Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα protein targets. The N terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by the delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide with three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has an as-yet-undefined function, and C3 catalyzes the deamidation of a specific active-site glutamine residue in Gα protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates the delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers nonnative green fluorescent protein (GFP) cargo into the cytosol, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for the cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105 to 1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

Application of CRISPR-Based Human and Bacterial Ribosomal RNA Depletion for SARS-CoV-2 Shotgun Metagenomic Sequencing.

OBJECTIVES: The aim of this study is to evaluate the effectiveness of a CRISPR-based human and bacterial ribosomal RNA (rRNA) depletion kit (JUMPCODE Genomics) on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shotgun metagenomic sequencing in weakly positive respiratory samples. METHODS: Shotgun metagenomics was performed on 40 respiratory specimens collected from solid organ transplant patients and deceased intensive care unit patients at UCLA Medical Center in late 2020 to early 2021. Human and bacterial rRNA depletion was performed on remnant library pools prior to sequencing by Illumina MiSeq. Data quality was analyzed using Geneious Prime, whereas the identification of SARS-CoV-2 variants and lineages was determined by Pangolin. RESULTS: The average genome coverage of the rRNA-depleted respiratory specimens increased from 72.55% to 93.71% in overall samples and from 29.3% to 83.3% in 15 samples that failed to achieve sufficient genome coverage using the standard method. Moreover, rRNA depletion enhanced genome coverage to over 85% in 11 (73.3%) of 15 low viral load samples with cycle threshold values up to 35, resulting in the identification of genotypes. CONCLUSION: The CRISPR-based human and bacterial rRNA depletion enhanced the sensitivity of SARS-CoV-2 shotgun metagenomic sequencing, especially in low viral load samples.

On variants and vaccines: The effectiveness of Covid-19 monoclonal antibody therapy during two distinct periods in the pandemic.

BACKGROUND: While Covid-19 monoclonal antibody therapies (Mab) have been available in the outpatient setting for over a year and a half, little is known about the impact of emerging variants and vaccinations on the effectiveness of Mab therapies. We sought to determine the effectiveness of Covid-19 Mab therapies during the first two waves of the pandemic in Los Angeles County and assess the impact of vaccines, variants, and other confounding factors. METHODS AND FINDINGS: We retrospectively examined records for 2209 patients of with confirmed positive molecular SARS-CoV2 test either referred for outpatient Mab therapy or receiving Mab treatment in the emergency department (ED) between December 2020 and 2021. Our primary outcome was the combined 30-day incidence of ED visit, hospitalization, or death following the date of referral. Additionally, SARS-CoV2 isolates of hospitalized patients receiving Mabs were sequenced. The primary outcome was significantly reduced with combination therapy compared to bamlanivimab or no treatment (aHR 0·60; 95% CI ·37, ·99), with greater benefit in unvaccinated, moderate-to-high-risk patients (aHR ·39; 95% CI ·20, ·77). Significant associations with the primary outcome included history of lung disease (HR 7·13; 95% CI 5·12, 9·95), immunocompromised state (HR 6·59; 95% CI 2·91-14·94), and high social vulnerability (HR 2·29, 95% CI 1·56-3·36). Two predominant variants were noted during the period of observation: the Epsilon variant and the Delta variant. CONCLUSIONS: Only select monoclonal antibody therapies significantly reduced ED visits, hospitalizations, and death due to COVID-19 during. Vaccination diminished effectiveness of Mabs. Variant data and vaccination status should be considered when assessing the benefit of novel COVID-19 treatments.

Selective Membrane Redistribution and Depletion of Gαq-Protein by Pasteurella multocida Toxin.

Pasteurella multocida toxin (PMT), the major virulence factor responsible for zoonotic atrophic rhinitis, is a protein deamidase that activates the alpha subunit of heterotrimeric G proteins. Initial activation of G alpha-q-coupled phospholipase C-beta-1 signaling by PMT is followed by uncoupling of G alpha-q-dependent signaling, causing downregulation of downstream calcium and mitogenic signaling pathways. Here, we show that PMT decreases endogenous and exogenously expressed G alpha-q protein content in host cell plasma membranes and in detergent resistant membrane (DRM) fractions. This membrane depletion of G alpha-q protein was dependent upon the catalytic activity of PMT. Results indicate that PMT-modified G alpha-q redistributes within the host cell membrane from the DRM fraction into the soluble membrane and cytosolic fractions. In contrast, PMT had no affect on G alpha-s or G beta protein levels, which are not substrate targets of PMT. PMT also had no affect on G alpha-11 levels, even though G alpha-11 can serve as a substrate for deamidation by PMT, suggesting that membrane depletion of PMT-modified G-alpha-q has specificity.

Membrane interaction of Pasteurella multocida toxin involves sphingomyelin.

Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.