Rapid Membrane Disruption by aPerforin-Like Protein Facilitates Parasite

Exit from Host Cells

Björn F. C. Kafsack, Janethe D. O. Pena, Isabelle Coppens,Sandeep Ravindran, John C. Boothroyd, Vern B. Carruthers

1st demonstration of mode of action for a pathogen PLP1st demonstration of mode of action for a pathogen PLP

Toxoplasma Egress Paper Discussion

Hypothesis

Techniques used

For each Figure we will address the following:

Major Finding(s)

MACPF are eukaryoticCholesterol dependent cytolysins

MACPF = Membrane Attack Complex Pore Forming

Previously, no mode of action or pore-forming activity had beendemonstrated for microbial Perforin-like proteins.

Superfamily of proteins containing acommon domain - MACPF

Original members - components ofthe MAC (C6, C7, C8α, C8β, C9) andperforin

Perforin-like proteins (PLPs) areexpressed by many bacterial andprotozoan pathogens.

Little is known about their functions.

T. gondii PLP1 has a conservedMACPF domain

P. luminescens γ-proteo Toxoplasma gondii

MACPF Proteins - Pore Forming Ability

MACPF-domain proteins of the mammalian immune system induce death byoligomerizing on the surface of target cells and inserting into the membrane

to for large diameter pores. - remember membrane attack complex?

The Membrane Attack Complex

Supplemental – MACPF domain

charged (blue),polar (green),

aliphatic (yellow),aromatic (red) or

high turn propensity(orange).

Core elements are conserved

MACPF signature motif(Y/W)-X6-(F/Y)GTH(F/Y)-X6-GG

Critical residues important forpore insertion are conserved

Fig 1: TgPLP1 is secreted from micronemes

PLP1 localizes to micronemes

PLP1 is secreted in a Ca2+ dep manner

Figure 2 – plp1ko phenotypes

48 hour post infection

PLP1KO – sphericalstructures accumulate

PLP1KO – PVM and host cellmembrane intact

Fig 2 - plp1KOphenotypes

Motility is essential for egressPLP1KO motility is normal

PLP1 can act on both luminal andcytoplasmic side of vacuole

Figure 3 – Evidence of pore formation

Fig 3 - Evidence of pore formation

Motility was inhibited with cytochalasin D.Parasitophorous vacuole was loaded with RFP.

Monitored PVM permeabilization in real time.

Time lapse images from movies

Fig 3 - Effect of plp1KO on virulence

Class Summary - think aboutthe techniques used

Fig 1: Hypothesis or questionIf TgPLP1 is a perforin-like pore forming protein, it is likely

to be localized the the apical organelles (secrete proteins).

Conclusions:PLP1 localizes to the micronemes (colocalizes with MIC2),

and is secreted in a Ca2+ dependent fashion like other micronemalproteins

Fig 2: Hypothesis or questionSecreted TgPLP1 may play a role in egress.

Conclusions:PLP1 Knockout phenotypes include spherical structures

containing motile parasites that have greatly delayed egress.

Class Summary - think aboutthe techniques used

Fig 3: Hypothesis or questionPLP1 is assisting in egress by forming pores in the

parasitophorous vacuole.

Conclusions:When parasites are immobilized, the PLP1 play a role in

pore formation (what is the evidence?).

Fig 3: Hypothesis or questionDoes PLP1 play a role in virulence?

Conclusions:PLP1 Knockout parasites do not cause host death even at

extremely high infection rates (5 fold higher).