Protein unfolding is an essential requirement for transport across the parasitophorous vacuolar membrane of Plasmodium falciparum

Plasmodium falciparum traffics a large number of proteins to its host cell, the mature human erythrocyte. How exactly these proteins gain access to the red blood cell is poorly understood. Here we have investigated the effect of protein folding on the transport of model substrate proteins to the host cell. We find that proteins must pass into the erythrocyte cytoplasm in an unfolded state. Our data strongly support the presence of a protein-conducing channel in the parasitophorous vacoular membrane, and additionally imply an important role for molecular chaperones in keeping parasite proteins in a 'translocation competent' state prior to membrane passage.     

Visual impairment from fibrous dysplasia in a middle-aged African man: a case report

Introduction

Fibrous dysplasia is a benign tumour of the bones and is a disease of unknown aetiology. This report discusses a case of proptosis and visual deterioration with associated bony mass involving the right orbit.

Case presentation

A 32-year-old Nigerian man of Yoruba ethnic origin presented to the eye clinic of our hospital with right-eye proptosis and visual deterioration of 7-year duration. Presentation was preceded by a history of trauma. Proptosis was preceded by trauma but was non-pulsatile with no thrill or bruit but was associated with bony orbital mass. The patient reported no weight loss. Examination of his right eye showed visual acuity of 6/60 with relative afferent pupillary defect. Fundal examination revealed optic atrophy. Computed tomography showed an expansile bony mass involving all the walls of the orbit. The bony orbital mass was diagnosed histologically as fibrous dysplasia. Treatment included orbital exploration and orbital shaping to create room for the globe and relieve pressure on the optic nerve.

Conclusion

Fibrous dysplasia should be considered in the differential diagnosis of slowly developing proptosis with associated visual loss in young adults.

     

An Unusual ERAD-Like Complex Is Targeted to the Apicoplast of Plasmodium falciparum

Many apicomplexan parasites, including Plasmodium falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event. The exact molecular mechanisms directing the transport of nuclear-encoded proteins to the apicoplast of P. falciparum are not well understood. Recently, in silico analyses revealed a second copy of proteins homologous to components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) system in organisms with secondary plastids, including the malaria parasite P. falciparum. These proteins are predicted to be endowed with an apicoplast targeting signal and are suggested to play a role in the transport of nuclear-encoded proteins to the apicoplast. Here, we have studied components of this ERAD-derived putative preprotein translocon complex in malaria parasites. Using transfection technology coupled with fluorescence imaging techniques we can demonstrate that the N terminus of several ERAD-derived components targets green fluorescent protein to the apicoplast. Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues of yeast ERAD components) localize to the apicoplast, where PfsDer1-1 tightly associates with membranes. Conversely, PfhDer1-1 (a host-specific copy of the Der1-1 protein) localizes to the ER. Our data suggest that ERAD components have been “rewired” to provide a conduit for protein transport to the apicoplast. Our results are discussed in relation to the nature of the apicoplast protein transport machinery.The apicomplexan parasite Plasmodium falciparum is the etiological agent of malaria tropica, the most severe form of human malaria, responsible for over 250 million infections and 1 million deaths annually (61). Many apicomplexan parasites, including P. falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event (27, 58). Although during the course of evolution this plastid organelle has lost the ability to carry out photosynthesis, it is still the site of several important biochemical pathways, including isoprenoid and heme biosynthesis, and as such is essential for parasite survival (60). As in other plastids, the vast majority of genes originally encoded on the plastid genome have been transferred to the nucleus of the host. As a result, their gene products (predicted to constitute up to 10% of all nucleus-encoded proteins) must be imported back into the apicoplast (12). The apicoplast is surrounded by four membranes (55), and this protein import process thus represents a major cell biological challenge and has attracted much research interest, not least due to the importance of P. falciparum as a human pathogen (16, 50).The signals directing transport of nucleus-encoded proteins to complex plastids, including the apicomplexan apicoplast, have been studied in great detail in recent years, and reveal that such proteins are endowed with specific N-terminal targeting sequences, referred to as a bipartite topogenic signals (BTS), that direct their transport to this compartment (50). BTS are composed of an N-terminal endoplasmic reticulum (ER)-type signal sequence, which initially allows proteins to enter the secretory system via the Sec61 complex (59). Following this, proteins are carried via a Golgi complex-independent transport step to the second outermost membrane, from where they are then translocated across the remaining three apicoplast membranes, directed by the second part of the BTS, the transit peptide (51). Based on evolutionary considerations, it has long been suggested that transport across the inner two apicoplast membranes occurs via a Toc/Tic-like (where Toc and Tic are translocons of the outer and inner chloroplast envelopes, respectively) protein translocase machinery, and this is supported by a recent publication that provides evidence for an essential role of a Toxoplasma gondii Tic20 homologue in this transport process (50, 57). Despite this progress, it is still unclear how proteins travel across the second and third outer apicoplast membranes. Several models have been discussed to account for this transport step, including vesicular shuttle and translocon-based mechanisms (recently reviewed in reference 19), but until recently no actual molecular equipment had been found which could account for these membrane translocation events. To address this question, Sommer et al. screened the nucleomorph genome of the chromalveolate cryptophyte Guillardia theta (which, similar to P. falciparum, contains a four-membrane-bound plastid organelle) for genes encoding potential translocon-related proteins (49). Surprisingly, the authors identified genes encoding proteins usually involved in the ER-associated protein degradation pathway (ERAD), which recognizes incorrectly folded protein substrates and retrotranslocates them to the cell cytosol for degradation by the ubiquitin (Ub)-proteasome system (35, 44). As such, the ERAD system functions as a translocation complex, capable of transporting proteins across a biological membrane. Further characterization of one of these proteins (G. theta Der1-1, a homologue of yeast Der1p, a component of the ERAD system) provided strong evidence for a plastid localization. These data suggested an attractive solution to the mechanistic problem of transport across the second and third outermost membrane of complex plastids by hypothesizing a role for an ERAD-derived protein translocon complex. Intriguingly, this study also identified several members of this ERAD-derived translocon complex (apicoplast ERAD [apERAD]) in the nuclear genome of P. falciparum endowed with an N-terminal BTS (49). The BTS derived from one of these proteins, P. falciparum sDer1-1 [PfsDer1-1], was sufficient to direct transport of green fluorescent protein (GFP) to the apicoplast of P. falciparum, suggesting that this ERAD-like machinery is ubiquitous among chromalveolates with four membrane-bound plastids (49). In this current report we extend our study of the P. falciparum apERAD complex.     

Distinct subcellular localization in the cytosol and apicoplast,unexpected dimerization and inhibition of Plasmodium falciparum glyoxalases

The ubiquitous glyoxalase system removes methylglyoxal as a harmful by‐product of glycolysis. Because malaria parasites have drastically increased glycolytic fluxes, they could be highly susceptible to the inhibition of this detoxification pathway. Here we analysed the intracellular localization, oligomerization and inhibition of the glyoxalases from Plasmodium falciparum. Glyoxalase I (GloI) and one of the two glyoxalases II (cGloII) were located in the cytosol of the blood stages. The second glyoxalase II (tGloII) was detected in the apicoplast pointing to alternative metabolic pathways. Using a variety of methods, cGloII was found to exist in a monomer–dimer equilibrium that might have been overlooked for homologues from other organisms and that could be of physiological importance. The compounds methyl‐gerfelin and curcumin, which were previously shown to inhibit mammalian GloI, also inhibited P. falciparum GloI. Inhibition patterns were predominantly competitive but were complicated because of the two different active sites of the enzyme. This effect was neglected in previous inhibition studies of monomeric glyoxalases I, with consequences for the interpretation of inhibition constants. In summary, the present work reveals novel general glyoxalase properties that future research can build on and provides a significant advance in characterizing the glyoxalase system from P. falciparum.     

Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.     

Short-term plant species impact on microbial community structure in soils with long-term agricultural history

The goal of our study was to capture the short-term effects of individual plant species on an established microbial community in a soil with a well-defined agricultural history. Using biochemical and molecular techniques we quantified the effects of plant species on changes in the soil microbial community over an 8-week time-course. We conducted a greenhouse experiment using field soil from a site that was managed as a Zea mays monoculture for over 50 years. The conditioned soil provided a baseline from which changes in microbial community composition through the effects of newly introduced plants could be determined. Within a short time frame (8 weeks), introduced plants influenced the soil microbial community in ways unique to each plant. Some species (Fagopyrm esculentum and xTriticosecale spp.) resulted in an increase of total microbial community richness, diversity and the stimulation of new microbial species not associated with the legacy vegetation. Other plants (Vicia villosa and Lolium multiflorum) tended to reduce community diversity. We suggest root surface area is good general predictor of rhizosphere microbial community diversity, but in some cases other plant traits may have dominant influence on plant-induced changes in microbial community composition.     

Parasite‐encoded Hsp40 proteins define novel mobile structures in the cytosol of the P. falciparum‐infected erythrocyte

Plasmodium falciparum is predicted to transport over 300 proteins to the cytosol of its chosen host cell, the mature human erythrocyte, including 19 members of the Hsp40 family. Here, we have generated transfectant lines expressing GFP‐ or HA‐Strep‐tagged versions of these proteins, and used these to investigate both localization and other properties of these Hsp40 co‐chaperones. These fusion proteins labelled punctate structures within the infected erythrocyte, initially suggestive of a Maurer's clefts localization. Further experiments demonstrated that these structures were distinct from the Maurer's clefts in protein composition. Transmission electron microscopy verifies a non‐cleft localization for HA‐Strep‐tagged versions of these proteins. We were not able to label these structures with BODIPY–ceramide, suggesting a lower size and/or different lipid composition compared with the Maurer's clefts. Solubility studies revealed that the Hsp40–GFP fusion proteins appear to be tightly associated with membranes, but could be released from the bilayer under conditions affecting membrane cholesterol content or organization, suggesting interaction with a binding partner localized to cholesterol‐rich domains. These novel structures are highly mobile in the infected erythrocyte, but based on velocity calculations, can be distinguished from the ‘highly mobile vesicles’ previously described. Our study identifies a further extra‐parasitic structure in the P. falciparum‐infected erythrocyte, which we name ‘J‐dots’ (as their defining characteristic so far is the content of J‐proteins). We suggest that these J‐dots are involved in trafficking of parasite‐encoded proteins through the cytosol of the infected erythrocyte.     

Primate-specific evolution of noncoding element insertion into PLA2G4C and human preterm birth

Background

The use of biological annotation such as genes and pathways in the analysis of gene expression data has aided the identification of genes for follow-up studies and suggested functional information to uncharacterized genes. Several studies have applied similar methods to genome wide association studies and identified a number of disease related pathways. However, many questions remain on how to best approach this problem, such as whether there is a need to obtain a score to summarize association evidence at the gene level, and whether a pathway, dominated by just a few highly significant genes, is of interest.

Methods

We evaluated the performance of two pathway-based methods (Random Set, and Binomial approximation to the hypergeometric test) based on their applications to three data sets of Crohn's disease. We consider both the disease status as a phenotype as well as the residuals after conditioning on IL23R, a known Crohn's related gene, as a phenotype.

Results

Our results show that Random Set method has the most power to identify disease related pathways. We confirm previously reported disease related pathways and provide evidence for IL-2 Receptor Beta Chain in T cell Activation and IL-9 signaling as Crohn's disease associated pathways.

Conclusions

Our results highlight the need to apply powerful gene score methods prior to pathway enrichment tests, and that controlling for genes that attain genome wide significance enable further biological insight.