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.