Isolation and Identification of a Rare Spike Gene Double-Deletion SARS-CoV-2 Variant From the Patient With High Cycle Threshold Value

Coronavirus disease 2019 (COVID-19) is an emerging life-threatening pulmonary disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, Hubei Province, China, in December 2019. COVID-19 develops after close contact via inhalation of respiratory droplets containing SARS-CoV-2 during talking, coughing, or sneezing by asymptomatic, presymptomatic, and symptomatic carriers. This virus evolved over time, and numerous genetic variants have been reported to have increased disease severity, mortality, and transmissibility. Variants have also developed resistance to antivirals and vaccination and can escape the immune response of humans. Reverse transcription polymerase chain reaction (RT–PCR) is the method of choice among diagnostic techniques, including nucleic acid amplification tests (NAATs), serological tests, and diagnostic imaging, such as computed tomography (CT). The limitation of RT–PCR is that it cannot distinguish fragmented RNA genomes from live transmissible viruses. Thus, SARS-CoV-2 isolation by using cell culture has been developed and makes important contributions in the field of diagnosis, development of antivirals, vaccines, and SARS-CoV-2 virology research. In this research, two SARS-CoV-2 strains were isolated from four RT–PCR-positive nasopharyngeal swabs using VERO E6 cell culture. One isolate was cultured successfully with a blind passage on day 3 post inoculation from a swab with a Ct > 35, while the cells did not develop cytopathic effects without a blind passage until day 14 post inoculation. Our results indicated that infectious SARS-CoV-2 virus particles existed, even with a Ct > 35. Cultivable viruses could provide additional consideration for releasing the patient from quarantine. The results of the whole genome sequencing and bioinformatic analysis suggested that these two isolates contain a spike 68-76del+spike 675-679del double-deletion variation. The double deletion was confirmed by amplification of the regions spanning the spike gene deletion using Sanger sequencing. Phylogenetic analysis revealed that this double-deletion variant was rare (one per million in public databases, including GenBank and GISAID). The impact of this double deletion in the spike gene on the SARS-CoV-2 virus itself as well as on cultured cells and/or humans remains to be further elucidated.

A unified model for the biogenesis of mitochondrial outer membrane multi-span proteins

The mitochondrial outer membrane (MOM) harbors proteins that traverse the membrane via several helical segments, so-called multi-span proteins. Two contradicting mechanisms were suggested to describe their integration into the MOM. The first proposes that the mitochondrial import (MIM) complex facilitates this process and functions as an insertase, whereas the second suggests that such proteins can integrate into the lipid phase without the assistance of import factors in a process that is enhanced by phosphatidic acid. To resolve this discrepancy and obtain new insights on the biogenesis of these proteins, we addressed this issue using yeast mitochondria and the multi-span protein Om14. Testing different truncation variants, we show that only the full-length protein contains all the required information that assure targeting specificity. Employing a specific insertion assay and several single and double deletion strains, we show that neither the import receptor Tom70 nor any other protein with a cytosolically exposed domain have a crucial contribution to the biogenesis process. We further demonstrate that Mim1 and Porin are required for optimal membrane integration of Om14 but none of them is absolutely required. Unfolding of the newly synthesized protein, its optimal hydrophobicity, as well as higher fluidity of the membrane dramatically enhanced the import capacity of Om14. Collectively, our findings suggest that MOM multi-span proteins can follow different biogenesis pathways in which proteinaceous elements and membrane behavior contribute to a variable extent to the combined efficiency.

A double deletion prevents replication of the pestivirus bovine viral diarrhea virus in the placenta of pregnant heifers

In contrast to wild type bovine viral diarhea virus (BVDV) specific double deletion mutants are not able to establish persistent infection upon infection of a pregnant heifer. Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection. Viral RNA was detected via qRT-PCR and RNAscope analyses in maternal tissues for both viruses from day 6 p.i. on. However, the double deletion mutant was not detected in placenta and was only found in samples from animals infected with the wild type virus. Similarly, high levels of wild type viral RNA were present in fetal tissues whereas the genome of the double deletion mutant was not detected supporting the hypothesis of a specific inhibition of mutant virus replication in the placenta. We compared the induction of gene expression upon infection of placenta derived cell lines with wild type and mutant virus via gene array analysis. Genes important for the innate immune response were strongly upregulated by the mutant virus compared to the wild type in caruncle epithelial cells that establish the cell layer on the maternal side at the maternal–fetal interface in the placenta. Also, trophoblasts which can be found on the fetal side of the interface showed significant induction of gene expression upon infection with the mutant virus although with lower complexity. Growth curves recorded in both cell lines revealed a general reduction of virus replication in caruncular epithelial cells compared to the trophoblasts. Compared to the wild type virus this effect was dramtic for the mutant virus that reached only a TCID50 of 1.0 at 72 hours post infection.

Long-term labeling and imaging of synaptically-connected neuronal networks in vivo using nontoxic, double-deletion-mutant rabies viruses

SummaryThe highly specific and complex connectivity between neurons is the hallmark of nervous systems, but techniques for identifying, imaging, and manipulating synaptically-connected networks of neurons are limited. Monosynaptic tracing, or the gated replication and spread of a deletion-mutant rabies virus to label neurons directly connected to a targeted population of starting neurons1, is the most widely-used technique for mapping neural circuitry, but the rapid cytotoxicity of first-generation rabies viral vectors has restricted its use almost entirely to anatomical applications. We recently introduced double-deletion-mutant second-generation rabies viral vectors, showing that they have little or no detectable toxicity and are efficient means of retrogradely targeting neurons projecting to an injection site2, but they have not previously been shown to be capable of gated replication in vivo, the basis of monosynaptic tracing. Here we present a complete second-generation system for labeling direct inputs to genetically-targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Spread of the viruses requires complementation of both of the deleted viral genes in trans in the starting postsynaptic cells; suppressing the expression of these viral genes following an initial period of viral replication, using the Tet-Off system, reduces toxicity to the starting cells without decreasing the efficiency of viral spread. Using longitudinal two- photon imaging of live monosynaptic tracing in visual cortex, we found that 94.4% of all labeled cells, and an estimated 92.3% of starting cells, survived for the full twelve-week course of imaging. Two-photon imaging of calcium responses in labeled networks of neurons in vivo over ten weeks showed that labeled neurons’ visual response properties remained stable for as long as we followed them. This nontoxic labeling of inputs to genetically-targeted neurons in vivo is a long-held goal in neuroscience, with transformative applications including nonperturbative transcriptomic and epigenomic profiling, long-term functional imaging and behavioral studies, and optogenetic and chemogenetic manipulation of synaptically-connected neuronal networks over the lifetimes of experimental animals.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system

The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly.

Deletion of two genes improves heterologous secretion of fungal lignin-degrading heme peroxidases in S. cerevisiae

Efficient, large-scale heterologous production of enzymes is a crucial component of the biomass valorization industry. Whereas cellulose utilization has been successful in applications such as bioethanol, its counterpart lignin remains significantly underutilized despite being an abundant potential source of aromatic commodity chemicals. Fungal lignin-degrading heme peroxidases are thought to be the major agents responsible for lignin depolymerization in nature, but their large-scale production remains inaccessible due to the genetic intractability of basidiomycete fungi and the challenges in the heterologous production of these enzymes. In this study, we employ a strain engineering approach based on functional genomics to identify mutants of the model yeast Saccharomyces cerevisiae with enhanced heterologous production of lignin-degrading heme peroxidases. We show that our screening method coupling an activity-based readout with fluorescence-assisted cell sorting enables identification of two single null mutants of S. cerevisiae, pmt2 and cyt2, with up to 11-fold improved secretion of a versatile peroxidase from the lignin-degrading fungus Pleurotus eryngii. We demonstrate that the double deletion strain pmt2cyt2 displays positive epistasis, improving and even enabling production of members from all three classes of lignin-degrading fungal peroxidases. We anticipate that these mutant strains will be broadly applicable for improved heterologous production of this biotechnologically important class of enzymes.

Evaluation of Safety and Protective Efficacy of a waaJ and spiC Double Deletion Korean Epidemic Strain of Salmonella enterica Serovar Gallinarum

With an aim to develop a highly attenuated and strongly immunogenic distinguishable vaccine candidate, a waaJ (a gene involved in the synthesis of lipopolysaccharide) and spiC (a virulence gene) double deletion Korean epidemic strain of S. enterica ser. Gallinarum (SG005) was constructed. Our results showed that the growth and biochemical characteristics were not altered by this double deletion. The double deletion strain contained dual markers. One was a bacteriological marker (rough phenotype) and the other was a serological marker helping distinguish infected chickens from vaccinated chickens. The double deletion strain showed good genetic stability and reduced resistance to environmental stresses in vitro; furthermore, it was extremely safe and highly avirulent in broilers. Single intramuscular or oral immunization of 7-day-old broilers with the double deletion strain could stimulate the body to produce antibody levels similar to the conventional vaccine strain SG9R. In addition, against a lethal wild-type challenge, it conferred effective protection that was comparable to that seen in the group vaccinated with SG9R. In conclusion, this double deletion strain may be an effective vaccine candidate for controlling S. enterica ser. Gallinarum infection in broilers.

Effects of dual deletion of glnR and mtrA on expression of nitrogen metabolism genes in Streptomyces venezuelae

GlnR activates nitrogen metabolism genes under nitrogen-limited conditions whereas MtrA represses these genes under nutrient-rich conditions in Streptomyces. In this study, we compared the transcription patterns of nitrogen metabolism genes in a double deletion mutant (ΔmtrA-glnR) lacking both mtrA and glnR and in mutants lacking either mtrA (ΔmtrA) or glnR (ΔglnR). The nitrogen metabolism genes were expressed similarly in ΔmtrA-glnR and ΔglnR under both nitrogen-limited and nutrient-rich conditions, with patterns distinctly different from that of ΔmtrA, suggesting a decisive role for GlnR in the control of nitrogen metabolism genes and further suggesting that regulation of these genes by MtrA is GlnR-dependent. MtrA and GlnR utilize the same binding sites upstream of nitrogen metabolism genes, and we showed stronger in vivo binding of MtrA to these sites under nutrient-rich conditions and of GlnR under nitrogen-limited conditions, consistent with the higher levels of MtrA or GlnR under those respective conditions. In addition, we showed that both mtrA and glnR are auto-regulatory. Our study provides new insights into the regulation of nitrogen metabolism genes in Streptomyces.

Endometrial PTEN Deficiency Leads to SMAD2/3 Nuclear Translocation

TGF-β has a dichotomous function, acting as tumor suppressor in premalignant cells but as a tumor promoter for cancerous cells. These contradictory functions of TGF-β are caused by different cellular contexts, including both intracellular and environmental determinants. The TGF-β/SMAD and the PI3K/PTEN/AKT signal transduction pathways have an important role in the regulation of epithelial cell homeostasis and perturbations in either of these two pathways’ contributions to endometrial carcinogenesis. We have previously demonstrated that both PTEN and SMAD2/3 display tumor-suppressive functions in the endometrium, and genetic ablation of either gene results in sustained activation of PI3K/AKT signaling that suppresses TGF-β-induced apoptosis and enhances cell proliferation of mouse endometrial cells. However, the molecular and cellular effects of PTEN deficiency on TGF-β SMAD2/3 signaling remain controversial. Here, using an in vitro and in vivo model of endometrial carcinogenesis, we have demonstrated that loss of PTEN leads to a constitutive SMAD2/3 nuclear translocation. To ascertain the function of nuclear SMAD2/3 downstream of PTEN deficiency, we analyzed the effects of double deletion PTEN and SMAD2/3 in mouse endometrial organoids. Double PTEN/SMAD2/3 ablation results in a further increase of cell proliferation and enlarged endometrial organoids compared to those harboring single PTEN, suggesting that nuclear translocation of SMAD2/3 constrains tumorigenesis induced by PTEN deficiency.