Formation of ER-lumenal intermediates during export of Plasmodium proteins containing transmembrane-like hydrophobic sequences

During the blood stage of a malaria infection, malaria parasites export both soluble and membrane proteins into the erythrocytes in which they reside. Exported proteins are trafficked via the parasite endoplasmic reticulum and secretory pathway, before being exported across the parasitophorous vacuole membrane into the erythrocyte. Transport across the parasitophorous vacuole membrane requires protein unfolding, and in the case of membrane proteins, extraction from the parasite plasma membrane. We show that trafficking of the exported Plasmodium protein, Pf332, differs from that of canonical eukaryotic soluble-secreted and transmembrane proteins. Pf332 is initially ER-targeted by an internal hydrophobic sequence that unlike a signal peptide, is not proteolytically removed, and unlike a transmembrane segment, does not span the ER membrane. Rather, both termini of the hydrophobic sequence enter the ER-lumen and the ER-lumenal species is a productive intermediate for protein export. Furthermore, we show in intact cells, that two other exported membrane proteins, SBP1 and MAHRP2, assume a lumenal topology within the parasite secretory pathway. Although the addition of a C-terminal ER-retention sequence, recognised by the lumenal domain of the KDEL receptor, does not completely block export of SBP1 and MAHRP2, it does enhance their retention in the parasite ER. This indicates that a sub-population of each protein adopts an ER-lumenal state that is an intermediate in the export process. Overall, this suggests that although many exported proteins traverse the parasite secretory pathway as typical soluble or membrane proteins, some exported proteins that are ER-targeted by a transmembrane segment-like, internal, non-cleaved hydrophobic segment, do not integrate into the ER membrane, and form an ER-lumenal species that is a productive export intermediate. This represents a novel means, not seen in typical membrane proteins found in model systems, by which exported transmembrane-like proteins can be targeted and trafficked within the lumen of the secretory pathway

Repetitive sequences in malaria parasite proteins.

Five species of parasite cause malaria in humans with the most severe disease caused by Plasmodium falciparum. Many of the proteins encoded in the P. falciparum genome are unusually enriched in repetitive low-complexity sequences containing a limited repertoire of amino acids. These repetitive sequences expand and contract dynamically and are among the most rapidly changing sequences in the genome. The simplest repetitive sequences consist of single amino acid repeats such as poly-asparagine tracts that are found in approximately 25% of P. falciparum proteins. More complex repeats of two or more amino acids are also common in diverse parasite protein families. There is no universal explanation for the occurrence of repetitive sequences and it is possible that many confer no function to the encoded protein and no selective advantage or disadvantage to the parasite. However, there are increasing numbers of examples where repetitive sequences are important for parasite protein function. We discuss the diverse roles of low-complexity repetitive sequences throughout the parasite life cycle, from mediating protein-protein interactions to enabling the parasite to evade the host immune system

Amphibian chytridiomycosis outbreak dynamics are linked with host skin bacterial community structure

© The Author(s).Host-associated microbes are vital for combatting infections and maintaining health. In amphibians, certain skin-associated bacteria inhibit the fungal pathogen Batrachochytrium dendrobatidis (Bd), yet our understanding of host microbial ecology and its role in disease outbreaks is limited. We sampled skin-associated bacteria and Bd from Pyrenean midwife toad populations exhibiting enzootic or epizootic disease dynamics. We demonstrate that bacterial communities differ between life stages with few shared taxa, indicative of restructuring at metamorphosis. We detected a significant effect of infection history on metamorph skin microbiota, with reduced bacterial diversity in epizootic populations and differences in community structure and predicted function. Genome sequencing of Bd isolates supports a single introduction to the Pyrenees and reveals no association between pathogen genetics and epidemiological trends. Our findings provide an ecologically relevant insight into the microbial ecology of amphibian skin and highlight the relative importance of host microbiota and pathogen genetics in predicting disease outcome.K.A.B. was funded by a CASE studentship from NERC; M.C.F. and T.W. J.G. were funded by the NERC award NE/E006701/1 and the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity.Peer Reviewe

Zemiology: reconnecting crime and social harm

This book challenges the given dichotomies between crime and harm, and criminology and zemiology. The main aim of the volume is to highlight the inexorable interconnectedness between systemically induced social harm and the corrosive flows of everyday crime both perpetrated and endured by those victimised by the capitalist system and its hegemonic vicissitudes. Drawing attention not only to various structurally imbedded harms, the chapters also outline the wider consequences of such harms, as they extend beyond immediate victims and contribute towards the further perpetuation of criminogenic and zemiogenic conditions. Comprising two parts, the first explores the relationship between crime and harm and criminology and zemiology, and the second explores the intersections of crime and harm through various lenses, including those trained on probation; global mobility; sexuality and gender; war and gendered violence; fashion counterfeiting; and the harms of the service economy. An exciting and wide-reaching volume written by world-renowned scholars, this collection is a must-read for students, academics, and policy makers in the fields of law, criminology, sociology, social policy, criminal justice, and social justice

Fluorescence microscopy analysis of split-GFP expressing parasites.

(A-B) Cartoon representation of plasmepsin V with a C-terminal S11 tag (plasmepsinV:3xHA:C-S11), and phase contrast and green fluorescence images of parasites expressing GFP1-10 fragments together with plasmepsinV:3xHA:C-S11 are shown. (C-D) Images of parasites co-expressing cytoplasmic mCherry that has a C-terminal S11 tag with either ER-lumenal GFP1-10 or cytoplasmic GFP1-10 are shown. For increased clarity and comparison to figures in the main text, two brightness ranges are shown for each image, as indicated. For GFP and mCherry images in the main text brightness settings of 0–1000 and 0–800 were used, respectively. In the images shown here, 0–1000 and 0–800 are shown for GFP and mCherry, respectively, but a brightness setting of 0–4095 is also shown for both channels. (E-F) Images of parasites co-expressing ER-lumenal mCherry (ER-lumenal mCherry comprises the N-terminal signal peptide derived from PF3D7_0827900, mCherry, a C-terminal S11 tag, and a STREP tag, followed by an SDEL sequence) with either ER-lumenal GFP1-10 or cytoplasmic GFP1-10, are shown. (TIF)</p

High contrast GFP images of MAHRP2-expressing parasites.

(A-C) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP1-10 or cytoplasmic GFP1-10, as indicated. Images are identical to those in the main text Fig 6 except that high contrast images of the GFP channel are shown. Contrast settings for GFP images are set at 0–200 to show weak GFP signal. Scale bar: 2 μm. (TIF)</p

Mass spectrometry methods.

During the blood stage of a malaria infection, malaria parasites export both soluble and membrane proteins into the erythrocytes in which they reside. Exported proteins are trafficked via the parasite endoplasmic reticulum and secretory pathway, before being exported across the parasitophorous vacuole membrane into the erythrocyte. Transport across the parasitophorous vacuole membrane requires protein unfolding, and in the case of membrane proteins, extraction from the parasite plasma membrane. We show that trafficking of the exported Plasmodium protein, Pf332, differs from that of canonical eukaryotic soluble-secreted and transmembrane proteins. Pf332 is initially ER-targeted by an internal hydrophobic sequence that unlike a signal peptide, is not proteolytically removed, and unlike a transmembrane segment, does not span the ER membrane. Rather, both termini of the hydrophobic sequence enter the ER-lumen and the ER-lumenal species is a productive intermediate for protein export. Furthermore, we show in intact cells, that two other exported membrane proteins, SBP1 and MAHRP2, assume a lumenal topology within the parasite secretory pathway. Although the addition of a C-terminal ER-retention sequence, recognised by the lumenal domain of the KDEL receptor, does not completely block export of SBP1 and MAHRP2, it does enhance their retention in the parasite ER. This indicates that a sub-population of each protein adopts an ER-lumenal state that is an intermediate in the export process. Overall, this suggests that although many exported proteins traverse the parasite secretory pathway as typical soluble or membrane proteins, some exported proteins that are ER-targeted by a transmembrane segment-like, internal, non-cleaved hydrophobic segment, do not integrate into the ER membrane, and form an ER-lumenal species that is a productive export intermediate. This represents a novel means, not seen in typical membrane proteins found in model systems, by which exported transmembrane-like proteins can be targeted and trafficked within the lumen of the secretory pathway.</div

ER-lumenal location of the C-terminus of MAHRP2.

(A) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP1-10 or cytoplasmic GFP1-10, as indicated. (B) Images of parasites expressing MAHRP2:C-S11:DSLE expressed alone, co-expressed with ER-lumenal GFP1-10 or cytoplasmic GFP1-10, as indicated. Parasites were treated with 1μg/ml Brefeldin A for four hours prior to imaging. (C) Images of parasites expressing MAHRP2:C-S11:SDEL and the indicated GFP1-10 proteins, are shown. Scale bar: 2 μm. (D) The fraction of total mCherry fluorescence located within the parasite is shown for each parasite line expressing the indicated MAHRP2 and GFP1-10 proteins. Forty individual trophozoite stage parasites, from four independent experiments, were analysed for each parasite line. Data points for individual parasites, mean, and standard deviation are shown. P-values were determined using a one-way ANOVA test, P 1-10 proteins, the total mCherry fluorescence and total GFP fluorescence levels are plotted. Forty individual trophozoite stage parasites, from four independent experiments, were analysed for each parasite line.</p

Characterisation of GFP_1-10 protein expression in parasites.

Characterisation of GFP1-10 protein expression in parasites.</p

The putative TM segment of Pf332 translocates into the ER lumen.

(A-B) Phase contrast and fluorescence images of parasites expressing the indicated proteins. (C) Phase and fluorescence images of parasites expressing REX3AQLSE:Pf332:C-S11:DSLE alone, or co-expressing either ER-lumenal GFP1-10 or cytoplasmic GFP1-10, as indicated. Scale bar: 2 μm. (D) Western blot of parasites expressing the indicated proteins (probed with an anti-mCherry antibody). (E) Cartoon representation of plasmepsin V cleavage of the PEXEL sequence in REX3RQLSE:Pf332:C-S11:SDEL within the ER and recognition of the C-terminal SDEL sequence in the Golgi lumen.</p