Structural characterization of Type III Secretion System related translocator and effector from Pseudomonas aeruginosa

Pseudomonas aeruginosa, a Gram-negative pathogen utilizes a specialized set of Type III Secretion System (T3SS) translocator and effector proteins to establish virulence in the host cell. An understanding of these pathogenic factors that play a key role in the establishment and maintenance of bacterial pathogenicity are thus, of immense importance, and are likely the interest of study of several research groups. The T3SS encoding “translocator operon” of P. aeruginosa consists of a major translocator protein PopB, minor translocator protein PopD and their cognate chaperone PcrH. We present a comprehensive study of PopB structure, its interaction with PcrH and their associated pH-based structural and functional changes. The pH-dependent studies indicate that PcrH not only provides structural support to the ordered molten globule PopB in complex but also undergoes conformational change to assist PopB to pass through the needle complex of T3SS and form pores in the host cell membrane. In addition, we report an abinitio model of PopB docked to PcrH, which together with PCR-based deletion mutations and SPR studies determine the terminal domains of PopB to be involved in hydrophobic interaction with PcrH as well as maintain the oligomerisation of translocator complex. Amongst all effector toxin proteins of T3SS present in P. aeruginosa, both ExoT and ExoS are considered to be actual virulence determinants. Here, we report the first X-ray crystal structure of the amino-terminal fragment of effector toxin ExoT, in complex with full-length homodimeric chaperone SpcS at 2.1Å resolution. The full-length dimeric chaperone SpcS has the conserved α-β-β-β-α-β-β-α fold of class I chaperones, the characteristic hydrophobic patches for binding effector proteins and a conserved polar cavity at the dimeric interface. The stable crystallized amino-terminal fragment of ExoT consists of a chaperone binding domain and a membrane localization domain that wraps around the dimeric chaperone. Sitedirected mutagenesis experiments and molecular dynamics study complement each other in revealing Asn65, Phe67 and Trp88 as critical dimeric interfacial residues that can strongly influence the effector-chaperone interactions. This structural and functional T3SS study can thus serve to be promising drug candidates to combat host cell infections