Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion


  • Editor: Craig Winstanley

Correspondence: Matthew S. Francis, Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden. Tel.: +46 90 7852536; fax: +46 90 771420; e-mail: matthew.francis@molbiol.umu.se


The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.


Pseudomonas aeruginosa is a frequent cause of life-threatening infections in patients with compromised immune systems (Lyczak et al., 2000). Underlying the severity of P. aeruginosa infections are multiple mechanisms for adaptation to various host niches, such as quorum sensing (Passador et al., 1993), resistance to antimicrobials (Hancock, 1998; Kriengkauykiat et al., 2005) and numerous secreted and cell-surface-associated virulence determinants like exo- and endotoxins, proteases, pili and a type III secretion system (T3SS) (Salyers & Whitt, 2002). This T3SS can directly translocate effector proteins, called exoenzymes, into eukaryotic cells, where they subvert host signalling and enable the bacteria to overcome the immune system (Vance et al., 2005).

The T3SS translocator proteins PopB and PopD are absolutely essential for toxin translocation by virtue of their pore-forming activity in target cell plasma membranes (Dacheux et al., 2001; Sundin et al., 2002; Schoehn et al., 2003). In part, this activity is governed by intrabacterial PcrH, which controls the presecretory stability of both PopB and PopD (Bröms et al., 2003b). Like other class II T3SS chaperones, PcrH interacts with translocator substrates to allow formation of a functional translocon to ensure effector translocation (Page & Parsot, 2002; Parsot et al., 2003).

Class II chaperones possess three internal tetratricopeptide repeats (TPRs) (Pallen et al., 2003). These TPRs are important for at least one chaperone, LcrH from Yersinia pseudotuberculosis (Edqvist et al., 2005), the function of which is complicated by additional roles in system regulation (Francis et al., 2001; Anderson et al., 2002; Bröms et al., 2003b, 2005). TPRs are not found in class I T3SS chaperones that target secreted effector proteins. These are rather composed of similar α/β folds (Birtalan & Ghosh, 2001; Luo et al., 2001; Stebbins & Galan, 2001; Evdokimov et al., 2002; Phan et al., 2004; Van Eerde et al., 2004; Buttner et al., 2005; Locher et al., 2005; Yip et al., 2005).

Although present in proteins from all origins, most information about TPR function has arisen from studies on eukaryotic proteins. An individual TPR module consists of 34 residues that are usually poorly conserved, with the exception of canonical positions at 8, 20 and 27 occupied by small and sometimes hydrophobic residues. However, alternation between small and large amino acids is a common feature, allowing individual TPRs – each consisting of two antiparallel α-helices – to tightly pack into an all α-helical array revealing distinct concave substrate-binding grooves accommodating various protein–protein interactions (Lamb et al., 1995; Blatch & Lassle, 1999; D'Andrea & Regan, 2003). This fold is also predicted for the class II chaperones (Pallen et al., 2003).

Given the critical roles of class II chaperones in T3S, and that no structure is yet solved, we further investigated the role of the TPRs in this group of proteins. This study focused on PcrH of P. aeruginosa, which, in contrast to LcrH (Edqvist et al., 2005), does not influence system regulation (Bröms et al., 2003b, 2005). Therefore, PcrH could be a good model to study TPR function. TPR residues within PcrH were randomly selected for mutagenesis, revealing their crucial role in chaperone function towards the translocators PopB and PopD.

Materials and methods

Bacterial strains, plasmids and growth conditions

Bacterial strains and plasmids used are listed in Table 1. Routine culturing of Pseudomonas aeruginosa and Escherichia coli occurred in Luria–Bertani (LB) broth or agar (LA) at 37°C with aeration. When necessary, antibiotics of indicated final concentrations were added: gentamicin (Gm; 20 μg mL−1), kanamycin (Km; 50 μg mL−1) or ampicillin (Ap; 100 μg mL−1).

Table 1.   Strains and plasmids used in this study
Strain or plasmidRelevant genotype or phenotypeSource or reference
 Escherichia coli
  TOP10F-mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74,recA1, deoR, araD139, Δ(ara-leu)7679, galU, galK, rpsL (StrR), endA1, nupGInvitrogen
  S17-1λpirrecA, thi, pro, hsdR-M+, SmR, <RP4:2-Tc:Mu:Ku:Tn7>TpRSimon et al. (1983)
 Pseudomonas aeruginosa
  PAKWild-type clinical isolateD. Bradley
  PAKpcrHPAK, pcrH in frame full-length deletion of codons 7-149Bröms et al. (2003b)
  PAKpcrH1PAK, PcrHL63EThis study
  PAKpcrH2PAK, PcrHY41A/F45AThis study
  PAKpcrH3PAK, PcrHC80A/Q82AThis study
  PAKpcrH4PAK, PcrHL77A/R81AThis study
  PAKpcrH5PAK, PcrHA112EThis study
  PAKpcrH6PAK, PcrHA90EThis study
  PAKpcrH7PAK, PcrHE113A/L116AThis study
  PAKpcrH8PAK, PcrHF109A/H110AThis study
  PAKpcrH9PAK, PcrHL75A/G76AThis study
  PAKpcrH10PAK, PcrHG78EThis study
  PAKpcrH11PAK, PcrHI102AThis study
  PAKpcrH12PAK, PcrHA131EThis study
 Saccharomyces cerevisiae
  AH109MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UASGAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ, MEL1Clontech Laboratories
  pCR®4-TOPO®TA cloning vector, KmR, ApRInvitrogen
  pEX18GmSuicide plasmid carrying sacBR, GmRHoang et al. (1998)
  pJEB226pEX18Gm encoding mutant pcrH (PcrHL63E), GmRThis study
  pJEB227pEX18Gm encoding mutant pcrH (PcrHL77A/R81A), GmRThis study
  pJEB228pEX18Gm encoding mutant pcrH (PcrHA90E), GmRThis study
  pJEB229pEX18Gm encoding mutant pcrH (PcrHF109A/H110A), GmRThis study
  pJEB230pEX18Gm encoding mutant pcrH (PcrHA112E), GmRThis study
  pJEB231pEX18Gm encoding mutant pcrH (PcrHY41A/F45A), GmRThis study
  pJEB232pEX18Gm encoding mutant pcrH (PcrHC80A/Q82A), GmRThis study
  pJEB233pEX18Gm encoding mutant pcrH (PcrHE113A/L116A), GmRThis study
  pJEB243pEX18Gm encoding mutant pcrH (PcrHL75A/G76A), GmRThis study
  pJEB244pEX18Gm encoding mutant pcrH (PcrHG78E), GmRThis study
  pJEB245pEX18Gm encoding mutant pcrH (PcrH102A), GmRThis study
  pJEB246pEX18Gm encoding mutant pcrH (PcrHA131E), GmRThis study
  pGADT7LEU2, ApRClontech Laboratories
  pJEB56pGADT7 encoding wild-type pcrH, LEU2, ApRBröms et al. (2003b)
  pJEB251pGADT7 encoding mutant pcrH (PcrHL63E), LEU2, ApRThis study
  pJEB252pGADT7 encoding mutant pcrH (PcrHL77A/R81A), LEU2, ApRThis study
  pJEB253pGADT7 encoding mutant pcrH (PcrHA90E), LEU2, ApRThis study
  pJEB254pGADT7 encoding mutant pcrH (PcrHF109A/H110A), LEU2, ApRThis study
  pJEB255pGADT7 encoding mutant pcrH (PcrHA112E), LEU2, ApRThis study
  pJEB256pGADT7 encoding mutant pcrH (PcrHY41A/F45A), LEU2, ApRThis study
  pJEB257pGADT7 encoding mutant pcrH (PcrHC80A/Q82A), LEU2, ApRThis study
  pJEB258pGADT7 encoding mutant pcrH (PcrHE113A/L116A), LEU2, ApRThis study
  pJEB259pGADT7 encoding mutant pcrH (PcrHG78E), LEU2, ApRThis study
  pJEB260pGADT7 encoding mutant pcrH (PcrHA131E), LEU2, ApRThis study
  pJEB261pGADT7 encoding mutant pcrH (PcrHI102A), LEU2, ApRThis study
  pJEB262pGADT7 encoding mutant pcrH (PcrHL75A/G76A), LEU2, ApRThis study
  pGBKT7TRP1, KmRClontech Laboratories
  pJEB58pGBKT7 encoding wild-type popD, TRP1, KmRBröms et al. (2003b)
  pJEB60pGBKT7 encoding wild-type popB, TRP1, KmRBröms et al. (2003b)
  pMMB67EHgmptac expression plasmid, GmRFürste et al. (1986)
  pJEB317pMMB67EHgm encoding FLAG-tagged PcrHL63E, GmRThis study
  pJEB318pMMB67EHgm encoding FLAG-tagged PcrHY41A/F45A, GmRThis study
  pJEB319pMMB67EHgm encoding FLAG-tagged PcrHC80A/Q82A, GmRThis study
  pJEB320pMMB67EHgm encoding FLAG-tagged PcrHL77A/R81A, GmRThis study
  pJEB321pMMB67EHgm encoding FLAG-tagged PcrHA112E, GmRThis study
  pJEB322pMMB67EHgm encoding FLAG-tagged PcrHA90E, GmRThis study
  pJEB323pMMB67EHgm encoding FLAG-tagged PcrHE113A/L116A, GmRThis study
  pJEB324pMMB67EHgm encoding FLAG-tagged PcrHF109A/H110A, GmRThis study
  pJEB325pMMB67EHgm encoding FLAG-tagged PcrHL75A/G76A, GmRThis study
  pJEB326pMMB67EHgm encoding FLAG-tagged PcrHG78E, GmRThis study
  pJEB327pMMB67EHgm encoding FLAG-tagged PcrHI102A, GmRThis study
  pJEB328pMMB67EHgm encoding FLAG-tagged PcrHA131E, GmRThis study
  pJEB329pMMB67EHgm encoding FLAG-tagged wild-type PcrH, GmRThis study

Site-directed mutagenesis of pcrH

Primer pairs used for site-directed mutant construction are listed in Table S1 (supplementary material). By overlap PCR (Horton & Pease, 1991), ∼1430 bp DNA fragments containing sequence flanking a specific mutation in pcrH were amplified from wild-type P. aeruginosa PAK template DNA. Fragments were confirmed by routine sequence analysis after introduction into PCR® 4-TOPO® (Invitrogen AB, Stockholm, Sweden) before subcloning into EcoRI-digested suicide mutagenesis vector, pEX18Gm (Hoang et al., 1998). Escherichia coli S17-1λpir was used as the donor strain in conjugal mating experiments with the ΔpcrH null mutant PAKpcrH of P. aeruginosa (Bröms et al., 2003b). Selection of the allelic exchange events that reconstituted the respective full-length deletion mutants followed established methods (Milton et al., 1996; Bröms et al., 2003b).

Chaperone stability

Stability analysis of each C-terminal FLAG-tagged PcrH substitution mutant expressed in trans was by the method of Feldman et al. (2002). Protein fractions were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting using mouse α-FLAG monoclonal antiserum (Sigma, Stockholm, Sweden).

Analysis of substrate synthesis and secretion

Induction of T3S by P. aeruginosa has been described elsewhere (Bröms et al., 2003b). Intrabacterial protein levels were determined using pelleted bacteria (expression fraction) and cleared supernatants used to assess protein secretion levels (secretion fraction). Analysis was by Western blotting using rabbit polyclonal antisera raised against the P. aeruginosa T3S substrates PopB, PopD or ExoS.

Yeast two-hybrid assay

To analyse protein–protein interactions in yeast between PcrH and translocator substrates, PcrH variants were cloned into the GAL4 activation domain plasmid pGADT7 (Clontech Laboratories, Palo Alto, CA), while cognate substrate alleles were fused to the GAL4 DNA-binding domain plasmid pGBKT7 (Clontech Laboratories). Transformation of Saccharomyces cerevisiae reporter strain AH109 and analysis of protein–protein interactions was performed as previously documented (Francis et al., 2000, 2001).

The Ras modification assay

Handling and infection of HeLa cells as well as the analysis of ExoS-mediated Ras modification followed an established method (Sundin et al., 2002).

Results and discussion

Mutagenesis of tetratricopeptide repeat residues

Like all known TPR motifs, those of class II chaperones contain canonical residues at positions 8, 20 and 27 expected to form part of the hydrophobic core (Pallen et al., 2003). To determine whether these were necessary for maintaining the TPR fold in PcrH, a subset of canonical residues – Leu-63 (in TPR-A), Gly-78, Ala-90 (TPR-B), and Ala-112, Ala-131 (TPR-C) (Fig. 1a) – was replaced with glutamic acid. This larger residue would likely prevent the tight packing of the TPR helices and lead to protein misfolding and instability. If not however, some might specifically interfere with binding of the cognate PopB and PopD substrates on the inner, convex side of the PcrH molecule from where the canonical residue side chains are predicted to project (Pallen et al., 2003).

Figure 1.

 Sequence comparison of the tetratricopeptide repeats (TPRs) in class II chaperones PcrH and LcrH (a). Alignments were formed using the ClustalW 1.82 web server (http://www.ebi.ac.uk/clustalw/) with areas of amino-acid identity (*) or similarity (: or .) indicated. The three tandem TPR motifs (Pallen et al., 2003) are shown in various shades of grey. The intra-bacterial stability of TPR mutants in PcrH (b). Escherichia coli harbouring expression plasmids were grown at 37°C in the presence of 0.1 mM isopropyl-β-d-thiogalactopyranoside. After inhibition of new protein synthesis (time 0 min), aliquots were taken at different time points and protein amount was detected by Western blot using monoclonal anti-FLAG antiserum. The experiment was repeated at least two times with a representative shown. Mutants are indicated by open squares (□), alanine substitutions of surface located residues in the putative substrate binding groove; open triangles (Δ), alanine substitutions of residues predominantly conserved in all TPR containing type III chaperones that are predicted to lie outside the putative binding groove; closed circles (•), glutamate substitutions of the key canonical residues located in all known TPRs at positions 8, 20 and 27 (Pallen et al., 2003).

Structural modelling of several translocator chaperones also revealed a conserved structure with a similar convex, putative peptide-binding groove, which was lined with residues from all three TPRs (Pallen et al., 2003). Therefore, we randomly selected the residues Tyr-41, Tyr-45 (TPR-A), Leu-75, Gly-76, Cys-80, Gln-82 (TPR-B) and Phe-109, His-110, Glu-113 and Leu-116 (TPR-C) (Fig. 1a) for alanine substitution to determine whether this putative-binding groove affects PcrH function.

Finally, alanine substitutions were generated in the residues Leu-77, Arg-81 and Ile-102 from TPR-B (Fig. 1a). Although not always hydrophobic in PcrH, these residues are among those in TPRs at positions 4, 7, 11, 24 and 32 that are predominantly hydrophobic in all TPR containing class II chaperones and, from the related chaperone models (Pallen et al., 2003), would map to the concave region outside of the putative-binding groove.

In a parallel study, the same substitutions were generated in the multifunctional chaperone LcrH of Yersinia pseudotuberculosis (Fig. 1a) (Edqvist et al., 2006). This enabled phenotypic comparisons between the two chaperones to highlight any universal requirement for TPRs in class II chaperone function. Residues in PcrH were selected without prior knowledge of the LcrH mutant phenotypes. PcrH mutants were phenotypically screened for stability of both chaperone and substrates, substrates secretion, chaperone-substrate binding and exoenzyme translocation. We also attempted to determine if PcrH formed homodimers, because this might be relevant to class II chaperone function (Darwin & Miller, 2001; Tengel et al., 2002) and could contribute to the phenotypes of one or more of our PcrH mutants. However, even after several attempts with approaches such as the yeast two-hybrid system and overlay assays, we were unable to demonstrate PcrH homodimers (data not shown).

Effect of mutation on chaperone stability

To analyse the stability of each PcrH mutant, an intrabacterial protein stability assay was performed. Although not all misfolded proteins are degraded quickly by the cell machinery (Sekijima et al., 2005), this assay is routinely used to assess the half-lives of assorted proteins inside bacteria (Sassanfar & Roberts, 1990; Geuskens et al., 1992; Frank et al., 1996; Gonzalez et al., 1998; Feldman et al., 2002; Jackson et al., 2004; Losada & Hutcheson, 2005). After antibiotic-mediated inhibition of de novo protein synthesis, protein samples were collected from bacteria at various time points and analysed with immunoblot and α-FLAG antisera. We observed that substitution mutants of conserved hydrophobic residues predicted to lie outside of the putative binding groove were detectable after 60 min postantibiotic addition, reminiscent of wild-type PcrH stability (Fig. 1b). However, mutants in residues predicted to form the substrate-binding groove or reside in canonical positions were less stable, although detectable after 10 min postantibiotic addition (Fig. 1b). A decrease in half-life of proteins with canonical residue substitutions is consistent with their conservation among all TPR-containing proteins and their likeliness to be embedded in the protein structure (Magliery & Regan, 2004). Predictably, their replacement with the larger glutamic acid must prevent tight packing of the TPR helices leading to misfolding. We observed similar effects in a study of LcrH from Y. pseudotuberculosis (Edqvist et al., 2006). These dual findings support the model where TPR packing defines the structural folds in class II T3S chaperones (Pallen et al., 2003).

PcrH mutants devoid of chaperone function

Our interest was to investigate if any TPR residue influenced the ability of PcrH to ensure presecretory stabilization and/or secretion of the translocator substrates PopB and PopD (Bröms et al., 2003b). Therefore, expression and secretion profiles of PopB and PopD from mutant bacteria were scrutinized. Mutants PcrH3 (C80A/Q82A), PcrH4 (L77A/R81A) and PcrH11 (I102A) all produced and secreted PopB and PopD at levels comparable to the wild type (Fig. 2) and successfully engaged both substrates in the yeast two-hybrid assay (Table 2). We therefore classified these mutants as ‘wild type-like’ and concluded that the conserved Leu-77, Arg-81 and Ile-102 residues outside the putative-binding groove and the Cys-80 and Gln-82 residues inside the binding groove play no direct role in chaperone function.

Figure 2.

 Secretion and expression analysis of Pops and ExoS produced from Pseudomonas aeruginosa. Bacteria were grown either with (+) or without (−) Ca2+. Proteins recovered from the culture supernatants (secretion fraction) or bacterial pellets (expression fraction), were analysed by immunoblot using polyclonal rabbit anti-PopB, anti-PopD or anti-ExoS antiserum. Analysis of ExoS was included to ensure that PcrH had no effect on stability or secretion of an independent substrate. Strain PAK is wild type and ΔpcrH is the isogenic pcrH deletion mutant PAKpcrH. Additional strains listed represent PcrH mutants, each containing an amino-acid substitution in the tetratricopeptide repeat region. All mutants have been divided into three phenotypic groups (‘wild type-like’, ‘diminished PopB’ and ‘null mutant-like’) according to the expression and secretion profiles of the PcrH substrates PopB and PopD. The asterisk indicating mutant L75A/G76A signifies an intermediate stability and secretion phenotype. See Fig. 1 for definitions of (•), (Δ) and (□). The experiment was repeated at least three times and a representative is shown.

Table 2.   Yeast two-hybrid data for interaction of PcrH with PopB and PopD
PcrH variant*Yeast two-hybrid assay
  • *

    Each group is based upon the substrate expression and secretion profiles observed in Fig. 2.

  • All PcrH variants were expressed fused to the GAL4 activation domain from plasmid pGADT7, while the cognate substrates PopB and PopD were expressed fused to the GAL4 DNA-binding domain from plasmid pGBKT7. Containing HIS3 and ADE2 as reporter genes, the yeast strain Saccharomyces cerevisiae AH109 was used as host for the two-hybrid assay. HIS3+ and ADE2+ represent strong growth (++++) to no growth (−) on minimal medium devoid of histidine or adenine, respectively, recorded after day 4. Results reflect trends in growth from three independent experiments, in which several individual transformants were tested on each separate occasion. Yeast containing only PcrH, PopB or PopD routinely failed to grow on the assay medium.

Wild type++++++++++++++
Vector alone (pGADT7)
Wild type-like
Diminished PopB
Null mutant-like

In contrast, we identified several site-specific mutants that were devoid of chaperone function (denoted ‘null mutant-like’). Not surprisingly, substitution of the canonical residues at position 27 of TPR-A (Leu-63), positions 8 and 20 of TPR-B (Gly-78, Ala-90) and positions 8 and 27 of TPR-C (Ala-112 and Ala-131) not only resulted in less stable chaperone, but both substrates were also destabilized and not secreted (Fig. 2). Moreover, only mutant L63E bound substrate in yeast, but this was only detected by the less stringent reporter system (HIS3+) (Table 2). Furthermore, at least one of the two aromatic residues from TPR-A (Tyr-41 and Phe-45) that are predicted to line the binding groove were also essential for full chaperone effect, because mutant PcrH2 (Y41A/F45A) failed to bind (Table 2), stabilize or secrete (Fig. 2) either substrate.

One explanation for the defective function of these six chaperone mutants is that they were less stable. However, we could verify that they were stable in yeast (data not shown), but still could not bind cognate substrates. Furthermore, mutant PcrH3 (C80A/Q82A), which is also relatively unstable (Fig. 1b), maintained full chaperone activity, suggesting that this degree of stability is sufficient for function. Thus, while important in PcrH folding, it remains possible that these residues directly influence substrate binding. In this respect, it is interesting that five of the nine canonical residues mutated in the homologous LcrH chaperone were required for full binding and stability of the YopB substrate (Edqvist et al., 2006). Naturally, solving a solution structure of a class II chaperone in the presence of substrate would shed light on this issue. Nevertheless, such a structure would only represent a single snapshot of a highly dynamic process. Therefore, mutagenesis studies like this one make available much needed information for interpretation of a future structure.

Some PcrH mutants show diminished chaperone function towards PopB

Residues Glu-113, Leu-116, Phe-109 and His-110 from TPR-B are predicted to line the binding groove and were necessary for stable production and secretion of PopB, but not PopD (designated as ‘Diminished PopB’ in Fig. 2), although curiously we could not detect a defect in PopB binding in yeast (Table 2). Nonetheless, these residues must preferentially cross-talk with the PopB substrate, implying that the translocator substrates may possess different interaction sites on the PcrH molecule. In fact, this was also demonstrated for the LcrH chaperone, in that residues specifically necessary for stable production and secretion of YopB lined the putative binding groove, while residues required for YopD stability and/or efficient secretion, mapped to another location outside the binding groove to the broadly conserved hydrophobic residues at positions 4, 7, 11, 24 and 32 (Edqvist et al., 2006). Collectively, this suggests that the primary chaperone-binding site for PopB/YopB substrate is within the PcrH/LcrH convex-binding groove. Furthermore, just as YopD possesses a unique binding site on LcrH (outside the binding groove), so also may PopD for PcrH, especially since we have demonstrated reciprocal binding between PcrH/LcrH and PopD/YopD (Bröms et al., 2003a). Thus, hydrophobic residues in LcrH that specifically cross-talk with YopD (Edqvist et al., 2006) may play a similar role for PcrH-PopD binding. Our prediction therefore is that all translocator-class chaperones would possess unique binding sites for each substrate.

Analysis of translocon function

ExoenzymeS (ExoS)-dependent ADP-ribosylation of eukaryotic Ras provides a sensitive method for assaying the direct translocation of the effector protein ExoS into target cells (Sundin et al., 2002; Bröms et al., 2003c; Olsson et al., 2004). Therefore, it can be used to measure how well our PcrH mutants assemble a functional translocon. To assess the level of Ras modification in vivo, HeLa cells were infected for various times with the PcrH mutants and harvested in sample buffer. Lysates were analysed by immunoblot with monoclonal anti-Ras antibody (Fig. 3). Like wild type, mutants that could efficiently bind and stabilize both PopB and PopD were able to modify a significant proportion of the total cellular Ras population (seen as a slower migrating product) already after 45 min, with full modification occurring after 90 min (‘wild type-like’) (Fig. 3, compare lanes b to d with a). Interestingly, this translocation efficiency was also seen for members of the group ‘diminished PopB’ that showed a specific defect in stability and secretion of PopB during in vitro conditions, as well as the phenotypically intermediate mutant PcrH9 (L75A/G76A) (Fig. 3, compare lanes e, f and n with a). Not surprisingly, the majority of PcrH mutants that were severely defective for binding, synthesis and secretion of both substrates in vitro (‘null mutant-like’) were also unable to translocate ExoS, similar to the pcrH null mutant (Fig. 3, compare lanes j to m with g). However, delayed ExoS translocation by the mutants PcrH1 (L63E) and PcrH2 (Y41A/F45A) was observed (Fig. 3, compare lanes h and i with a). This finding reinforces our observations in Yersinia (Bröms et al., 2003a) (P.J. Edqvist, unpublished) that very small amounts of secreted translocators are sufficient to ensure efficient translocation.

Figure 3.

 Ras-modification in HeLa cells infected with Pseudomonas aeruginosa. HeLa cells were harvested after infection at 45, 90 or 180 min and dissolved in sample buffer. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with anti-Ras monoclonal antibody. Lanes: a, PAK; b, PAKpcrH3 (C80A/Q82A); c, PAKpcrH4 (L77A/R81A); d, PAKpcrH11 (I102A); e, PAKpcrH7 (E113A/L116A); f, PAKpcrH8 (F109A/H110A); g, PAKpcrH; h: PAKpcrH1 (L63E); i, PAKpcrH2 (Y41A/F45A); j, PAKpcrH5 (A112E); k, PAKpcrH6 (A90E); l, PAKpcrH10 (G78E); m, PAKpcrH12 (A131E); n, PAKpcrH9 (L75A/G76A) and o, cells left uninfected. All mutants have been divided into three phenotypic groups (‘wild type-like’, ‘diminished PopB’ and ‘null mutant-like’) according to the expression and secretion profiles in Fig. 2. The asterisk indicating mutant L75A/G76A signifies an intermediate phenotype. The arrowheads identify ADP-ribosylated Ras. For definitions of (•), (Δ) and (□), see Fig. 1. The experiment was repeated at least three times and a representative is shown.

A role for TPRs in chaperone – substrate specificity

PcrH and LcrH are genetically similar chaperones that can efficiently complement for the loss of the other during cellular infections (Bröms et al., 2003a, b). However, unlike the straightforward role of PcrH in P. aeruginosa, we have recently discovered that the function of LcrH in Yersinia is multifaceted, being also a system regulator that requires additional protein–protein interactions (Bröms et al., 2005). We therefore wanted to compare the phenotypes of TPR residue substitutions made in the simpler PcrH model reported here with those equivalent mutations in the complex LcrH model (Edqvist et al., 2006). Strikingly, five out of 12 mutations exhibited the same general ‘wild type-like’ or ‘null mutant-like’ phenotypes (Table 3), consistent with the basic reciprocal functions of both chaperones (Bröms et al., 2003a, b). Intriguingly, however, the remaining seven mutations in counterpart residues produced different phenotypes in the respective systems (Table 3). While it is possible that these phenotypic discrepancies could be because of minor structural constraints, another speculation is that these TPRs confer the substrate specificity within each chaperone. This might explain the nonreciprocal binding of YopB/PopB with PcrH/LcrH (Bröms et al., 2003a). In fact, the inherent flexibility within the TPR protein–protein interaction motifs could also explain why class II chaperone-substrate interaction domains cannot be mapped to the same region of each substrate, like they can for class I chaperone substrate interactions (Page & Parsot, 2002; Parsot et al., 2003; Edqvist et al., 2006).

Table 3.   Comparative phenotypic summary resulting from identical substitution of equivalent PcrH/LcrH residues
PcrHSimilarity to
LcrH mutant
MutantPhenotypic group
  • *

    Results summarized from the study in Yersinia pseudotuberculosis where the cognate substrates for LcrH are YopB and YopD (Edqvist et al., 2006). Where phenotypes differed in the two models, those recorded in Yersinia are given in parentheses.

  • Intermediate phenotype with low, but similar stabilization, and secretion of both PopB and PopD substrates.

  • WT, wild type-like; Null, null mutant-like.

L77A/R81AWTNo (diminished YopD)
F109/H110ADiminished PopBNo (WT)
E113A/L116ADiminished PopBNo (WT)
L63ENullNo (diminished YopB)
A112ENullNo (diminished YopB)
A131ENullNo (diminished YopB)
L75A/G76AIntermediateNo (diminished YopB)


Using PcrH of Pseudomonas aeruginosa as a simpler model system, we have validated our recent discovery that class II chaperones possess three tandem TPR motifs that are critical for the chaperone's ability to engage its two translocator substrates (Pallen et al., 2003; Edqvist et al., 2006). In PcrH, residues important for preferential stability and secretion of one substrate over the other were identified and mapped to a spatially distinct region within the chaperone, a feature also shared by the homologous chaperone LcrH of pathogenic Yersinia (Edqvist et al., 2006). Thus, TPR motifs do allow a class II chaperone to engage its two translocator substrates differently. In addition, highly conserved residues among the chaperones, such as those found in LcrH and PcrH, often had different roles in substrate binding, stability and/or secretion, demonstrating that the TPR protein–protein interaction motif may determine substrate specificity within this translocator class of chaperones.


We thank Mark Pallen for constructive discussion throughout this project and Ina Attree for supplying anti-PopB antisera. Research support was obtained from the Carl Tryggers Foundation for Scientific Research (MSF), Swedish Research Council (ÅF and MSF), Foundation for Medical Research at Umeå University (MSF), Swedish Foundation for Strategic Research (ÅF), Swedish Cystic Fibrosis Association Research Fund (MSF), Swedish Medical Association (MSF) and the J C Kempes Memorial Fund (JEB and PJE).