variant did not interfere with the function of the endogenous EscK protein (Fig. 7C, top and “S” panels). As negative controls, we showed that wild-type EPEC secretion was not affected by the presence of the empty plasmid or the pTrc99A-based plasmid producing His-EscK (Fig. 7C, top and “S” panels). All plasmid constructs were found to produce stable proteins in nearly the same amounts (Fig. 7C, “P” panels). These results showed that only the nonfunctional EscK variants capable of interacting with EscQ had an inhibitory effect on secretion, suggesting that EscK incorporation into the C-ring structure is essential for type III secretion. Furthermore, it was recently reported that E. coli motility is significantly reduced upon expression of the LEE1 operon, suggesting that a protein encoded by this operon can potentially interfere with flagellar assembly or function (59). Therefore, we investigated if the LEE1-encoded EscK protein could be responsible for the inhibitory effect on motility. The EPEC wild-type strain was transformed with empty vector (pTrc99A) or with the plasmid expressing His-EscK and spotted onto soft-agar plates for motility assays. As shown in Fig. S6, overproduction of EscK strongly inhibited the motility of wild-type cells. Likewise, we tested the effect of EscK loss-of-function proteins on bacterial motility. In a way similar to the dominant negative effect on type III secretion described above, we found that the EscK variants D100A and R108A inhibited EPEC motility, while the EscK Y101A and Y107A variants did not significantly affect the motility of wild-type cells (Fig. S6). Although additional experiments are required to clarify the underlying cause of this motility inhibition, one possible explanation for these results is that the EscK protein and variants capable of interacting with EscQ are incorporated into the homologous flagellar export machinery, thereby interfering with bacterial motility. Substrate overproduction overcomes the secretion defect of sorting platform mutant strains. It was previously reported that the C-ring requirement for flagellar January 2017 Volume 199 Issue 1 e00538-16

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FIG 7 Identification of amino acid residues involved in the EscK-EscQ protein interaction. (A) EscK loss-of-function mutants were subcloned into the pGBKT7 vector, and their interaction with EscQ was tested by Y2H assays. (B) In vitro pulldown assays of MBP-EscK loss-of-function variants and GST-EscQ. Cleared lysates of BDP cells expressing equal amounts of MBP-EscK or its variants (input) were loaded onto a column of immobilized GST-EscQ glutathione-Sepharose resin. After washing, bound proteins were eluted (output) and analyzed by CBB-stained SDS-PAGE and immunoblotting with anti-MBP antibodies. (C) Dominant negative blockage of EPEC T3S by expression of EscK loss-of-function mutants. A secretion assay was performed with the EPEC WT strain carrying the empty plasmid pTrc99A (⫺), a pTrc99A plasmid expressing His-EscK, or its mutant derivatives. Secreted proteins from each strain were recovered from the supernatant (S), subjected to SDS-PAGE, and analyzed by CBB staining or immunodetection using the indicated antibodies. Immunoblot analysis of whole-cell lysates (P) showed comparable levels of Tir, EspA, and His-EscK proteins in the corresponding strains. Expression of His-EscK or His-EscK mutant derivatives was induced with 0.02 mM IPTG.

type III secretion can be bypassed by overproduction of the ATPase FliI (60), as well as by upregulation of the flagellar master operon flhDC (61). In order to gain a more complete understanding of the ΔescK mutant phenotype, we investigated whether the phenotypes of this strain and the other EPEC sorting platform mutants (ΔescL and ΔescQ) could be bypassed by a substrate excess. To this end, we evaluated the secretion of type III substrates overproduced from the high-copy-number plasmid pTOPO-2HA, which generates C-terminally double-HA-epitope-tagged proteins. The secretion profiles of the different strains overproducing LEE- or non-LEE-encoded effectors, as well as the translocator EspA, are shown in Fig. 8. All type III secretion substrates tested were consistently immunodetected in the supernatants of the ΔescK, ΔescL, and ΔescK ΔescL mutant strains, though in smaller amounts than the wild-type levels, and were not secreted in the ATPase and export apparatus negative-control ΔescN and ΔescU mutant strains (Fig. 8, “S” panels). As an additional control, we showed that substrates detected in the supernatant of the ΔescK strain were delivered specifically through the T3SS, as secretion was abolished in a ΔescN ΔescK double-null mutant (Fig. S7). Regarding the ΔescQ mutant, EscQ appears to be more critical for secretion, although it could be bypassed by overproduction of the translocator EspA, which was secreted at higher levels than the rest of the substrates (Fig. 8, “S” panels). All the recombinant substrates were produced at similar levels in the different mutant backgrounds (Fig. 8, “P” panels). The cytoplasmic chaperone DnaK was used as a loading control for whole cells as well as a cell lysis control in the supernatant (shown only for EspH). The autotransporter EspC was used as a supernatant loading control (shown only for EspH) (Fig. 8). These results indicate that the sorting platform is not entirely essential for type III secretion when substrates are overproduced, which suggests that it may act not only as a docking site for ordered sequential substrate secretion, as previously reported for Salmonella (49), but also as an affinity docking station for T3S substrates, increasing their local concentration around the export gate. January 2017 Volume 199 Issue 1 e00538-16

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FIG 8 Secretion assay of sorting platform-defective mutants with overproduced substrates. The EPEC WT strain and the indicated nonpolar mutants harboring pTOPO-2HA-based plasmids expressing the specified type III secreted substrates fused to a C-terminal double-HA tag were grown under type III secretion-inducing conditions. The presence of each tagged substrate in both the culture supernatant (S) and the bacterial pellet (P) was determined by immunoblotting using anti-HA antibodies. Anti-EspC and anti-DnaK served as loading controls for secreted and produced proteins, respectively. To avoid a saturated signal, the amount of secreted EspA-HA sample loaded in the WT lane was one-half of that loaded in the rest of the lanes.

DISCUSSION The injectisome is one of the most complex bacterial machines known. Recent advances in assembly, structure, and mechanistic studies have considerably increased our current understanding of the function of this macromolecular complex (62–65). Despite this, there are still several gaps in our knowledge on the molecular mechanisms underlying effector protein translocation into host cells. The cytosolic C-ring is a conserved and crucial component of the T3SS, although its precise composition and function are still not well understood (33, 46). In particular, the specific roles of members of the SctK protein family that form part of the sorting platform remain unclear. In this study, we performed the initial characterization of the EPEC EscK (formerly known as Orf4) protein, whose role in type III secretion has not previously been explored. 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). As shown in this work for EscK, proteins of the SctK family are needed for type III protein secretion (52, 69–71) and form a complex with the C-ring protein SctQ (YscQ, Spa33, or SpaO) (33, 49, 51, 52). Therefore, our bioinformatic analysis and experimental evidence support the hypothesis that EscK shares a common ancestry with the YscK/MxiK/OrgA family, and hence we propose that these proteins carry out similar roles in their respective T3SSs. Future January 2017 Volume 199 Issue 1 e00538-16

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structural studies will contribute to disclosing remote similarities among the SctK group of proteins. Moreover, as previously suggested (68, 72), it can be speculated that members of the SctK family are evolutionarily related to the flagellar FliG protein, given that its encoding gene is usually located between fliF (encoding an SctJ homolog) and fliH (encoding an SctL homolog) (73) and the fact that FliG interacts with the C-ring component FliM (an SctQ homolog), connecting the cytoplasmic ring to the flagellar basal body (44). Although in our Y2H screening we did not detect any interaction between EscK and a basal body T3SS component, we cannot rule out the possibility that EscK has a FliG-like function, aiding to dock the EscQ cytoplasmic ring to the injectisome base. In this regard, high-resolution cryo-electron tomography of the S. flexneri sorting platform revealed an unassigned density anchoring each one of the Spa33 (SctQ) pods to the cytoplasmic portion of the basal body formed by the N-terminal domain of the MxiG (SctD) protein (48). It is thus tempting to suggest that such density corresponds, at least partially, to the MxiK (SctK) protein. In this work, we uncovered a novel protein association between EscK and EscQ in the EPEC injectisome by using yeast two-hybrid and copurification experiments. However, due to the high rate of false-negative results with the yeast two-hybrid system (74), we do not discard the possibility of the existence of additional EscK binding partners that were missed in our screening or that were not examined. For S. flexneri, it has been shown that MxiK interacts with the Spa47 ATPase (52), although the role of this interaction is unclear. Alignment-guided mutagenesis allowed us to identify a set of residues that are critical for EscK function. Among these, the complementation defect of the EscK Y101A and Y107A protein variants could be attributed to their loss of association with EscQ, highlighting the importance of the EscK-EscQ interaction for T3S. In contrast, the loss of function of the EscK D100A and R108A variants may have been due to a different protein-protein interaction failure, suggesting the existence of additional EscK binding partners. Furthermore, we showed that EscK has an intrinsic affinity for membrane association, since its membrane subcellular localization does not depend on any component of the T3SS. This result is consistent with what has been observed for predicted soluble components of other T3SS machineries (58, 75). Also, it has been shown that EscQ interacts with EscN and EscL, but it does not require the presence of any of these proteins to be targeted to the bacterial membrane (34). In agreement, we demonstrated here that EscK is not required for EscQ membrane localization. However, the orthologous proteins in Yersinia enterocolitica, YscN, YscL, and YscK, are necessary for focal localization of YscQ at the base of the injectisome (35). It is thus possible that the intrinsic membrane targeting of the cytoplasmic components facilitates their rapid incorporation into the secretion machinery, where they require each other for assembly at the base of the export apparatus. For several T3SS structural components, it has been shown that expression of nonfunctional proteins in a wild-type background can lead to a dominant negative blockage of T3S (76–79). In wild-type EPEC, the production of loss-of-function EscK variants that retain the capacity to interact with EscQ (D100A and R108A substitutions) prevented substrate secretion through the injectisome. This dominant negative effect on T3S suggests that these mutant versions are assembled into the C-ring/sorting platform structure, thereby interfering with protein secretion. On the other hand, loss-of-function EscK variants that had lost the capacity to interact with EscQ did not interfere with the function of the wild-type copy of EscK. Interestingly, similar results were obtained for bacterial motility when the EscK protein variants were expressed in EPEC, as only the EscK protein and the EscK D100A and R108A variants inhibited motility. Although in vivo the EPEC LEE island and flagellar operons are not expected to be expressed simultaneously, due in part to the cross-regulation mediated by the GrlA-GrlR system on the flhDC master regulator (80), the interference caused by the EscK protein on flagellar function could be the result of a cross-interaction between EscK and the flagellar C-ring. January 2017 Volume 199 Issue 1 e00538-16

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In this study, we showed that EscK is an important component of the EPEC T3SS machinery, as it is necessary for substrate secretion and translocon-dependent hemolytic activity. These results are in agreement with previous observations showing that a C. rodentium Δorf4 mutant is defective for T3S and unable to infect a murine animal model (17). However, a more thorough examination of the ΔescK mutant phenotype revealed that the absence of the EscK protein could be partially alleviated by increasing T3S substrate levels. This was also the case for EscL, the other minor component of the sorting platform, since a substrate excess also compensated for the secretion defect of a ΔescL mutant. The similar bypass phenotypes seen for these two mutants suggest a functional linkage between the EscK and EscL proteins. Furthermore, overproduced substrates were similarly secreted in a ΔescK ΔescL double mutant background. On the other hand, the EscQ protein, which is proposed to be the major component of the sorting platform (48), appeared to be more important for secretion, although it could be bypassed by overproduction of the EspA translocator. The nonessential nature of the equivalent flagellar components (FliM, FliN, FliH, and FliG) has already been reported (60, 61, 81), unraveling a common mechanism of action of this cytoplasmic complex in type III secretion machineries. Importantly, it has been shown that the assembly of the core of the injectisome (basal body and export apparatus) is not affected in the absence of the cytoplasmic components (SctQ, SctL, and SctK) (33, 48, 52, 69), so it is possible that this primordial core is sufficient to drive protein secretion, providing a surplus of substrates. Members of both the SctK and SctL protein families have been implicated in the correct assembly of the C-ring complex formed by the oligomerization of SctQ (35). A possible explanation for the observed bypass phenotype is that in the absence of the EscK and EscL proteins, the probability of EscQ C-ring assembly is reduced but the C-ring occasionally can be formed, allowing (though inefficiently) the subsequent substrate secretion. Alternatively, EscK and EscL may directly participate as docking sites for T3S substrates. Indeed, EscL has been shown to bind EspA (82). Moreover, it is interesting that proteins of the SctK family are the less-conserved constituents of the sorting platform structure. The high evolutionary rate of members of this protein family may reflect a role in species-specific substrate recognition, which may also account for the noninterchangeability between EscK and its S. enterica SPI-2 homolog, STM1410. Taken together, these results suggest that the C-ring cytosolic structure serves as a docking site for type III substrates, increasing their local concentration around the export gate. To the best of our knowledge, this is the first report directly showing that an intact sorting platform is not required for T3S as long as substrates are present in excess. Despite its importance in the secretion process, the components forming the cytoplasmic sorting platform have scarcely been investigated. In this study, we deciphered the function of the EscK protein in the EPEC injectisome, which together with EscQ and EscL plays an essential role in the T3S process. Overall, our data contribute to a better understanding of the function of the sorting platform complex and provide a more complete picture of how the T3SS works. MATERIALS AND METHODS Bacterial strains and growth conditions. All strains and plasmids used in the present study are listed in Table 1. Bacterial strains were routinely grown in lysogeny broth (LB) medium at 37°C or, for T3S-inducing conditions, in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) under static conditions at 37°C in a 5% CO2 atmosphere. When required, antibiotics were used at the following concentrations: streptomycin (Sm), 25 ␮g/ml; kanamycin (Km), 50 ␮g/ml; ampicillin (Ap), 100 ␮g/ml; chloramphenicol (Cm), 25 ␮g/ml; and tetracycline (Tc), 25 ␮g/ml. Construction of EPEC null mutants and epitope-tagged strains encoding EscQ-3ⴛFLAG. Nonpolar EPEC mutants were generated using the Lambda Red recombinase system (␭-Red) (83). Briefly, a PCR fragment containing a kanamycin resistance cassette was amplified from the template plasmid pKD4 by using primers flanked by homologous sequences upstream and downstream of the gene to be deleted. The PCR product was electroporated into an EPEC strain carrying the pKD46 plasmid. Successful mutants were selected on LB-Km plates. EPEC strains expressing a C-terminally 3⫻FLAG-tagged EscQ protein in its chromosomal context were generated using a modification of the ␭-Red recombinase system employing the pSUB11 plasmid as the template (84). For double mutant strains, the kanamycin cassette was excised from the corresponding single mutant strain by using the helper plasmid pFLP2, January 2017 Volume 199 Issue 1 e00538-16

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TABLE 1 Strains and plasmids used in this study Strain or plasmid Strains EPEC strains E2348/69 ΔescN strain ΔescU strain ΔescK strain ΔescQ strain ΔescL strain ΔescK ΔescL strain ΔescK ΔescQ strain ΔescN ΔescK strain Δler strain escQ::3⫻FLAG strain ΔescK escQ::3⫻FLAG strain E. coli strains BL21(DE3)/pLysS XL1-Blue Top10 S. cerevisiae PJ69-4a/␣

Plasmids pET19b pTrc99A pMAL-c2x pGEX-4T-2 pTOPO-2HA pGBKT7 pGADT7 pKD46 pKD4 pFLP2 pSUB11 pAEo4 pAEo4-W26A pAEo4-L93A pAEo4-D100A pAEo4-Y101A pAEo4-F103A pAEo4-S104A pAEo4-Y107A pAEo4-R108A pAEo4-Q122A pAEo4-P171A pMTHo4 pMTHo4-D100A pMTHo4-Y101A pMTHo4-Y107A pMTHo4-R108A pSLo4 pSLo4-D100A pSLo4-Y101A pSLo4-Y107A pSLo4-R108A pArGsQ pJHnD pJHmap pJHtir pJHeH pJHnC pSHeA

Descriptiona

Source or reference

Wild-type EPEC O127:H6 strain; Smr E2348/69 escN in-frame deletion mutant; Smr E2348/69 escU in-frame deletion mutant; Smr Kmr E2348/69 escK in-frame deletion mutant; Smr Kmr E2348/69 escQ in-frame deletion mutant; Smr Kmr E2348/69 escL in-frame deletion mutant; Smr Kmr E2348/69 escK escL double in-frame deletion mutant; Smr Kmr E2348/69 escK escQ double in-frame deletion mutant; Smr Kmr E2348/69 escN escK double in-frame deletion mutant; Smr Kmr E2348/69 ler in-frame deletion mutant; Smr Kmr E2348/69 derivative expressing 3⫻FLAG-tagged EscQ; Smr Kmr E2348/69 escK mutant derivative expressing 3⫻FLAG-tagged EscQ; Smr Kmr

91 23 This This This This This This This 92 This This

Protein expression strain; Cmr Cloning strain; Tcr Cloning strain; Smr

Novagen Stratagene Invitrogen

MATa/␣ trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ

93

His10 tag expression vector; T7 promoter; Apr Expression vector; trc promoter; Apr MBP tag expression vector; Apr GST tag expression vector; Apr pCR2.1-TOPO derivative carrying C. rodentium espG coding region tagged with two HA epitopes at the C terminus; Apr Kmr Y2H vector containing the GAL4 DNA binding domain; TRP1 Y2H vector containing the GAL4 activation domain; LEU2 Red recombinase system plasmid; araB promoter; Apr Template plasmid for amplification of the kanamycin cassette used for the Red recombinase system; Apr Kmr Flp recombinase expression system; Apr Template plasmid for amplification of the FLAG epitope; Apr pET19b encoding His-EscK pAEo4 derivative expressing His-EscK W26A pAEo4 derivative expressing His-EscK L93A pAEo4 derivative expressing His-EscK D100A pAEo4 derivative expressing His-EscK Y101A pAEo4 derivative expressing His-EscK F103A pAEo4 derivative expressing His-EscK S104A pAEo4 derivative expressing His-EscK Y107A pAEo4 derivative expressing His-EscK R108A pAEo4 derivative expressing His-EscK Q122A pAEo4 derivative expressing His-EscK P171A pTrc99A encoding His-EscK pMTHo4 derivative expressing His-EscK D100A pMTHo4 derivative expressing His-EscK Y101A pMTHo4 derivative expressing His-EscK Y107A pMTHo4 derivative expressing His-EscK R108A pMAL-c2x encoding MBP-EscK pSLo4 derivative expressing MBP-EscK D100A pSLo4 derivative expressing MBP-EscK Y101A pSLo4 derivative expressing MBP-EscK Y107A pSLo4 derivative expressing MBP-EscK R108A pGEX-4T2 encoding GST-EscQ pTOPO-2HA carrying nleD with its native RBS pTOPO-2HA carrying map with its native RBS pTOPO-2HA carrying tir with its native RBS pTOPO-2HA carrying espH with its native RBS pTOPO-2HA carrying nleC with its native RBS pTOPO-2HA carrying espA with its native RBS

Novagen Amersham-Pharmacia New England Biolabs GE Healthcare 17

study study study study study study study study study

Clontech Clontech 83 83 85 84 This This This This This This This This This This This This This This This This This This This This This This 57 57 57 57 57 This

study study study study study study study study study study study study study study study study study study study study study study

study

(Continued on next page)

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TABLE 1 (Continued) Strain or plasmid pSHo4 pGBKT7-LAM pGBKT7-53 pGADT-T7 pSGBKo4 pSGBKo4-D100A pSGBKo4-Y101A pSGBKo4-Y107A pSGBKo4-R108A pSGADeQ aApr,

Descriptiona pTOPO-2HA carrying escK with its native RBS pGBKT7 encoding a fusion of the GAL4 DNA binding domain with human lamin C pGBKT7 encoding a fusion of the GAL4 DNA binding domain with murine p53 pGADT7 encoding a fusion of the GAL4 activation domain with the simian virus 40 (SV40) large T antigen pGBKT7 encoding EscK pSGBKo4 derivative expressing EscK D100A pSGBKo4 derivative expressing EscK Y101A pSGBKo4 derivative expressing EscK Y107A pSGBKo4 derivative expressing EscK R108A pGADT7 encoding EscQ

Source or reference This study Clontech Clontech Clontech This This This This This This

study study study study study study

ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance; Smr, streptomycin resistance; Tcr, tetracycline resistance.

which expresses the FLP recombinase (85). The second allelic exchange was performed as described above. Mutant and tagged strains were confirmed by PCR. Importantly, the mutant strains used in this study were shown to be nonpolar, as the wild-type secretion phenotype was restored by introducing the plasmid(s) expressing the corresponding deleted gene(s). Plasmid construction and site-directed mutagenesis. The escK gene was amplified from genomic DNA of the EPEC wild-type strain by use of primers containing NdeI and BamHI restriction sites. The escK fragment was cloned into the pET19b vector to generate plasmid pAEo4 and subsequently subcloned as an NdeI/BamHI fragment into the pGBKT7 vector and as an NcoI/BamHI fragment into the pTrc99A vector to generate plasmids pSGBKo4 and pMTHo4, respectively. To create a plasmid expressing an N-terminally MBP-tagged EscK protein, the escK gene was amplified and ligated into the pMAL-c2x vector as a BamHI/HindIII fragment. To generate plasmids expressing proteins fused to a C-terminal double-HA tag, the coding regions of escK and espA without the stop codon, but including the native ribosome binding site (RBS), were amplified and cloned into the pTOPO-2HA vector as HindIII/XhoI fragments. The coding region of escQ was PCR amplified, cloned into the PCR-Blunt II-TOPO vector, and then subcloned as a BamHI/EcoRI fragment into the pGEX-4T-2 vector to produce plasmid pArGsQ. Site-directed mutagenesis was performed following the QuikChange protocol (Stratagene). Where possible, mutagenic primers were designed using SDM-Assist software (86) to introduce a silent restriction site along with the desired mutation to simplify its subsequent identification via enzymatic digestion. All constructs were verified by DNA sequencing. T3S assays. Overnight LB cultures of EPEC were used to inoculate 6 ml of DMEM (1:50), and growth was continued under T3S-inducing conditions until an optical density at 600 nm (OD600) of 0.8 to 1.0 was reached. The culture was centrifuged at 12,000 ⫻ g for 5 min, and the supernatant was collected. Proteins in the culture supernatant were precipitated by addition of trichloroacetic acid (TCA; 10% [vol/vol]) and overnight incubation at 4°C. Precipitated proteins were concentrated by centrifugation at 16,863 ⫻ g for 30 min and resuspended in SDS-PAGE sample buffer supplemented with saturated Tris according to the OD600 of each culture. Hemolysis assay. Bacterial T3SS-dependent hemolysis was carried out as previously described (54, 87). Red blood cells (RBCs) obtained from EDTA-treated blood were washed 3 times with saline solution (0.9% NaCl) by centrifugation at 1,000 ⫻ g for 10 min. EPEC strains from overnight LB precultures were diluted 1:50 in DMEM and grown to an OD600 of 0.4. A 4% RBC-DMEM solution (0.5 ml) was mixed with 0.5-ml aliquots of the different EPEC cultures. To favor the contact between bacteria and erythrocytes, the mixture was centrifuged at 2,500 ⫻ g for 1 min and then incubated in a 5% CO2 atmosphere for 4 h at 37°C. The bacterium-RBC mixture was gently resuspended, solid material was removed by centrifugation at 12,000 ⫻ g for 1 min, and hemoglobin release was measured by determining the OD450. Yeast two-hybrid assays. The Matchmaker GAL4 two-hybrid system 3 was used to screen for protein-protein interactions according to the user’s manual provided by the manufacturer (Clontech). pGADT7- or pGBKT7-based constructs were transformed into Saccharomyces cerevisiae strain PJ69-4a (MATa) or PJ69-4␣ (MAT␣), respectively, using the lithium acetate procedure (88). Transformed yeast strains were mated overnight on yeast extract-peptone-dextrose (YPD) medium to obtain diploids, which were selected on synthetically defined (SD) medium lacking both leucine and tryptophan (SD⫺Leu⫺Trp). Yeast cotransformants were grown overnight on liquid SD⫺Leu⫺Trp, and the cultures were 10-fold serially diluted. Dilutions were spotted onto high-stringency medium plates lacking leucine, tryptophan, histidine, and adenine (SD⫺Leu⫺Trp⫺His⫺Ade) to screen for the activity of the reporter genes. Pulldown assays. E. coli BL21(DE3)/pLysS (BDP) cells carrying the plasmid pGEX-4T-2, pArGsQ, or pMAL-c2x or a pSLo4 derivative (Table 1) were grown at 37°C in 200 ml of LB to an OD600 of 0.6. Protein production was then induced with 0.2 mM isopropyl-␤-D-thiogalactopyranoside (IPTG), and cells were grown for another 4 h at 30°C. Cultures were harvested by centrifugation, and the bacterial pellet was resuspended in 10 ml of ice-cold phosphate-buffered saline (PBS; pH 7.3) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were disrupted by sonication, and lysates were clarified by centrifugation (23,708 ⫻ g for 30 min at 4°C). Cleared lysates containing the GST-tagged proteins were incubated with 160 ␮l of glutathione-Sepharose 4B beads for 3 h at 4°C with gentle agitation. Next, the January 2017 Volume 199 Issue 1 e00538-16

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mixture was loaded onto a column and washed with 20 ml of ice-cold PBS containing 5 mM dithiothreitol (DTT), followed by the addition of a cleared lysate containing MBP-tagged proteins. Unbound proteins were extensively washed with 50 ml of ice-cold PBS. Finally, bound proteins were eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 20 mM reduced glutathione). EPEC fractionation. Bacteria were fractionated as described by Gauthier et al. (23), with slight modifications. Briefly, EPEC strains from overnight cultures were diluted 1:50 in 100 ml of prewarmed DMEM. Cultures were grown at 37°C and 5% CO2 to an OD600 of 0.4 to 0.5. Protein production of EPEC strains carrying the plasmid pMTHo4 was then induced with 0.1 mM IPTG, and growth was continued to an OD600 of 0.8 to 1.0. Bacterial cells were collected by centrifugation and washed once with PBS. To generate spheroplasts, the bacterial pellet was carefully resuspended in 1 ml of osmotic shock buffer (50 mM Tris-HCl, pH 7.0, 20% [wt/vol] sucrose, 1 mM PMSF). EDTA and lysozyme were added to final concentrations of 10 mM and 100 ␮g/ml, respectively, and the mixture was incubated for 30 min at room temperature. The suspension was centrifuged at 8,000 ⫻ g for 10 min, and the supernatant, containing the periplasmic fraction, was recovered. Pelleted spheroplasts were resuspended in 10 ml of sonication buffer (20 mM Tris-HCl, pH 7.0, 1 mM PMSF), lysed by sonication, and centrifuged at 8,200 ⫻ g for 10 min to remove unbroken cells. Cleared supernatant, containing both the cytoplasmic and membrane fractions, was subjected to ultracentrifugation at 90,000 ⫻ g for 1 h (using a 60 Ti rotor in a Beckman XL-90 ultracentrifuge) to separate the soluble cytoplasmic fraction from the total membrane pellet. The supernatant, containing the cytoplasmic fraction, was once again ultracentrifuged in order to remove membrane remnants, and the membrane pellet was collected. The total membrane pellet was washed once and resuspended in 1 ml of sonication buffer. In addition, to separate the inner and outer membranes, the whole-membrane fraction was treated with 0.5% N-lauroylsarcosine, incubated with shaking for 30 min, and centrifuged at 50,000 ⫻ g for 1 h. The supernatant from the Sarkosyl-treated membrane fraction containing the inner membrane was collected, and the pellet, containing the outer membrane-enriched fraction, was washed again with 0.5% N-lauroylsarcosine, resuspended in 250 ␮l of sonication buffer supplemented with 0.5% N-lauroylsarcosine, and stored for further examination. The same fractionation protocol was used for LB-grown BDP cells carrying the plasmid pMTHo4. Total protein concentration was measured using the DC protein assay (Bio-Rad). Western blot analysis. Protein samples were subjected to 12 or 15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membranes. Membranes were blocked with Trisbuffered saline (TBS) containing 5% nonfat dry milk overnight at 4°C. Immunoblotting was carried out using rabbit polyclonal anti-Tir, anti-EspF, anti-EspB, anti-EspA, anti-EscJ, anti-EscC, anti-EspC, and anti-ArcB antibodies. The commercial antibodies anti-DnaK (MBL International), horseradish peroxidase (HRP)-conjugated anti-HA (Roche), anti-MBP (New England BioLabs), anti-His (Santa Cruz Biotechnology), and anti-FLAG (Sigma) were used according to the manufacturers’ instructions. When necessary, an HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (Santa Cruz Biotechnology) was used. Detection was carried out with an Immobilon Western chemiluminescent HRP substrate kit (Millipore). Motility experiments. Freshly transformed colonies of the wild-type EPEC strain carrying the corresponding pTrc99A-based plasmids were picked onto the surfaces of soft tryptone agar plates (0.25% Bacto agar) containing 0.2 mM IPTG. The swimming plates were incubated at 37°C for 7 h. In silico procedures. The amino acid sequence of the EscK protein was used as the query in a DELTA-BLAST search against the nonredundant GenBank database. Multiple-sequence alignment was carried out using MUSCLE (89). The genomic context comparison was plotted using the R package genoPlotR (90).

SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/ JB.00538-16. TEXT S1, PDF file, 0.8 MB. ACKNOWLEDGMENTS We thank all laboratory members for helpful discussions and support. We are grateful to Ana Karen Mojica Ávila for help with preliminary experiments. We acknowledge Luis Ángel Fernández, Ángel Manjarrez-Hernández, and Dimitris Georgellis for kind gifts of anti-EscC, anti-EspC, and anti-ArcB antibodies. We thank Santiago Vargas for T3SS cartoon design. We acknowledge Teresa Ballado, Javier de la Mora, and Juan Barbosa for excellent technical assistance. E.S. and M.O.G. were supported by fellowships (349951 and 262002) from the Consejo Nacional de Ciencia y Tecnología (CONACyT). This work was supported by grants from the Dirección General de Asuntos del Personal Académico, UNAM (grant IN209514), and from CONACyT (grant 180460) to B.G.-P.


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