Oligomerization of PcrV and LcrV, Protective Antigens of Pseudomonas aeruginosa and Yersinia pestis

Abstract

Protective antigens of Pseudomonas aeruginosa (PcrV) and Yersinia pestis (LcrV) are key elements of specialized machinery, the type III secretion system (T3SS), which enables the injection of effector molecules into eukaryotic cells. Being positioned at the injectisome extremity, V proteins participate in the translocation process across the host cell plasma membrane. In this study, we demonstrate the assembly of V proteins into oligomeric doughnut-like complexes upon controlled refolding of the proteins in vitro. The oligomeric nature of refolded PcrV was revealed by size exclusion chromatography, native gel electrophoresis, and native mass spectrometry, which ascertain the capacity of the protein to multimerize into higherorder species. Furthermore, transmission electron microscopy performed on oligomers of both PcrV and LcrV revealed the presence of distinct structures with approximate internal and external diameters of 3-4 and 8 -10 nm, respectively. The C-terminal helix, ␣12, of PcrV and notably the hydrophobic residues Val 255 , Leu 262 , and Leu 276 located within this helix, were shown to be crucial for oligomerization. Moreover, the corresponding mutant proteins produced in P. aeruginosa were found to be non-functional in in vivo type III-dependent cytotoxicity assays by directly affecting the correct assembly of PopB/D translocon within the host cell membranes. The detailed understanding of structure-function relationships of T3SS needle tip proteins will be of value in further developments of new vaccines and antimicrobials.

Biophysical Characterization of Chlamydia trachomatis CT584 Supports Its Potential Role as a Type III Secretion Needle Tip Protein

“…This molecular mass and assembly of oligomers are very similar to those observed for natively purified T3SS needle tip proteins, including IpaD (29, 46). To provide further support for the hypothesis that CT584 is a Chlamydia T3SS needle tip protein, biophysical characterization of this protein was performed and the results were compared to prior studies that revealed protein subfamilies on the basis of their response to pH and temperature (34–36).…”

“…Some tip proteins are known to oligomerize in their native state and are reported to form pentamers or hexamers when localized to the needle tip (29, 46, 53, 54). Samples of CT584 were examined in both sedimentation velocity and equilibrium modes using interference and absorption optical systems.…”

“…Many of the T3SS tip proteins have been shown to oligomerize in vitro and in vivo, and it is believed that, on the needle tip, they are pentamers or hexamers (29, 46, 53, 54). AUC sedimentation velocity and equilibrium analysis demonstrate that in aqueous solution, CT584 oligomerizes most probably to a pentamer with some higher-order aggregates present.…”

Amino acid and structural variability of Yersinia pestis LcrV protein

“…It is a well-established fact that recombinant LcrV may exist in solution as a dimer and higher order oligomers (Derewenda et al , 2004; Hamad & Nilles, 2007; Lawton et al , 2002). Moreover, a controlled refolding of this protein led to the assembly of LcrV into oligomeric doughnut-like complexes, and the C-terminal helix α12 (a.a. 279–317) was crucial for this in vitro oligomerization (Caroline et al , 2008). Previously, it was established that the helix α7 (a.a. 148–182) is involved in oligomerization as well (Lawton et al , 2002).…”

Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

“…For antibody calibrations, purified proteins 6His-PcrV, PopB and PopD [16], [19] were diluted in PBS buffer and 0.3 ng of each was loaded on the same gel. The antibodies were previously described [18].…”