α-Synuclein inhibits Snx3-retromer retrograde trafficking of the conserved membrane-bound proprotein convertase Kex2 in the secretory pathway of Saccharomyces cerevisiae

We tested the ability of alpha-synuclein (α-syn) to inhibit Snx3-retromer mediated retrograde trafficking of Kex2 and Ste13 between late endosomes and the trans-Golgi (TGN) using a Saccharomyces cerevisiae model of Parkinson’s disease (PD). Kex2 and Ste13 are a conserved, membrane-bound proprotein convertase and dipeptidyl aminopeptidase, respectively, that process pro-α-factor and pro-killer toxin. Each of these proteins contains a cytosolic tail that binds to sorting nexin Snx3. Using a combination of techniques, including fluorescence microscopy, western blotting and a yeast mating assay, we found that α-syn disrupts Snx3-retromer trafficking of Kex2-GFP and GFP-Ste13 from the late endosome to the TGN, resulting in these two proteins transiting to the vacuole by default. Using three α-syn variants (A53T, A30P, and α-synΔC, which lacks residues 101–140), we further found that A53T and α-synΔC, but not A30P, reduce Snx3-retromer trafficking of Kex2-GFP, which is likely to be due to weaker binding of A30P to membranes. Degradation of Kex2 and Ste13 in the vacuole should result in the secretion of unprocessed, inactive forms of α-factor, which will reduce mating efficiency between MATa and MATα cells. We found that wild-type α-syn but not A30P significantly inhibited the secretion of α-factor. Collectively, our results support a model in which the membrane-binding ability of α-syn is necessary to disrupt Snx3-retromer retrograde recycling of these two conserved endopeptidases.

Whole genome sequencing reveals the genomic diversity, taxonomic classification, and evolutionary relationships of the genus Nocardia

Nocardia is a complex and diverse genus of aerobic actinomycetes that cause complex clinical presentations, which are difficult to diagnose due to being misunderstood. To date, the genetic diversity, evolution, and taxonomic structure of the genus Nocardia are still unclear. In this study, we investigated the pan-genome of 86 Nocardia type strains to clarify their genetic diversity. Our study revealed an open pan-genome for Nocardia containing 265,836 gene families, with about 99.7% of the pan-genome being variable. Horizontal gene transfer appears to have been an important evolutionary driver of genetic diversity shaping the Nocardia genome and may have caused historical taxonomic confusion from other taxa (primarily Rhodococcus, Skermania, Aldersonia, and Mycobacterium). Based on single-copy gene families, we established a high-accuracy phylogenomic approach for Nocardia using 229 genome sequences. Furthermore, we found 28 potentially new species and reclassified 16 strains. Finally, by comparing the topology between a phylogenomic tree and 384 phylogenetic trees (from 384 single-copy genes from the core genome), we identified a novel locus for inferring the phylogeny of this genus. The dapb1 gene, which encodes dipeptidyl aminopeptidase BI, was far superior to commonly used markers for Nocardia and yielded a topology almost identical to that of genome-based phylogeny. In conclusion, the present study provides insights into the genetic diversity, contributes a robust framework for the taxonomic classification, and elucidates the evolutionary relationships of Nocardia. This framework should facilitate the development of rapid tests for the species identification of highly variable species and has given new insight into the behavior of this genus.

Search for Viral Infections in Cerebrospinal Fluid From Patients With Autoimmune Encephalitis

Background It has been reported that virus-mediated brain tissue damage can lead to autoimmune encephalitis (AE) characterized by the presence of antibodies against neuronal surface antigens. In the study, we investigate the presence of viruses in cerebrospinal fluid (CSF) from patients with AE using reverse transcription polymerase chain reaction (RT-PCR)/PCR and shotgun metagenomics. Methods CSF samples collected from 200 patients with encephalitis were tested for the presence of antibodies against antiglutamate receptor (NMDAR), contactin-associated protein 2 (CASPR2), glutamate receptors (type AMPA1/2), leucine-rich glioma-inactivated protein 1 (LGI1), dipeptidyl aminopeptidase-like protein 6 (DPPX), and GABA B receptor, and those found positive were further analyzed with real-time RT-PCR/PCR for common viral neuroinfections and shotgun DNA- and RNA-based metagenomics. Results Autoantibodies against neuronal cells were detected in CSF from 8 individuals (4% of all encephalitis patients): 7 (3.5%) had anti-NMDAR and 1 (0.5%) had anti-GABA B. RT-PCR/PCR identified human herpes virus type 1 (HSV-1; 300 copies/mL) and the representative of Enterovirus genus (550 copies/mL) in 1 patient each. Torque teno virus (TTV) was found in another patient using metagenomic analysis, and its presence was confirmed by specific PCR. Conclusions We detected the presence of HSV, TTV, and Enterovirus genus in CSF samples from 3 out of 8 AE patients. These findings support the concept of viral involvement in the pathogenesis of this disease.