Measurement of Yersinia Translocon Pore Formation in Erythrocytes

Costa, Tiago; Francis, Monika K.; Farag, Salah I.; Edgren, Tomas; Francis, Matthew S.

doi:10.1007/978-1-4939-9541-7_15

Cited by 2 publications

(3 citation statements)

References 92 publications

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“…In order to corroborate this result, we also tested pore formation in sheep red blood cells, a wellestablished technique for measuring T3SS pore formation (37). Sheep erythrocytes were pre-treated with papain, which cleaves surface glycoproteins and exposes cryptic binding sites for P. aeruginosa hemagglutinin (38).…”

Section: Pore Formation Is Hindered By Disruption Of the Interaction Between The Popb And The Tip Domain Of Pcrvmentioning

confidence: 93%

“…Following the previously described protocol (37), sheep erythrocytes in sodium citrate buffer (QuadFive) were washed several times with PBS until the supernatant was clear, then treated with 0.1% w/v papain and 0.01% w/v cysteine for 30 minutes to promote adhesion by P. aeruginosa (38). Erythrocytes were washed again and resuspended in RPMI 1640 (without phenol red) to a concentration of 5×10 8 cells/mL.…”

Section: Methodsmentioning

confidence: 99%

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PopB-PcrV Interactions are Essential for Pore Formation in the Pseudomonas aeruginosa Type III Secretion System Translocon

Kundracik

Trichka

Aponte

et al. 2022

Preprint

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The type III secretion system (T3SS) is a syringe-like virulence factor which delivers bacterial proteins directly into the cytoplasm of host cells. An essential component of the system is the translocon, which creates a pore in the host cell membrane through which proteins are injected. In Pseudomonas aeruginosa, the translocation pore is formed by proteins PopB and PopD and attaches to the T3SS needle via the needle tip protein PcrV. The pore is multimeric, but the exact stoichiometry and structure of the pore are unknown. We took a genetic approach to map contact points within the system by taking advantage of the fact that the translocator proteins of Pseudomonas aeruginosa and the related Aeromonas hydrophila T3SS are incompatible and cannot be freely exchanged. We created chimeric versions of P. aeruginosa PopB and A. hydrophila AopB to intentionally disrupt and restore protein-protein interactions. We identified a chimeric B-translocator that specifically breaks an interaction with the needle tip protein and interferes with the formation of the translocation pore. Breaking the interaction did not disrupt membrane insertion, arguing that the needle tip protein chaperones formation of the translocation pore.

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Section: Pore Formation Is Hindered By Disruption Of the Interaction Between The Popb And The Tip Domain Of Pcrvmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

PopB-PcrV Interactions are Essential for Pore Formation in the Pseudomonas aeruginosa Type III Secretion System Translocon

Kundracik

Trichka

Aponte

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Following the previously described protocol ( 52 ), sheep erythrocytes in sodium citrate buffer (Quad Five) were washed several times with PBS until the supernatant was clear and then treated with 0.1% papain and 0.01% cysteine for 30 min to promote adhesion by P. aeruginosa ( 53 ). Erythrocytes were washed again and resuspended in RPMI 1640 (without phenol red) to a concentration of 5e8 cells/mL.…”

Section: Methodsmentioning

confidence: 99%