The Pseudomonas aeruginosa type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes

Abstract

Many Gram-negative bacterial pathogens use a type III secretion system to infect eukaryotic cells. The injection of bacterial toxins or protein effectors via this system is accomplished through a plasma membrane channel formed by two bacterial proteins, termed translocators, whose assembly and membrane-insertion mechanisms are currently unclear. Here, using purified proteins we demonstrate that the translocators PopB and PopD in assemble heterodimers in membranes, leading to stably inserted hetero-complexes. Using site-directed fluorescence labeling with an environment-sensitive probe, we found that hydrophobic segments in PopD anchor the translocator to the membrane, but without adopting a typical transmembrane orientation. A fluorescence dual-quenching assay revealed that the presence of PopB changes the conformation adopted by PopD segments in membranes. Furthermore, analysis of PopD's interaction with human cell membranes revealed that PopD adopts a distinctive conformation when PopB is present. An N-terminal region of PopD is only exposed to the host cytosol when PopB is present. We conclude that PopB assists with the proper insertion of PopD in cell membranes, required for the formation of a functional translocon and host infection.

The type III secretion system needle, tip, and translocon

“…When both PopB and PopD were added together, eight subunits each of PopB and PopD associated and formed into a 16‐subunit complex on membranes . Acidic pH changes the protonation of PopD residues 63–81, making this segment more hydrophobic, and together with the predicted transmembrane segment (residues 119–137), assists PopD in the formation of the translocon pore . Recently, it has been observed that the PopB‐PopD translocon remains attached to the host cell membrane even after bacterium has left the site of contact …”

Immunoglobulin for Treating Bacterial Infections: One More Mechanism of Action

“…The homologs of these P. aeruginosa proteins, which are encoded by pcrGVHpopBD, are PopB and PopD [43]. YopB and YopD from Yersinia [49] and PopB and PopD from P. aeruginosa [51,52] are involved in pore formation in the eukaryotic cell membrane. The structural mechanism involving LcrV, YopB, and YopD in Yersinia and PcrV, PopB, and PopD in P. aeruginosa involves a translocon (Figure 3(3)) [50,52].…”

“…YopB and YopD from Yersinia [49] and PopB and PopD from P. aeruginosa [51,52] are involved in pore formation in the eukaryotic cell membrane. The structural mechanism involving LcrV, YopB, and YopD in Yersinia and PcrV, PopB, and PopD in P. aeruginosa involves a translocon (Figure 3(3)) [50,52]. The structural position of the V-antigen proteins in the type III secretion system was unknown until 2005, when electron microscopy analysis showed that LcrV and PcrV are both cap-like structures located at the tip of the needle structure in the secretion apparatus [53,54].…”

“…The pcrGVHpopBD operon encodes five proteins associated with toxin translocation [46]. (3) PcrV is a cap-like structure located at the top of the secretion needle [53], and PopB and PopD are involved in pore formation in the eukaryotic cell membrane [51,52]. The structural mechanism involving three of the PcrV-PopB-PopD proteins involves a translocon.…”

“…Antibodies 2019, 8, 52 6 of 13 recent years, more researchers have reported the clinical application of anti-PcrV antibodies, which have been developed using different approaches [67][68][69][70].…”