Protective Anti‐V Antibodies InhibitPseudomonasandYersiniaTranslocon Assembly within Host Membranes

Abstract

Pathogenic Yersinia species and Pseudomonas aeruginosa share a similar type III secretion/translocation system. The translocation system consists of 3 secreted proteins, YopB/PopB, YopD/PopD, and LcrV/PcrV; the latter is known to be a protective antigen. In an in vitro assay, the translocation system causes the lysis of erythrocytes infected with wild-type (wt) P. aeruginosa. wt Y. enterocolitica is not hemolytic, but a multiknockout mutant deprived of all the effectors and of YopN ( Delta HOPEMN) is hemolytic. In the presence of antibodies against PcrV and Y. pestis LcrV, the hemolytic activity of P. aeruginosa was inhibited. Similarly, the hemolytic activity of Delta HOPEMN was inhibited in the presence of anti-LcrV antibodies. The assembly of the translocon, composed of PopB/D and YopB/D proteins, was disturbed in immunoprotected erythrocyte membranes, mimicking the phenotypes of V knockout mutants. Thus, protective antibodies against the V antigens of Yersinia species and P. aeruginosa act at the level of the formation of the translocon pore in membranes of infected host cells by blocking the function of LcrV/PcrV. The hemolysis assay could be adapted for high-throughput screening of anti-infectious compounds that specifically target the type III translocon.

YopD Self-assembly and Binding to LcrV Facilitate Type III Secretion Activity by Yersinia pseudotuberculosis

“…Moreover, pore formation is a highly dynamic process responding to multiple molecular interactions between individual pore constituents as well as with the target cell plasma membrane. For instance, the YopD and YopB translocators of Yersinia probably do not function alone but require at least transient input from several additional components including LcrV, YopE, YopK, and YopN (23,24,48,51,58,59). However, the consequences of this molecular network essentially remain an enigma because it is difficult to reach meaningful interpretations of data collected from inadequate studies of target cell lysis induced by bacteria lacking individual translocon components.…”

“…These proteins are encoded on the lcrGVHyopBD translocon operon (21), which is similar in gene organization and sequence to operons in other pathogens, including the pcrGVHpopBD operon from the opportunist Pseudomonas aeruginosa. Experiments to compare and contrast Yersinia and P. aeruginosa have proven useful in probing the functions of each operon component (6,(22)(23)(24)(25)(26).…”

Mutations in the Yersinia pseudotuberculosis Type III Secretion System Needle Protein, YscF, That Specifically Abrogate Effector Translocation into Host Cells

“…YopB and YopD are found in the host cell plasma membrane and thus are likely to form the translocation pore (21,48,72). LcrV is required for pore assembly (24) but is not found in the host membrane (20). LcrV was recently localized to the distal ends of needles (47), where it multimerizes to form a "tip complex."…”

High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement

“…Knock-out of the pcrV gene leads to aberrant leakage of the exotoxins to the bacterial growth medium and to impeded exotoxin translocation into the host cell cytoplasm, resulting in nearly complete abolition of in vitro and in vivo cytotoxicity (16,(22)(23)(24)(25)(26). More so, PcrV is essential for the correct assembly of the PopB/PopD T3SS translocon into the host cell membrane (23,27).…”

“…Anti-PcrV antibodies and derived Fab fragments have been shown to block PopB/PopD translocon assembly in the host cell membrane and subsequent exotoxin translocation as well as T3SS-dependent oncosis (27,29,30).…”