Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

Oncogenic epithelial cell-derived exosomes containing Rac1 and PAK2 induce angiogenesis in recipient endothelial cells.

The metastatic cascade describes the escape of primary tumour cells to distant secondary sites. Cells at the leading tumour edge are thought to undergo epithelial-mesenchymal transition (EMT), to enhance their motility and invasion for spreading. Whether EMT cells directly promote tumour angiogenesis, and the role of exosomes (30-150 nm extracellular vesicles) remains largely unknown. We examined the functional effects of exosomes from MDCK cells, MDCK cells stably expressing YBX1 (MDCKYBX1, intermediate EMT), and Ras-transformed MDCK cells (21D1 cells, complete EMT). 2F-2B cell motility and tube formation (length and branching) was significantly increased following supplementation with MDCKYBX1 or 21D1 exosomes, but not MDCK exosomes. Next, Matrigel™ plugs containing exosome-supplemented 2F-2B cells were subcutaneously injected into mice. Systemic perfusion was only observed for plugs supplemented with MDCKYBX1 or 21D1 exosomes. Comparative proteomics revealed that 21D1 exosomes contained VEGF-associated proteins, while MDCKYBX1 exosomes were enriched with activated Rac1 and PAK2. To validate, 2F-2B cells and HUVECs were pre-treated with PAK inhibitors prior to exosome supplementation. PAK inhibition nullified the effects of MDCKYBX1 exosomes by reducing the tube length and branching to baseline levels. By contrast, the effects of 21D1 exosomes were not significantly decreased. Our results demonstrate for the first time that oncogenic cells undergoing EMT can communicate with endothelial cells via exosomes, and establish exosomal Rac1/PAK2 as angiogenic promoters that may function from early stages of the metastatic cascade.

Inhibition of hIAPP Amyloid Aggregation and Pancreatic ß-Cell Toxicity by OH-Terminated PAMAM Dendrimer.

Human islet amyloid polypeptide (hIAPP, or amylin) forms amyloid deposits in the islets of Langerhans, a phenomenon that is associated with type-2 diabetes impacting millions of people worldwide. Accordingly, strategies against hIAPP aggregation are essential for the prevention and eventual treatment of the disease. Here, it is shown that generation-3 OH-terminated poly(amidoamine) dendrimer, a polymeric nanoparticle, can effectively halt the aggregation of hIAPP and shut down hIAPP toxicity in pancreatic MIN6 and NIT-1 cells as well as in mouse islets. This finding is supported by high-throughput dynamic light scattering experiment and thioflavin T assay, where the rapid evolution of hIAPP nucleation and elongation processes is halted by the addition of the dendrimer up to 8 h. Discrete molecular dynamics simulations further reveal that hIAPP residues bound strongly with the dendrimer near the c-terminal portion of the peptide, where the amyloidogenic sequence (residues 22-29) locates. Furthermore, simulations of hIAPP dimerization reveal that binding with the dendrimer significantly reduces formation of interpeptide contacts and hydrogen bonds, thereby prohibiting peptide self-association and amyloidosis. This study points to a promising nanomedicinal strategy for combating type-2 diabetes and may have broader implications for targeting neurological disorders whose distinct hallmark is also amyloid fibrillation.

Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells.

During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined sub-domains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process.

Thermoflavifilum aggregans gen. nov., sp. nov., a thermophilic and slightly halophilic filamentous bacterium from the phylum Bacteroidetes.

A strictly aerobic, thermophilic, moderately acidophilic, non-spore-forming bacterium, strain P373(T), was isolated from geothermally heated soil at Waikite, New Zealand. Cells were filamentous rods, 0.2-0.4 µm in diameter and grew in chains up to 80 µm in length. On the basis of 16S rRNA gene sequence similarity, strain P373(T) was shown to belong to the family Chitinophagaceae (class Sphingobacteriia) of the phylum Bacteroidetes, with the most closely related cultivated strain, Chitinophaga pinensis UQM 2034(T), having 87.6 % sequence similarity. Cells stained Gram-negative, and were catalase- and oxidase-positive. The major fatty acids were i-15 : 0 (10.8 %), i-17 : 0 (24.5 %) and i-17 : 0 3-OH (35.2 %). Primary lipids were phosphatidylethanolamine, two unidentified aminolipids and three other unidentified polar lipids. The presence of sulfonolipids (N-acyl-capnines) was observed in the total lipid extract by mass spectrometry. The G+C content of the genomic DNA was 47.3 mol% and the primary respiratory quinone was MK-7. Strain P373(T) grew at 35-63 °C with an optimum temperature of 60 °C, and at pH 5.5-8.7 with an optimum growth pH of 7.3-7.4. NaCl tolerance was up to 5 % (w/v) with an optimum of 0.1-0.25 % (w/v). Cell colonies were non-translucent and pigmented vivid yellow-orange. Cells displayed an oxidative chemoheterotrophic metabolism. The distinct phylogenetic position and the phenotypic characteristics separate strain P373(T) from all other members of the phylum Bacteroidetes and indicate that it represents a novel species in a new genus, for which the name Thermoflavifilum aggregans gen. nov., sp. nov. is proposed. The type strain of the type species is P373(T) ( = ICMP 20041(T) = DSM 27268(T)).