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Journal of Bacteriology, November 2005, p. 7738-7752, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7738-7752.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mapping of a YscY Binding Domain within the LcrH Chaperone That Is Required for Regulation of Yersinia Type III Secretion

Jeanette E. Bröms,1,2 Petra J. Edqvist,2 Katrin E. Carlsson,2 Åke Forsberg,1,2 and Matthew S. Francis2*

Department of Medical Countermeasures, Swedish Defence Research Agency, FOI NBC-Defence, SE-901 82 Umeå,1 Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden2

Received 7 April 2005/ Accepted 29 August 2005


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ABSTRACT
 
Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a {Delta}yscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.


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INTRODUCTION
 
Many animal and plant pathogenic bacteria utilize a common type III secretion system (T3SS) to cause disease (26, 41). A syringe-like translocon extending from a bacterium is thought to inject toxic proteins directly into host cells (38, 44). Infected cells become disarmed of their innate defenses, and this enables establishment of often-lethal infections (16, 65, 83). A unique feature of all T3SSs is their requirement for dedicated cytosolic accessory proteins (chaperones) to specifically bind one, or at most a few, cognate substrates to ensure their presecretory stabilization and/or efficient targeting to the type III secretion machinery (22, 53, 55). Recent high-resolution structural analysis suggests that these chaperones maintain their cargo in a partially nonfolded conformation, ensuring their efficient secretion (64). However, there is a clear structural demarcation between chaperones of the effector class (those that bind one or more substrates, which are destined for translocation into target cells) and chaperones of the translocator class (those that bind two substrates that are essential for translocation of the effectors), since only this latter class contains tetratricopeptide repeat (TPR) motifs (54). Not only are these TPRs required for chaperone function, but their inherent flexibility allows the chaperones to recognize the two cognate translocator substrates differently (21a).

LcrH (also termed SycD) of pathogenic Yersinia spp. is a translocator class chaperone responsible for the presecretory stabilization and efficient secretion of the translocator proteins YopB and YopD (24, 51, 75). YopD possesses two distinct LcrH binding domains, one spanning the N terminus and one encompassing the C-terminal amphipathic domain (24), while no discrete binding domains were observed in YopB (51). Interestingly, LcrH (2, 25) and other similar chaperones, like SicA of Salmonella enterica (14, 71), IpgC of Shigella flexneri (46), and SycB of Y. enterocolitica (73), are involved in regulation of gene expression and the ordered secretion of type III substrates. In Yersinia, LcrH-YopD complex formation is an important regulatory event (2, 25, 52), as is binding to the T3SS component YscY (25). However, another LcrH homologue, PcrH of Pseudomonas aeruginosa (1, 6), does not influence system regulation in this pathogen, nor can it complement the regulatory defect of an {Delta}lcrH null mutant of Yersinia, despite its capacity to bind, stabilize, and promote efficient secretion of the YopD regulatory element (6). This suggests that LcrH contains a distinct regulatory domain, not present in PcrH, which is required for maintenance of controlled type III secretion in Yersinia.

In this study we sought to locate key domains of LcrH that define it as an important T3SS regulator. We established chimeras between LcrH and PcrH in which fusion points were determined by the borders of the recently defined TPR sequences (54). This enabled the mapping of a distinct regulatory domain, independent of YopD binding, to the N terminus of LcrH, located just upstream of the first TPR. This region also contributed to the YscY binding ability of the chaperone. Moreover, since PcrH was unable to bind to either YscY or the homologue Pcr4 of P. aeruginosa, and since Pcr4 could not complement a {Delta}yscY null mutant, we envisage the LcrH-YscY complex to be a specific regulatory mechanism of type III secretion in pathogenic Yersinia.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Unless otherwise indicated, bacteria were routinely cultivated in Luria-Bertani (LB) agar or broth at either 26°C (Y. pseudotuberculosis) or 37°C (Escherichia coli, P. aeruginosa) with aeration. Where appropriate, antibiotics were added at the following final concentrations: ampicillin (Amp; 100 µg per ml), chloramphenicol (Cm; 25 µg per ml), gentamicin (Gm; 20 µg per ml), kanamycin (Km; 50 µg per ml), and tetracycline (Tet; 15 µg per ml).


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  		TABLE 1. Bacterial strains and plasmids used in this study

DNA amplification by PCR. All primer combinations used to amplify wild-type Y. pseudotuberculosis YPIII/pIB102- or P. aeruginosa PAK-specific DNA are listed in Table 2. Amplified DNA was confirmed by sequence analysis using the DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences, Uppsala, Sweden) by first cloning into the pCR4-TOPO TA cloning vector (Invitrogen AB, Stockholm, Sweden).


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  		TABLE 2. Oligonucleotides used in this study

Construction of mutants in P. aeruginosa and Y. pseudotuberculosis. Overlap PCR (39) was used in the construction of five suicide plasmids, pJEB297, pJEB298, pMF535, pMF534, and pJEB342, used to create the respective {Delta}pcr3 and {Delta}pcr4 mutations in wild-type P. aeruginosa PAK and the {Delta}yscX {Delta}yscY and {Delta}yscX yscY mutations in wild-type Y. pseudotuberculosis YPIII/pIB102. The resulting 1,037-, 1,026-, 640-, 550-, and 557-bp PCR fragments containing sequence flanking the pcr3, pcr4, yscX, and yscY genes, respectively, were introduced into EcoRI-HindIII-digested pEX18Gm (37) to give pJEB297 and pJEB298 or XhoI-XbaI-digested pDM4 (49) to give pMF535, pMF534, and pJEB342. Conjugal mating experiments using S17-1{lambda}pir as the donor strain allowed for the allelic exchange of the pJEB297 and pJEB298 suicide plasmids within regions of complementary sequences on the PAK chromosome and the pMF535, pMF534, and pJEB342 plasmids within regions of complementary sequences on the Yersinia virulence plasmid as described previously (49). The resulting mutants were denoted PAKpcr3 (a near-full-length in-frame deletion of codons 7 to 116 in Pcr3), PAKpcr4 (7 to 101 in Pcr4), YPIII/pIB880 (24 to 106 in YscX), YPIII/pIB890 (14 to 90 in YscY), and YPIII/pIB881 (from codon 24 of YscX to codon 90 of YscY). Finally, the {Delta}yscX yscY lcrH triple mutant (YPIII/pIB881-650) was constructed by the same allelic exchange procedure after introducing the mutagenesis vector pMF160, which contained a gene deletion of lcrH that removed codons 2 to 157 (24), into YPIII/pIB881.

Generation of trans-complementing expression plasmids. DNA fragments encoding FLAG-tagged chaperone chimeras were generated by overlap PCR (39) and subsequently introduced into HindIII-SalI-digested pMMB66HE (30). For construction of the PcrHNEISS variant, the Altered Sites II in vitro mutagenesis system (Promega) was used as specified by the manufacturer. pcrH (pJEB85)-specific template for mutagenesis is a derivative of pALTER-Ex1. The resulting mutagenized gene pcrHRGLSE(30-34)NEISS in pALTER-Ex1 (pJEB87) was used as PCR template to generate a FLAG-tagged HindIII-SalI fragment for cloning into pMMB66HE. To clone yscX, yscY, pcr3, and pcr4 with their native ribosome binding sites, amplified products were introduced into EcoRI-HindIII- or EcoRI-PstI-digested pMMB67EHgm (30). Plasmids were transferred into Y. pseudotuberculosis and P. aeruginosa by conjugation.

Protein stability. Intrabacterial protein stability was assessed by the method of Feldman and colleagues (23). Protein fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting using {alpha}-FLAG M2 monoclonal antiserum to detect chaperone chimeras (Sigma-Aldrich Sweden AB, Stockholm, Sweden) or a rabbit polyclonal antisera specifically recognizing YopD (AgriSera AB, Vännäs, Sweden) in combination with the enhanced chemiluminescence system (Amersham Biosciences).

Growth phenotypes and the MOX test. Determination of Yersinia plating frequencies and subsequent growth phenotypes under high- and low-Ca2+ conditions at 37°C were assessed using the MOX (magnesium oxalate) test (3, 32). In some cases, the growth phenotype was assessed after growth at 37°C in liquid TMH medium under high- and low-Ca2+ conditions (25, 52, 67). For definitions of growth phenotypes, see Table 3.


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  		TABLE 3. Growth phenotypes and plating frequencies of Yersinia pseudotuberculosis strains

Synthesis and secretion of type III-secreted substrates. Induction of type III substrate synthesis and secretion from Y. pseudotuberculosis and P. aeruginosa was performed as previously described (6, 24, 25). Total protein levels were assessed by sampling directly from the bacterial culture suspension containing a mix of proteins secreted to the culture medium and within intact bacteria. Sampling the cleared supernatant allowed assessment of secreted protein levels. All protein fractions were separated by SDS-PAGE and subjected to immunoblotting. Detection of specific proteins on membrane support was achieved by the use of rabbit polyclonal antisera raised against all secreted Yops or antisera specifically recognizing YopH, LcrV, or YopD (Yersinia substrates) as well as ExoS or PcrV (P. aeruginosa substrates) (AgriSera AB).

Cultivation and infection of HeLa cells. The human epithelial cell line HeLa was used in all in vitro infection experiments. Culture maintenance and infections with Yersinia (68) or P. aeruginosa (69) followed our standard methods, except that isopropyl-ß-D-thiogalactopyranoside (IPTG) (0.4 mM) was added to both bacteria and cell monolayers prior to infection. The cytotoxicity of infected HeLa cells was monitored by light microscopy, and images were collected at successive time points.

Yeast plasmid construction and transformation and the two-hybrid assay. Interaction studies in yeast between YscY (25) and full-length or hybrid chaperones required plasmid construction of hybrid alleles essentially generated as described in the section above on generation of trans-complementing expression plasmids by cloning of amplified DNA without the C-terminal FLAG epitope into the EcoRI/BamHI-digested GAL4 activation domain plasmid pGAD424 (Clontech Laboratories, Palo Alto, CA). Generation of PcrHNEISS fused to the GAL4 activation domain required initial amplification by PCR using the template pJEB87. For YscY and YscX interaction studies, EcoRI/BamHI-digested yscX was lifted from plasmid pSL122 (unpublished data) and introduced into the GAL4 activation domain plasmid pGADT7 (Clontech Laboratories) to give pPJE026. To investigate Pcr4-Pcr3 binding, PCR-amplified pcr4 was cloned into BamHI/PstI-digested pGBKT7, forming pPJE024, while pcr3 was cloned into EcoRI/XhoI-digested pGADT7, giving pPJE025.

Transformation of the Saccharomyces cerevisiae reporter strain PJ69-4A was performed as described earlier (24). Protein interactions from multiple independent transformations were determined by measuring the activation of the ADE2 reporter gene and the HIS3 reporter gene. For the latter assays, 1 mM 3-aminotriazole was used in the growth medium to overcome any risk of false positives (43). Analysis of protein stability in yeast was performed as previously described (24). However, it necessitated the lifting of GAL4 activation domain (GAL4-AD) fusions from pGAD424 into pGADT7 and GAL4 DNA binding domain (GAL4-BD) fusions from pGBT9 to pGBKT7, since these vectors are more suitable for protein expression studies in yeast (6, 24, 25).

Nucleotide sequence accession number. The nucleotide sequence incorporating pcr3 and pcr4 has been deposited in GenBank (accession number DQ000666).


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RESULTS
 
PcrH is unable to restore regulatory control in the {Delta}lcrH null mutant of Y. pseudotuberculosis. Although the PcrH chaperone of P. aeruginosa can substitute for LcrH in Yersinia to ensure correct assembly of a functional translocon, it is not able to restore yop-regulatory control, consistent with PcrH being dispensable for control of P. aeruginosa type III secretion (6). To investigate this difference in regulatory potential, we first compared the expression levels of C-terminally FLAG-tagged pcrH and lcrH under the control of the Ptac IPTG-inducible promoter from pMMB66HE. As detected by the anti-FLAG antibody, the PcrH levels produced in Yersinia when grown in media supplemented with 0.4 mM of IPTG was roughly equivalent to the level of LcrH recovered from 0.01 mM IPTG induction (Fig. 1). To determine if this low level of PcrH could explain the lack of complementation of the regulatory defect of the {Delta}lcrH null mutant (6), we examined the minimal level of LcrH required to complement this same mutant. As determined by a MOX plate analysis (3, 32), an {Delta}lcrH null mutant of Y. pseudotuberculosis did not grow at 37°C (termed the temperature-sensitive [TS] growth phenotype) regardless of Ca2+ concentration in the medium (Table 3). Reflecting a loss of regulatory control, this TS phenotype mirrored constitutive Yop synthesis during these same growth conditions (6, 25). In contrast, wild-type Y. pseudotuberculosis required Ca2+ to grow at 37°C (termed the calcium-dependent [CD] growth phenotype) (Table 3) and reflected normal yop-regulatory control (3, 56, 81). Significantly, as little as 0.004 mM IPTG was sufficient to induce expression of enough LcrH to restore regulatory control to the {Delta}lcrH null mutant by virtue of a change in growth phenotype from TS to CD (Fig. 1). However, the amount of LcrH was considerably less than the levels of noncomplementing PcrH resulting from 0.4 mM IPTG induction (Fig. 1). Therefore, we conclude that low expression levels do not account for the inability of PcrH to complement the regulatory defect of the {Delta}lcrH null mutant.



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  		FIG. 1. Comparative levels of PcrH and LcrH produced in trans by the Y. pseudotuberculosis {Delta}lcrH null mutant. Immunoblot of chaperone protein prepared from bacteria grown in Yop-inducing medium (BHI minus Ca2+) supplemented with a range of IPTG concentrations (0 to 0.4 mM). Both PcrH and LcrH were identified using monoclonal anti-FLAG antiserum in combination with enhanced chemiluminescence detection. Also indicated is the concentration of IPTG required to generate enough LcrH for trans-complementation of the {Delta}lcrH null mutant with respect to regulation. Definitions of TS (temperature sensitive) and CD (calcium dependent) can be found in Table 3.

Generation and expression analysis of LcrH/PcrH chaperone chimeras. These findings indicate that LcrH might possess a distinct regulatory domain lacking in PcrH. We therefore generated a series of LcrH/PcrH chimeras to identify regulatory domains specific to LcrH. The fusion point for the chimeras was placed at junctions of the recurring TPR motif (Fig. 2A) (54), resulting in hybrid chaperones that initiated with LcrH and contained increasing amounts of LcrH extending from the N terminus (Hyb1 to Hyb4) or initiated with PcrH and contained increasing amounts of C-terminal LcrH (Hyb5 to Hyb8) (Fig. 2B). Alleles were cloned under the control of the Ptac promoter of pMMB66HE with a C-terminal FLAG tag to facilitate immunodetection. We used an intrabacterial protein stability assay (23, 52) to investigate the stability of each chaperone chimera when expressed in an {Delta}lcrH null mutant background grown in non-secretion-permissive medium (brain heart infusion medium [BHI] plus Ca2+) in the presence of IPTG. Although some hybrids were slightly more susceptible to endogenous proteases, all hybrids were essentially stable (Fig. 3A). Levels of synthesis of each hybrid in secretion-permissive medium (BHI minus Ca2+) were also examined. N-terminal PcrH chaperone variants behaved like full-length PcrH, in that they were produced at lower levels compared to those of variants initiated by LcrH (Fig. 3B). We interpret these results to indicate that the hybrid chaperones appear to behave in a manner similar to that of the parental chaperones.



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  		FIG. 2. Schematic representation of the chaperone hybrids used in this study. Shown is a sequence alignment between LcrH of Y. pseudotuberculosis and PcrH of P. aeruginosa (A), where the positions of the three tetratricopeptide repeat (TPR) regions are indicated by various shades of gray. The fusion points at positions Hyb1 to Hyb10 are indicated for chaperone hybrids derived from LcrH (dark gray) and PcrH (light gray) (B). Block diagrams are not drawn to scale.



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  		FIG. 3. Stability and expression analysis of chaperone hybrids and their effect on substrate stability. (A) The intrabacterial stability of chaperone hybrids (left panel) and YopD substrate (right panel) produced by Y. pseudotuberculosis grown at 37°C in BHI supplemented with 2.5 mM CaCl2 (type III secretion repressed) and 0.4 mM IPTG was examined. At time zero, chloramphenicol was added in order to stop de novo protein synthesis. Samples from pelleted bacteria were taken at different time intervals, and the amount of protein was detected by Western blot. (B) Immunoblot of synthesized chaperone hybrids prepared from pelleted bacteria grown in Yop inducing medium (BHI minus Ca2+) supplemented with 0.4 mM IPTG. Hybrids were identified using monoclonal anti-FLAG antiserum and YopD by a polyclonal rabbit anti-YopD antiserum.

The N terminus of LcrH contains a unique regulatory domain. To examine the ability of these chaperone chimeras to restore yop-regulatory control in the {Delta}lcrH null mutant, a MOX test was performed in combination with a Western blot analysis of the total levels of Yops and LcrV synthesized (total samples) and secreted (cleared supernatants) during growth in inductive (BHI without Ca2+) and noninductive (BHI with Ca2+) media. Significantly, trans-production of full-length LcrH and chaperone chimeras Hyb1 to Hyb4 were all able to efficiently restore the growth phenotype of the null mutant from TS to a wild-type-like CD growth phenotype (Table 3) such that elevated levels of YopH and LcrV were produced and secreted only in inductive medium (Fig. 4). In contrast, the lcrH null mutant alone and that harboring full-length PcrH or the chaperone chimeras Hyb5 to Hyb8 all remained sensitive to the temperature up-shift (TS) (Table 3) and constitutively produced proteins in both inductive and noninductive media (Fig. 4, upper panel). In addition, these same strains also specifically secreted LcrV in non-secretion-competent medium (BHI with Ca2+) (Fig. 4, lower panel), which is a reproducible phenomenon observed in all mutants of lcrH or yopD exhibiting a TS phenotype (5, 6, 24, 25, 27, 63, 76).



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  		FIG. 4. Analysis of Yops and LcrV synthesis and secretion from Y. pseudotuberculosis strains grown either with (+) or without (–) Ca2+. Yops and LcrV in the total protein fraction (a mix of proteins secreted to the culture medium and contained within intact bacteria) (upper panel) or secreted to the extracellular medium (cleared culture supernatants) (lower panel) were separated by SDS-PAGE and identified by immunoblot analysis using polyclonal rabbit anti-YopH, anti-LcrV, and anti-YopD antiserum. Where indicated, IPTG was added at a final concentration of 0.4 mM upon temperature shift. Lanes: a and b, wild-type YPIII/pIB102; c and d, lcrH null mutant YPIII/pIB650; e and f, complemented YPIII/pIB650, pJEB133 (LcrH+); g and h, complemented YPIII/pIB650, pJEB121 (Hyb1+); i and j, complemented YPIII/pIB650, pJEB122 (Hyb2+); k and l, complemented YPIII/pIB650, pJEB123 (Hyb3+); m and n, complemented YPIII/pIB650, pJEB124 (Hyb4+); o and p, complemented YPIII/pIB650, pJEB125 (Hyb5+); q and r, complemented YPIII/pIB650, pJEB126 (Hyb6+); s and t, complemented YPIII/pIB650, pJEB127 (Hyb7+); u and v, complemented YPIII/pIB650, pJEB128 (Hyb8+); x and y, complemented YPIII/pIB650, pJEB129 (Hyb9+); z and aa, complemented YPIII/pIB650, pJEB199 (Hyb10+); bb and cc, complemented YPIII/pIB650, pJEB132 (PcrHNEISS+); dd and ee, complemented YPIII/pIB650, pJEB130 (PcrH+). Molecular masses shown in parentheses are deduced from primary sequences.

These data highlight a key regulatory domain specific to LcrH that resides within its N-terminal 35 amino acids (i.e., prior to the junction in Hyb1). To better define this regulatory region, two additional chaperone hybrids were generated (Hyb9 and Hyb10 in Fig. 2B). Hyb9 contained 17 and Hyb10 contained 28 N-terminal LcrH residues, followed by the remainder of PcrH. Both hybrids displayed a similar degree of stability (Fig. 3A) and were expressed at levels comparable to that of wild-type LcrH (Fig. 3B). However, when expressed in an {Delta}lcrH null mutant, neither hybrid could alter the temperature sensitivity of this mutant (Table 3) or its failure to regulate Yop synthesis and secretion (Fig. 4). Thus, the region of LcrH important for yop regulation is found between the boundaries of Hyb10 and Hyb1.

Closer inspection of this small region revealed that 5 residues, NEISS, located at positions 29 to 33 of LcrH, differ significantly from PcrH, which encodes RGLSE. We had previously identified the glutamate moiety at position 30 of LcrH as an important regulatory requirement (25). To further investigate this amino acid stretch, we replaced the RGLSE residues of PcrH with the NEISS residues of LcrH (Fig. 2B). This PcrHNEISS variant behaved like wild-type PcrH with respect to stability (Fig. 3A) and expression (Fig. 3B). In addition, it was unable to restore regulatory control when expressed in Yersinia defective for LcrH (Table 3, Fig. 4). We conclude that N-terminal LcrH is essential for controlled Yop synthesis; however, (an)other residue(s) in addition to NEISS at position 29 to 33 must also contribute to this function.

Loss of regulatory control is not due to alterations in YopD levels. One consequence of manipulating LcrH might be altered stability of YopD (24, 75), another key regulatory element in Yop synthesis (27, 76). To investigate whether the N-terminal-dependent regulatory function of LcrH is independent of YopD, we analyzed the ability of each chaperone hybrid to maintain normal stability and secretion of YopD when expressed in the {Delta}lcrH null mutant of Y. pseudotuberculosis. Bacteria were grown in non-secretion-permissive medium (BHI with Ca2+) in the presence of IPTG, and protein synthesis was blocked by the addition of chloramphenicol after 1 h. No major difference in the stability of YopD over time was observed for strains expressing the different hybrid chaperones, although regulatory competent hybrids Hyb1 and Hyb4 resulted in slightly less stable YopD (Fig. 3A). Furthermore, growth in secretion-permissive conditions (BHI without Ca2+) resulted in a comparable level of YopD secretion from all strains, except for the noncomplemented {Delta}lcrH null mutant used as a control (Fig. 4, lower panel). Since YopD is also a key element of the Yersinia translocon (27, 52, 76), we used the HeLa cell infection assay to determine whether the chimera expressing mutants were competent for translocation of the cytotoxin YopE. All mutants efficiently translocated YopE into infected HeLa cells, as visualized by a rapid cell rounding up (data not shown), further supporting maintenance of functional YopD. Thus, the failure to regain regulatory control in the {Delta}lcrH null mutant harboring PcrH, PcrHNEISS, or Hyb5 to Hyb10 is not due to destabilization of the YopD regulatory element, confirming that the N terminus of LcrH does contain a unique YopD-independent regulatory domain(s) directly involved in yop-regulatory control.

LcrH specifically establishes a regulatory complex with YscY of the Yersinia type III secretion machine. We have recently proposed a regulatory role for LcrH that involves binding a component of the Ysc (Yersinia secretion) secretion machine, YscY (25). Having defined an N-terminal region of LcrH that is important for yop regulation, we wondered whether this regulatory domain was related to the ability of LcrH to bind YscY. Using two independent promoter reporter assays (GAL2-ADE2 and LYS2::GAL1-HIS3) in the yeast two-hybrid system, we could confirm our earlier report that LcrH does interact with YscY (Table 4) (25). Furthermore, we observed that Hyb2 to Hyb4 and Hyb8 could also strongly bind to YscY, while Hyb1 displayed weaker binding. In contrast, PcrH, PcrHNEISS, and all remaining hybrids were all unable to form this complex as implied from a lack of growth on the dropout plates (Table 4). This was not due to instability of these chaperone variants in yeast, as most were readily detected in yeast protein lysates (Fig. 5A). Therefore, with one exception (Hyb8), these results highlight an intriguing correlation between the ability of a given chaperone hybrid to regulate Yop synthesis and its capacity to bind YscY of the type III secretion machine.


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  		TABLE 4. Protein-protein interactions in the yeast two-hybrid assaya



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  		FIG. 5. Expression of chaperone hybrids and other type III secretion components in Saccharomyces cerevisiae PJ69-4A. Protein extracts were generated from yeast harboring (A) lcrH (pLcrH+), hybrids (pHyb1+ to pHyb10+), and wild-type or mutated pcrH (pPcrH+ and pPcrHNEISS+) fused to the GAL4 activation domain of pGADT7, respectively; (B) yscX (pYscX+) and pcr3 (pPcr3+) fused to the GAL4 activation domain of pGADT7; and (C) yscY (pYscY+) and pcr4 (pPcr4+) fused to the GAL4 DNA binding domain plasmid pGBKT7. Samples were separated by SDS-PAGE, and recombinant proteins were identified by immunoblot analysis using a mouse hemagglutinin (HA) monoclonal antibody (mAb) (clone 12CA5) (Roche AB, Stockholm, Sweden) (A and B) or a GAL4-BD monoclonal antibody (Clontech Laboratories) (C). In each case, a protein extract from PJ69-4A harboring pGADT7 (A and B) or pGBKT7 (C) alone (vector) was included as a negative control. The arrowhead indicates the lower-molecular-weight protein band of interest.

The LcrH N terminus is obviously important for these functions. We therefore asked whether the first 35 residues of LcrH were sufficient to bind YscY in yeast. However, no interaction was observed (Table 4). This is likely explained by the phenotype of Hyb8, which could readily interact with YscY, even though control of Yop synthesis was not established when expressed in Yersinia. As Hyb8 lacks the NEISS domain required for yop-regulatory control, perhaps this indicates that full YscY binding actually requires two regions: the NEISS regulatory domain between the borders of Hyb10 and Hyb1 and also a flanking domain between the borders of Hyb8 and Hyb7 (encompassing the first TPR motif of LcrH). This is further supported by the phenotype of Hyb1, which lacks this latter domain and only moderately interacts with YscY.

The LcrH-YscY complex appears to be a strategic regulatory mechanism in Yersinia type III secretion. If so, one prediction would be that PcrH would not interact with the YscY homologue, Pcr4 of P. aeruginosa (80). Indeed, we could not detect this interaction using the two-hybrid system (Table 4), although both proteins were stably expressed (Fig. 5A and C). While this does not rule out the possibility of a weak interaction occurring in vivo, the LcrH-YscY complex does seem unique to Yersinia.

Functional analysis of pcr3 and pcr4 in P. aeruginosa type III secretion. Little is known about the role of YscY in Yersinia type III secretion, except that it might function as a chaperone aiding in the presecretory stabilization and efficient secretion of YscX, a component promoting functional type III secretion (15, 42). The homologs of these two proteins in P. aeruginosa T3SS are 49% (Pcr4) and 48% (Pcr3) identical to their respective Yersinia counterparts (80). We generated {Delta}pcr3 and {Delta}pcr4 null mutants and found both Pcr3 and Pcr4 to be essential for P. aeruginosa type III secretion, since ExoS, ExoT, and PcrV were not secreted by mutant bacteria grown in low-calcium conditions (Fig. 6, compare lanes d and j with b). Accordingly, these mutants were also unable to deliver effector substrates (exoenzymes) into infected HeLa cells (Fig. 7, compare panels B and E with A). As expected, type III secretion (Fig. 6, compare lanes f and l with b) and effector translocation (Fig. 7, compare panels C and F with A) could be restored by providing Pcr3 or Pcr4 in trans under an IPTG-inducible promoter. Furthermore, Pcr4 specifically bound Pcr3 in the yeast two-hybrid system (Table 4). Therefore, Pcr4 probably chaperones Pcr3 for efficient secretion in P. aeruginosa, which in turn is necessary for building up a functional T3SS.



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  		FIG. 6. Analysis of type III protein secretion from P. aeruginosa strains grown either with (+) or without (–) Ca2+. Proteins secreted to the culture medium were separated by SDS-PAGE and identified by immunoblotting using polyclonal rabbit anti-ExoS (cross-reacts with ExoT) or anti-PcrV antisera. Where indicated, IPTG was added at a final concentration of 0.4 mM. Lanes: a and b, wild-type PAK; c and d, pcr3 null mutant PAKpcr3; e and f, complemented PAKpcr3, pJEB295 (Pcr3+); g and h, complemented PAKpcr3, pJEB291 (YscX+); i and j, pcr4 null mutant PAKpcr4; k and l, complemented PAKpcr4, pJEB296 (Pcr4+); m and n, complemented PAKpcr4, pJEB292 (YscY+). Molecular masses shown in parentheses are deduced from primary sequences.



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  		FIG. 7. Infection of HeLa cells by P. aeruginosa. Strains were allowed to infect a monolayer of growing HeLa cells. At 3 h after infection, the effect of the bacteria on the HeLa cells was recorded by phase-contrast microscopy. Note the extensive rounding up of the ExoS-dependent, cytotoxically affected HeLa cells (A, C, D, F, and G). HeLa cells infected with the {Delta}pcr3 or {Delta}pcr4 mutant show normal cell morphology (compare B and E with H), even after prolonged infection for up to 6 h (data not shown). Shown are phase-contrast images of cells infected with wild-type PAK (A), {Delta}pcr3 mutant PAKpcr3 (B), trans-complemented PAKpcr3, pJEB295 (Pcr3+) (C), trans-complemented PAKpcr3, pJEB291 (YscX+) (D), {Delta}pcr4 mutant PAKpcr4 (E), trans-complemented PAKpcr4, pJEB296 (Pcr4+) (F), trans-complemented PAKpcr4, pJEB292 (YscY+) (G), or cells left uninfected (H).

yscX and yscY trans-complement P. aeruginosa pcr3 and pcr4 mutants. We further assessed the functional similarities between YscX/Pcr3 and YscY/Pcr4. Using the two-hybrid system, reciprocal binding for Pcr4 with YscX and YscY with Pcr3 was observed (Table 4). In agreement with these findings, YscX and YscY efficiently restored type III secretion (Fig. 6, compare lanes h and n with b) and effector translocation (Fig. 7, compare panels D and G with A) when expressed in P. aeruginosa pcr3 and pcr4 mutants, respectively. Intriguingly, however, expression of pcr3 and pcr4 could not restore the defects in yop regulation, secretion, or translocation caused by depletion of yscX or yscY in Yersinia. Like the noncomplemented mutants, these strains displayed calcium-independent growth phenotypes (Table 3), were severely down-regulated for yop expression and secretion (Fig. 8, compare lanes h and n with d and j), and consequently failed to translocate Yop effectors into HeLa cells during infection (Fig. 9, compare panels D and G with B and E). Importantly, these same mutants could be complemented by the native components (Table 3, Fig. 8, compare lanes f and l with b, and 9, compare panels C and F with A). Thus, the ability to efficiently bind YscY and YscX, respectively, is not sufficient to permit Pcr3 and Pcr4 to participate in Yersinia yop regulation.



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  		FIG. 8. Analysis of Yop synthesis and secretion from Y. pseudotuberculosis strains grown either with (+) or without (–) Ca2+. Yops in the total protein fraction (a mix of proteins secreted to the culture medium and contained within intact bacteria) (upper panel) or secreted to the extracellular medium (cleared culture supernatants) (lower panel) were separated by SDS-PAGE and identified by immunoblot analysis using a polyclonal rabbit antiserum recognizing secreted Yops. Where indicated, IPTG was added at a final concentration of 0.4 mM upon temperature shift. Lanes: a and b, wild-type YPIII/pIB102; c and d, yscX null mutant YPIII/pIB880; e and f, complemented YPIII/pIB880, pJEB291 (YscX+); g and h, complemented YPIII/pIB880, pJEB295 (Pcr3+); i and j, yscY null mutant YPIII/pIB890; k and l, complemented YPIII/pIB890, pJEB292 (YscY+); m and n, complemented YPIII/pIB890, pJEB296 (Pcr4+). Molecular masses shown in parentheses are deduced from primary sequences.



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  		FIG. 9. Infection of HeLa cells by Y. pseudotuberculosis. Strains were allowed to infect a monolayer of growing HeLa cells. At 3 h after infection, the effect of the bacteria on the HeLa cells was recorded by phase-contrast microscopy. Note the extensive rounding up of the YopE-dependent, cytotoxically affected HeLa cells (A, C, and F). HeLa cells infected with the {Delta}yscX or {Delta}yscY mutant show normal cell morphology (compare B and E with H), even after prolonged infection for up to 6 h (data not shown). Shown are phase-contrast images of cells infected with wild-type YPIII/pIB102 (A); yscX null mutant YPIII/pIB880 (B); complemented YPIII/pIB880, pJEB291 (YscX+) (C); complemented YPIII/pIB880, pJEB295 (Pcr3+) (D); yscY null mutant YPIII/pIB890 (E); complemented YPIII/pIB890, pJEB292 (YscY+) (F); complemented YPIII/pIB890, pJEB296 (Pcr4+) (G); and cells left uninfected (H).

Our earlier work determined that some P. aeruginosa components, such as PcrV and PopD, function poorly in Yersinia type III secretion unless they are coexpressed (5, 7). This made us wonder whether coexpression of Pcr3 and Pcr4 would make them able to complement the corresponding {Delta}yscX yscY double mutant of Yersinia. Not surprisingly, this mutant is unable to secrete or translocate Yops and is down-regulated for Yop synthesis (Table 3, Fig. 10, and data not shown). Although it could be trans-complemented by coexpression of native YscX and YscY, similarly expressed Pcr3 and Pcr4 still could not alter the mutant phenotypes (Table 3, Fig. 10, and data not shown). Importantly, this Pcr3 and Pcr4 coexpression construct is functional in P. aeruginosa, capable of complementing both pcr3 and pcr4 null mutants (data not shown). Furthermore, a {Delta}yscX yscY lcrH triple mutant in which Yop synthesis is constitutively up-regulated due to the loss of LcrH, even when in the absence of Yop secretion (Table 3, Fig. 10), could not be complemented by coexpression of PcrH, Pcr3, and Pcr4. Oddly, Yop synthesis was repressed in this strain (Fig. 10), which was consistent with a calcium-independent growth phenotype (Table 3). However, despite the technical challenges associated with two-plasmid expression in bacteria, controlled Yop synthesis, secretion and translocation, and also wild-type growth were restored in this mutant by the coexpression of native LcrH, YscX, and YscY (Table 3, Fig. 10, and data not shown). Thus, these collective data add further credence to our notion of different regulatory networks within these two pathogens. In Yersinia, a tight link between LcrH, YscY, and YscX is required for regulatory control, whereas PcrH-independent mechanisms appear to ensure regulatory control in P. aeruginosa.



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  		FIG. 10. Analysis of Yop synthesis and secretion from Y. pseudotuberculosis strains grown either with (+) or without (–) Ca2+. The protocol is essentially the same as that described in the legend to Fig. 8. Lanes: a and b as well as i and j, wild-type YPIII/pIB102; c and d, yscX yscY null mutant YPIII/pIB881; e and f, complemented YPIII/pIB881, pJEB340 (YscX, YscY+); g and h, complemented YPIII/pIB881, pJEB335 (Pcr3, Pcr4+); k and l, lcrH yscX yscY null mutant YPIII/pIB881-650; m and n, complemented YPIII/pIB881-650, pPJE020 (LcrH+), pJEB340 (YscX YscY+); o and p, complemented YPIII/pIB881-650, pKEC005 (PcrH+), pJEB335 (Pcr3 Pcr4+). Molecular masses shown in parentheses are deduced from primary sequences.


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DISCUSSION
 
The recent discovery that type III chaperones are regulatory molecules is a key development, representing an ingenious mechanism by which bacteria tightly couple protein synthesis with an ordered secretion of substrates (28, 48). In Yersinia, the LcrH chaperone, when in complex with the YopD translocator substrate, represses synthesis of substrates predestined for type III secretion (25) by a posttranscriptional mechanism that involves binding directly to untranslated regions of yop gene mRNA (2, 10). This is consistent with Yop synthesis being derepressed in the absence of LcrH (3, 25, 56) or YopD (27, 52, 76). LcrH utilizes internal TPRs to engage both the YopB and YopD translocators, although this occurs differently (21a). This TPR-mediated binding also impacts on regulation via the formation of LcrH-YopD regulatory complexes. However, we have now identified a unique N-terminal region in LcrH, positioned outside of the TPRs, that is also required for system regulation. A complex with YscY is important for this effect, which therefore represents a new capacity in which LcrH regulates type III secretion. This is based upon several lines of evidence: (i) LcrH-PcrH chaperone hybrids lacking a domain of LcrH within the N terminus do not maintain regulatory control when expressed in an {Delta}lcrH null mutant of Yersinia, (ii) YopD function is unperturbed in these hybrids, (iii) hybrids with the capacity to regulate Yops in Yersinia also bind YscY, (iv) reciprocal binding between Pcr4 with Pcr3 or YscX and YscY with YscX or Pcr3 is not sufficient for Pcr4 or Pcr3 to complement the corresponding null mutants of Yersinia, and (v) PcrH bound neither to Pcr4 nor YscY. Taken together, these data suggest an important role for the LcrH-YscY complex in Yersinia T3SS regulation. It is therefore fascinating how a small molecule like LcrH combines different complexes with YopD and YscY to achieve this regulatory outcome.

Attempts to understand the molecular effect(s) of the LcrH-YscY regulatory complex are thwarted by our inability to pinpoint the role(s) of YscY and its cognate secreted substrate YscX in type III secretion (15, 42). Whatever this role, it appears other homologs may act similarly, since Pcr4 and Pcr3 were found to interact and both were required for functional type III secretion in P. aeruginosa. However, the fact that only YscY and YscX were functional in both bacterial backgrounds reinforces the necessary regulatory cross-talk between YscY, YscX, and LcrH in Yersinia. Furthermore, the role of other known YscY, YscX, and LcrH homologs found in Aeromonas spp. (8, 9, 82), Photorhabdus luminescens (18, 74), Vibrio spp. (36, 45), and Desulfovibrio vulgaris Hildenborough (35) remains unknown. Interestingly, as the regulatory role of LcrH involves LcrQ (10), a molecule unique to Yersinia (60), the LcrH-YscY-YscX regulatory loop is even more likely to be confined to Yersinia. Of interest is whether this newly discovered loop marks a branching from the established LcrH-YopD-LcrQ regulatory network (2, 10, 25). If this were true, perhaps LcrH could be dissected into functionally distinct domains, whereby the N terminus is linked to YscY and the remainder is linked to the function of YopD. However, our finding that the LcrH N terminus alone was unable to interact with YscY in yeast would indicate that this region still needs to function in the context of full-length LcrH to facilitate controlled Yop synthesis. This suggests a more complex picture and might imply that all components constitute the one regulatory network.

Given that YscX is secreted via the T3SS (15), perhaps this is one regulatory outcome of an LcrH-YscY association. It is therefore important to determine if secretion of YscX is required to permit LcrH-YscY complex formation. Another point to address is the final destination of secreted YscX, whether it is associated with the external type III needle or released directly into the extracellular milieu. In addition, the ratios of LcrH, YscY, and YscX might influence the regulatory status of Yop synthesis, although we did not detect any direct regulatory effect from overexpression of YscY or LcrH in wild-type, lcrH, or yopD null mutant backgrounds (J.E. Bröms, unpublished data). Moreover, just as our detailed mutagenesis of both LcrH and YopD revealed important functional information about the LcrH-YopD complex (24, 25), we are now using a similar approach to try and elucidate the mode of interaction for YscY with LcrH and YscX. Of interest is whether the TPR module of LcrH or YscY (54) is important for complex formation. Also noteworthy is the recent observation that some type III chaperones physically dock to the T3SS at the inner face of the cytoplasmic membrane via an interaction with the ATPase energizer (31, 70), an evolutionarily conserved core component of T3SSs (26, 41). Thus, docking of either LcrH or YscY with the Yersinia-specific ATPase YscN (77) could be another effect of LcrH-YscY complex formation.

It is the N terminus of LcrH that sets it apart from PcrH with respect to maintenance of yop-regulatory control in Yersinia. Interestingly, this appears to occur at two levels. First, a regulatory domain that also contributes to YscY binding lies within a region encompassing residues NEISS at positions 29 to 33 of LcrH. However, two pieces of evidence indicate that this region apparently does not operate alone: PcrH containing NEISS (PcrHNEISS) is unable to restore yop-regulatory control or engage YscY, and the first 35 LcrH residues did not appear to bind YscY. Thus, we propose a second functional level of the LcrH N terminus that makes it distinct from PcrH. This involves a second YscY binding domain that is present in Hyb2 to Hyb4 and Hyb8. The common domain in these four hybrids is the first TPR motif of LcrH located between positions 36 and 69. This site is intriguing, because alone (i.e., in the absence of the NEISS region, such as in Hyb8) it is unable to promote yop-regulatory control despite strong YscY binding. Therefore, it seems that two regions between 29 to 33 and 36 to 69 act in concert to promote strong YscY binding and establish LcrH-dependent control of Yop synthesis. In turn, this would also explain why Hyb1 binds YscY weakly, because it lacks the second YscY binding site incorporating the first TPR of LcrH.

The realization that a PcrH-Pcr4 complex is not required for regulation of P. aeruginosa type III secretion, despite their high identity to components of the Yersinia plasmid-borne system (6, 7, 80), was another fascinating outcome of this study. It is pertinent that the genome of P. aeruginosa is uniquely large, expanded by the presence of many genes coding for putative regulatory factors (66). This is now reflected by a number of independent studies which connect several of these factors with a complex pattern of type III secretion regulation in P. aeruginosa (13, 33, 34, 40, 47, 58, 59, 72, 78, 79). Therefore, the need for a regulatory complex involving PcrH and Pcr4 is likely bypassed by one or more of these numerous regulatory factors.

It is curious that PcrH is poorly expressed in Yersinia. We cannot exclude that this is due to instability of mRNA derived from the construct pJEB130 (pPcrH+) or the poor accessibility of translation initiation signals to the ribosomes. However, since PcrH was still poorly produced when expressed under the LcrH-derived leader sequence and ribosome binding site (data not shown) and transcription initiates from the same promoter (Ptac), this appears unlikely. More important could be that P. aeruginosa has a genomic G+C content of 66.6% (66), compared to 48.9% in Y. pseudotuberculosis (11). As this would impact on the codon usage preference of each organism, the pcrH translational efficiency in Y. pseudotuberculosis might account for these low expression levels. However, we have similarly expressed other components of the P. aeruginosa pcrGVH-popBD operon in Yersinia without any obvious expression restriction (5-7, 29). From our work with the chaperone hybrids, however, the cause of low PcrH expression must reside in the extreme N terminus, since Hyb9, which essentially contained all of PcrH except for the first 17 LcrH amino acids, was expressed at roughly LcrH-like levels in Yersinia. It has been established that the 5' end of many E. coli genes is responsible for control of translation efficiency (12) as well as a wide variety of virulence determinants produced by numerous bacterial pathogens (17, 19, 20, 50, 57, 61). Therefore, minor codons in the extreme N terminus of LcrH might have evolved to control the translation efficiency of this important regulatory molecule, a concept we are currently exploring.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the Carl Tryggers Foundation for Scientific Research (M.S.F.), Swedish Research Council (Å.F. and M.S.F.), Foundation for Medical Research at Umeå University (M.S.F.), Swedish Foundation for Strategic Research (Å.F.), Swedish Cystic Fibrosis Association Research Fund (M.S.F.), Swedish Medical Association (M.S.F.), and the J. C. Kempes Memorial Fund (J.E.B. and P.J.E.).

We thank Sara Eriksson, Yingqi Tang, Rose Cherry, Richard Kneeling, and Peter Steggo for valuable technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden. Phone: 46-(0) 90-7852536. Fax: 46-(0)90-771420. E-mail: matthew.francis{at}molbiol.umu.se. Back


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Journal of Bacteriology, November 2005, p. 7738-7752, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7738-7752.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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