In situ structures of membrane-assisted assembly and selective autophagy of enteroviruses

SummaryEnteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. New virions are thought to be assembled near the genome replication sites and are released in vesicles through secretory autophagy. Here, we use cryo-electron tomography to show that poliovirus assembles directly on replication membranes. Assembly progression beyond a membrane-bound half-capsid intermediate requires the host lipid kinase VPS34, whereas inhibition of ULK1, the initiator of canonical autophagy, leads to accumulation of virions in vast intracellular arrays followed by an increased release at later time points. We further identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from a second class of autophagic membranes containing protein filaments bundles. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptide promotes clamp closing; their mature domain overcomes the rate limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly catch and release trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics and become secreted.

Itpr1 regulates the formation of anterior eye segment tissues derived from neural crest cells

Mutations in ITPR1 cause ataxia and aniridia in individuals with Gillespie syndrome (GLSP). However, the pathogenic mechanisms underlying aniridia remain unclear. We identified a de novo GLSP mutation hotspot in the 3′-region of ITPR1 in five individuals with GLSP. Furthermore, RNA-sequencing and immunoblotting revealed an eye-specific transcript of Itpr1 (218-aa isoform), encoding 218 amino acids (aa). This isoform is localized not only in the endoplasmic reticulum, but also in the nuclear and cytoplasmic membranes. Ocular-specific transcription was repressed by SOX9 and induced by c-MAF in the anterior eye segment (AES) tissues. Mice lacking seven base pairs of the last Itpr1 exon exhibited ataxia and aniridia, in which the iris lymphatic vessels, sphincter and dilator muscles, corneal endothelium and stroma were disrupted, but the neural crest cells persisted after the completion of AES formation. Our analyses revealed that the 218-aa isoform regulated the directionality of actin fibers and the intensity of focal adhesion. The isoform might control the nuclear entry of transcriptional regulators, such as YAP. It is possible that ITPR1 regulates both AES differentiation and muscle contraction in the iris.

Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic

Colistin is a polymyxin antibiotic of last resort for the treatment of infections caused by multi-drug resistant Gram-negative bacteria. By targeting lipopolysaccharide (LPS), the antibiotic disrupts both the outer and cytoplasmic membranes, leading to lysis and bacterial death. Colistin resistance in Escherichia coli occurs via mutations in the chromosome or the acquisition of mobilised colistin resistance (mcr) genes. Both these colistin resistance mechanisms result in chemical modifications to the LPS, with positively charged moieties added at the cytoplasmic membrane before the LPS is transported to the outer membrane. We have previously shown that MCR-1-mediated LPS modification protects the cytoplasmic but not the outer membrane from damage caused by colistin, enabling bacterial survival. However, it remains unclear whether this observation extends to colistin resistance conferred by other mcr genes, or resistance due to chromosomal mutations. Using a panel of clinical E. coli that had acquired mcr -1, -1.5, -2, -3, -3.2 or -5, or had acquired polymyxin resistance independently of mcr genes, we found that almost all isolates were susceptible to colistin-mediated permeabilisation of the outer, but not cytoplasmic, membrane. Furthermore, we showed that permeabilisation of the outer membrane of colistin resistant isolates by the polymyxin is in turn sufficient to sensitise bacteria to the antibiotic rifampicin, which normally cannot cross the LPS monolayer. These findings demonstrate that colistin resistance in E. coli is typically due to protection of the cytoplasmic but not outer membrane from colistin-mediated damage, regardless of the mechanism of resistance.

Bacterial cytoplasmic membranes synergistically enhance the antitumor activity of autologous cancer vaccines

Cancer vaccines based on resected tumors from patients have gained great interest as an individualized cancer treatment strategy. However, eliciting a robust therapeutic effect with personalized vaccines remains a challenge because of the weak immunogenicity of autologous tumor antigens. Utilizing exogenous prokaryotic constituents that act as adjuvants to enhance immunogenicity is a promising strategy to overcome this limitation. However, nonspecific stimulation of the immune system may elicit an undesirable immunopathological state. To specifically trigger sufficient antitumor reactivity without notable adverse effects, we developed an antigen and adjuvant codelivery nanoparticle vaccine based on Escherichia coli cytoplasmic membranes (EMs) and tumor cell membranes (TMs) from resected autologous tumor tissue. Introduction of the EM into the hybrid membrane nanoparticle vaccines (HM-NPs) induced dendritic cell maturation, thus activating splenic T cells. HM-NPs showed efficacy in immunogenic CT26 colon and 4T1 breast tumor mouse models and also efficiently induced tumor regression in B16-F10 melanoma and EMT6 breast tumor mouse models. Furthermore, HM-NPs provoked a strong tumor-specific immune response, which not only extended postoperative animal survival but also conferred long-term protection (up to 3 months) against tumor rechallenge in a CT26 colon tumor mouse model. Specific depletion of different immune cell populations revealed that CD8+ T and NK cells were crucial to the vaccine-elicited tumor regression. Individualized autologous tumor antigen vaccines based on effective activation of the innate immune system by bacterial cytoplasmic membranes hold great potential for personalized treatment of postoperative patients with cancer.

Genome-Wide Identification and Bioinformatics Characterization of Superoxide Dismutases in the Desiccation-Tolerant Cyanobacterium Chroococcidiopsis sp. CCMEE 029

A genome-wide investigation of the anhydrobiotic cyanobacterium Chroococcidiopsis sp. CCMEE 029 identified three genes coding superoxide dismutases (SODs) annotated as MnSODs (SodA2.1 and SodA2.2) and Cu/ZnSOD (SodC) as suggested by the presence of metal-binding motifs and conserved sequences. Structural bioinformatics analysis of the retrieved sequences yielded modeled MnSODs and Cu/ZnSOD structures that were fully compatible with their functional role. A signal-peptide bioinformatics prediction identified a Tat signal peptide at the N-terminus of the SodA2.1 that highlighted its transport across the thylakoid/cytoplasmic membranes and release in the periplasm/thylakoid lumen. Homologs of the Tat transport system were identified in Chroococcidiopsis sp. CCMEE 029, and the molecular docking simulation confirmed the interaction between the signal peptide of the SodA2.1 and the modeled TatC receptor, thus supporting the SodA2.1 translocation across the thylakoid/cytoplasmic membranes. No signal peptide was predicted for the MnSOD (SodA2.2) and Cu/ZnSOD, thus suggesting their occurrence as cytoplasmic proteins. No FeSOD homologs were identified in Chroococcidiopsis sp. CCMEE 029, a feature that might contribute to its desiccation tolerance since iron produces hydroxyl radical via the Fenton reaction. The overall-overexpression in response to desiccation of the three identified SOD-coding genes highlighted the role of SODs in the antioxidant enzymatic defense of this anhydrobiotic cyanobacterium. The periplasmic MnSOD protected the cell envelope against oxidative damage, the MnSOD localized in the thylakoid lumen scavengered superoxide anion radical produced during the photosynthesis, while the cytoplasmic MnSOD and Cu/ZnSOD reinforced the defense against reactive oxygen species generated at the onset of desiccation. Results contribute to decipher the desiccation-tolerance mechanisms of this cyanobacterium and allow the investigation of its oxidative stress response during future space experiments in low Earth orbit and beyond.

Comparative Antimicrobial Activity of Hp404 Peptide and Its Analogs against Acinetobacter baumannii

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion Heterometrus petersii. Hp1404 exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus but is cytotoxic. In this study, we designed antimicrobial peptides by substituting amino acids at the 14 C-terminal residues of Hp1404 to reduce toxicity and improve antibacterial activity. The analog peptides, which had an amphipathic α-helical structure, were active against gram-positive and gram-negative bacteria, particularly multidrug-resistant Acinetobacter baumannii, and showed lower cytotoxicity than Hp1404. N-phenyl-1-naphthylamine uptake and DisC3-5 assays demonstrated that the peptides kill bacteria by effectively permeating the outer and cytoplasmic membranes. Additionally, the analog peptides inhibited biofilm formation largely than Hp1404 at low concentrations. These results suggest that the analog peptides of Hp1404 can be used as therapeutic agents against A. baumannii infection.

Effect of Low Temperature on Germination, Growth, and Seed Yield of Four Soybean (Glycine max L.) Cultivars

During germination at low temperatures, seeds rich in proteins may experience damage to their cytoplasmic membranes. The study aimed to investigate the influence of the germination temperature on growth, development, and yield of four cultivars of soybean, a typical thermophilic species. The seeds were germinated at 10, 15, and 25 °C in the dark. After 48 h, one part of the seeds was analyzed for their amylase and dehydrogenase activity, cell membrane permeability, and germination vigor. The other part was transferred into soil and cultivated up to yielding. Chlorophyll fluorescence, fresh (FW) and dry weight (DW) of shoots, pod and seed number, and seed DW were analyzed. The plants of cvs. ‘Abelina’, ‘Malaga’, and ‘Merlin’, germinating at low temperature, produced the highest number of seeds. Seed number negatively correlated with their DW and positively with the number of active reaction centers (RC/CSm) in all cultivars. In cvs. ‘Abelina’ and ‘Malaga’, the number of seeds also positively correlated with the index performance of photosystem II (PSII), which was the highest in all plants germinating at low temperature. We suggest cultivating cv. ‘Abelina’ in cooler regions, while cvs. ‘Petrina’ and ‘Malaga’ in warmer areas.

Safety of Antimetabolite 2-Deoxy-D-arabinohexose (2DG) as a Coadjuvant Metabolic Intervention in 268 Cancer Patients

Although previously found to be quite safe and potentially useful as a metabolic sensitizer against a wide spectrum of cancer subtypes, 2-Deoxy-D-Arabinohexose, commonly known as 2-Deoxy-D-Glucose (2DG), has remained somewhat ignored in the clinical setting. As a glycolysis inhibitor, 2DG preferentially targets tumour cells, which densely overexpress glucose transporters (GLUTs) in their cytoplasmic membranes, as well as glycolytic and fermentation enzymes hexokinase 2 (HK-2) and lactic dehydrogenase isoenzyme A (LDH-A). The pronounced functional asymmetry, the distinct metabolic phenotypes that set apart neoplastic and normal cells offer a therapeutic window of opportunity to overcome multidrug resistance in the treatment of cancer. Nutripharmacological corrections of blood glucose, followed by the timely introduction of several nonmetabolizable structural analogues, is a cost-effective, minimally invasive coadjuvant treatment for solid tumours. Further, our research group has shown that a metabolic intervention with antimetabolites of glucose and pyruvate is strongly enhanced by a systemic suppression of the natural substrates of their catalyzing, rate-limiting enzymes within cancer cells. Here, we demonstrate that 2DG, perhaps the archetypal glucose antimetabolite, is very safe in humans.