Characterization of phage evolution and phage resistance in drug-resistant Stenotrophomonas maltophilia.

Phage therapy has become a viable antimicrobial treatment as an alternative to antibiotic treatment, with an increase in antibiotic resistance. Phage resistance is a major limitation in the therapeutic application of phages, and the lack of understanding of the dynamic changes between bacteria and phages constrains our response strategies to phage resistance. In this study, we investigated the changing trends of mutual resistance between Stenotrophomonas maltophilia (S. maltophilia) and its lytic phage, BUCT603. Our results revealed that S. maltophilia resisted phage infection through mutations in the cell membrane proteins, while the evolved phage re-infected the resistant strain primarily through mutations in structure-related proteins. Compared with the wild-type strain (SMA118), the evolved phage-resistant strain (R118-2) showed reduced virulence, weakened biofilm formation ability, and reduced resistance to aminoglycosides. In addition, the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant S. maltophilia in vitro and significantly improve the survival rate of S. maltophilia-infected mice. Altogether, these results suggest that in vitro characterization of bacteria-phage co-evolutionary relationships is a useful research tool to optimize phages for the treatment of drug-resistant bacterial infections.IMPORTANCEPhage therapy is a promising approach to treat infections caused by drug-resistant Stenotrophomonas maltophilia (S. maltophilia). However, the rapid development of phage resistance has hindered the therapeutic application of phages. In vitro evolutionary studies of bacteria-phage co-cultures can elucidate the mechanism of resistance development between phage and its host. In this study, we investigated the resistance trends between S. maltophilia and its phage and found that inhibition of phage adsorption is the primary strategy by which bacteria resist phage infection in vitro, while phages can re-infect bacterial cells by identifying other adsorption receptors. Although the final bacterial mutants were no longer infected by phages, they incurred a fitness cost that resulted in a significant reduction in virulence. In addition, the combination treatment with phage and aminoglycoside antibiotics could prevent the development of phage resistance in S. maltophilia in vitro. These findings contribute to increasing the understanding of the co-evolutionary relationships between phages and S. maltophilia.

Mammalian Commensal Streptococci Utilize a Rare Family of Class VI Lanthipeptide Synthetases to Synthesize Miniature Lanthipeptide-type Ribosomal Peptide Natural Products.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products with remarkable chemical and functional diversities. These peptides are often synthesized as signals or antibiotics and frequently associated with quorum sensing (QS) systems. With the increasing number of available genomes, many hitherto unseen RiPP biosynthetic pathways have been mined, providing new resources for novel bioactive compounds. Herein, we investigated the underexplored biosynthetic potential of Streptococci, prevalent bacteria in mammal-microbiomes that include pathogenic, mutualistic, and commensal members. Using the transcription factor-centric genome mining strategy, we discovered a new family of lanthipeptide biosynthetic loci under the control of potential QS. By in vitro studies, we investigated the reaction of one of these lanthipeptide synthetases and found that it installs only one lanthionine moiety onto its short precursor peptide by connecting a conserved TxxC region. Bioinformatics and in vitro studies revealed that these lanthipeptide synthetases (class VI) are novel lanthipeptide synthetases with a truncated lyase, a kinase, and a truncated cyclase domain. Our data provide important insights into the processing and evolution of lanthipeptide synthetase to tailor smaller substrates. The data are important for obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of lanthipeptides.

Transcriptome analysis of cepharanthine against a SARS-CoV-2-related coronavirus.

Antiviral therapies targeting the pandemic coronavirus disease 2019 (COVID-19) are urgently required. We studied an already-approved botanical drug cepharanthine (CEP) in a cell culture model of GX_P2V, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related virus. RNA-sequencing results showed the virus perturbed the expression of multiple genes including those associated with cellular stress responses such as endoplasmic reticulum (ER) stress and heat shock factor 1 (HSF1)-mediated heat shock response, of which heat shock response-related genes and pathways were at the core. CEP was potent to reverse most dysregulated genes and pathways in infected cells including ER stress/unfolded protein response and HSF1-mediated heat shock response. Additionally, single-cell transcriptomes also confirmed that genes of cellular stress responses and autophagy pathways were enriched in several peripheral blood mononuclear cells populations from COVID-19 patients. In summary, this study uncovered the transcriptome of a SARS-CoV-2-related coronavirus infection model and anti-viral activities of CEP, providing evidence for CEP as a promising therapeutic option for SARS-CoV-2 infection.

Computational design of ultrashort peptide inhibitors of the receptor-binding domain of the SARS-CoV-2 S protein.

Targeting the interaction between severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) is believed to be an effective strategy for drug design to inhibit the infection of SARS-CoV-2. Herein, several ultrashort peptidase inhibitors against the RBD-ACE2 interaction were obtained by a computer-aided approach based on the RBD-binding residues on the protease domain (PD) of ACE2. The designed peptides were tested on a model coronavirus GX_P2V, which has 92.2 and 86% amino acid identity to the SARS-CoV-2 spike protein and RBD, respectively. Molecular dynamics simulations and binding free energy analysis predicted a potential binding pocket on the RBD of the spike protein, and this was confirmed by the specifically designed peptides SI5α and SI5α-b. They have only seven residues, showing potent antiviral activity and low cytotoxicity. Enzyme-linked immunosorbent assay result also confirmed their inhibitory ability against the RBD-ACE2 interaction. The ultrashort peptides are promising precursor molecules for the drug development of Corona Virus Disease 2019, and the novel binding pocket on the RBD may be helpful for the design of RBD inhibitors or antibodies against SARS-CoV-2.

Omicron breakthrough infections in wild-type SARS-CoV-2 vaccinees elicit high levels of neutralizing antibodies against pangolin coronavirus GX_P2V.

Omicron BF.7 became the predominant SARS-CoV-2 variant in Beijing after the abolishment of Zero-COVID policy in December 2022. The ability of antibodies elicited by BF.7 infection to cross-react with SARS-CoV-2-like viruses is unknown. This study aimed to investigate the cross-reactive neutralizing antibodies against SARS-CoV-2-related pangolin coronavirus GX_P2V in sera from vaccinated and/or SARS-CoV-2-infected individuals. All vaccinated individuals who recovered from Omicron BF.7 breakthrough infections exhibited substantially higher levels of neutralizing antibodies against GX_P2V, compared to other subject groups, with a geometric mean titer (GMT) of 362. Uninfected individuals who received four-mixed-dose vaccines also demonstrated higher levels of neutralizing antibodies (GMT = 44) against GX_P2V than those uninfected individuals who received two- or three-dose vaccines and those unvaccinated convalescents of wild-type SARS-CoV-2. This study highlights the significance of prior vaccinations with wild-type SARS-CoV-2 vaccines in generating potent cross-protective immunity against future spillovers of SARS-CoV-2-like viruses.

Bovine lactoferrin inhibits SARS-CoV-2 and SARS-CoV-1 by targeting the RdRp complex and alleviates viral infection in the hamster model.

Breast milk has been found to inhibit coronavirus infection, while the key components and mechanisms are unknown. We aimed to determine the components that contribute to the antiviral effects of breastmilk and explore their potential mechanism. Lactoferrin (Lf) and milk fat globule membrane inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related coronavirus GX_P2V and transcription- and replication-competent SARS-CoV-2 virus-like particles in vitro and block viral entry into cells. We confirmed that bovine Lf (bLf) blocked the binding between human angiotensin-converting enzyme 2 and SARS-CoV-2 spike protein by combining receptor-binding domain (RBD). Importantly, bLf inhibited RNA-dependent RNA polymerase (RdRp) activity of both SARS-CoV-2 and SARS-CoV in vitro in the nanomolar range. So far, no biological macromolecules have been reported to inhibit coronavirus RdRp. Our result indicated that bLf plays a major role in inhibiting viral replication. bLf treatment reduced viral load in lungs and tracheae and alleviated pathological damage. Our study provides evidence that bLf prevents SARS-CoV-2 infection by combining SARS-CoV-2 spike protein RBD and inhibiting coronaviruses' RdRp activity, and may be a promising candidate for the treatment of coronavirus disease 2019.

Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3).

BACKGROUND: Identification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function. RESULTS: We expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3'-5' exonuclease activity, but does not have 5'-3' exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5'-3' polymerase activity and can amplify DNA at 15-35 °C and a pH range of 5.5-9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3'-5' exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10-6). CONCLUSIONS: A novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.

Characterization of the novel temperate Staphylococcus haemolyticus phage IME1365_01.

The presence of a novel functional prophage, IME1365_01, was predicted from bacterial high-throughput sequencing data and then successfully induced from Staphylococcus haemolyticus by mitomycin C treatment. Transmission electron microscopy showed that phage IME1365_01 has an icosahedral head (43 nm in diameter) and a long tail (172 nm long). This phage possesses a double-stranded DNA genome of 44,875 bp with a G+C content of 35.35%. A total of 63 putative open reading frames (ORFs) were identified in its genome. BLASTn analysis revealed that IME1365_01 is similar to Staphylococcus phage vB_SepS_E72, but with a genome homology coverage of only 26%. The phage genome does not have fixed termini. In ORF24 of phage IME1365_01, a conserved Toll-interleukin-1 receptor domain of the TIR_2 superfamily (accession no. c123749) is located at its N-terminus, and this might serve as a component of an anti-bacterial system. In conclusion, we developed a platform to obtain active temperate phage from prediction, identification, and induction from its bacterial host. After mass screening using this platform, numerous temperate phages and their innate anti-bacterial elements can provide extensive opportunities for therapy against bacterial (especially drug-resistant bacterial) infections.

Unveil the Secret of the Bacteria and Phage Arms Race.

Bacteria have developed different mechanisms to defend against phages, such as preventing phages from being adsorbed on the surface of host bacteria; through the superinfection exclusion (Sie) block of phage's nucleic acid injection; by restricting modification (R-M) systems, CRISPR-Cas, aborting infection (Abi) and other defense systems to interfere with the replication of phage genes in the host; through the quorum sensing (QS) enhancement of phage's resistant effect. At the same time, phages have also evolved a variety of counter-defense strategies, such as degrading extracellular polymeric substances (EPS) that mask receptors or recognize new receptors, thereby regaining the ability to adsorb host cells; modifying its own genes to prevent the R-M systems from recognizing phage genes or evolving proteins that can inhibit the R-M complex; through the gene mutation itself, building nucleus-like compartments or evolving anti-CRISPR (Acr) proteins to resist CRISPR-Cas systems; and by producing antirepressors or blocking the combination of autoinducers (AIs) and its receptors to suppress the QS. The arms race between bacteria and phages is conducive to the coevolution between bacteria and phages. This review details bacterial anti-phage strategies and anti-defense strategies of phages and will provide basic theoretical support for phage therapy while deeply understanding the interaction mechanism between bacteria and phages.

Genetic and Phenotypic Analysis of Phage-Resistant Mutant Fitness Triggered by Phage-Host Interactions.

The emergence of phage-resistant bacterial strains is one of the biggest challenges for phage therapy. However, the emerging phage-resistant bacteria are often accompanied by adaptive trade-offs, which supports a therapeutic strategy called "phage steering". The key to phage steering is to guide the bacterial population toward an evolutionary direction that is favorable for treatment. Thus, it is important to systematically investigate the impacts of phages targeting different bacterial receptors on the fitness of the bacterial population. Herein, we employed 20 different phages to impose strong evolutionary pressure on the host Pseudomonas aeruginosa PAO1 and examined the genetic and phenotypic responses of their phage-resistant mutants. Among these strains with impaired adsorptions, four types of mutations associated with bacterial receptors were identified, namely, lipopolysaccharides (LPSs), type IV pili (T4Ps), outer membrane proteins (OMPs), and exopolysaccharides (EPSs). PAO1, responding to LPS- and EPS-dependent phage infections, mostly showed significant growth impairment and virulence attenuation. Most mutants with T4P-related mutations exhibited a significant decrease in motility and biofilm formation ability, while the mutants with OMP-related mutations required the lowest fitness cost out of the bacterial populations. Apart from fitness costs, PAO1 strains might lose their resistance to antibiotics when counteracting with phages, such as the presence of large-fragment mutants in this study, which may inspire the usage of phage-antibiotic combination strategies. This work provides methods that leverage the merits of phage resistance relative to obtaining therapeutically beneficial outcomes with respect to phage-steering strategies.

Pharmacological Activity of Cepharanthine.

Cepharanthine, a natural bisbenzylisoquinoline (BBIQ) alkaloid isolated from the plant Stephania Cephalantha Hayata, is the only bisbenzylisoquinoline alkaloid approved for human use and has been used in the clinic for more than 70 years. Cepharanthine has a variety of medicinal properties, including signaling pathway inhibitory activities, immunomodulatory activities, and antiviral activities. Recently, cepharanthine has been confirmed to greatly inhibit SARS-CoV-2 infection. Therefore, we aimed to describe the pharmacological properties and mechanisms of cepharanthine, mainly including antitumor, anti-inflammatory, anti-pathogen activities, inhibition of bone resorption, treatment of alopecia, treatment of snake bite, and other activities. At the same time, we analyzed and summarized the potential antiviral mechanism of cepharanthine and concluded that one of the most important anti-viral mechanisms of cepharanthine may be the stability of plasma membrane fluidity. Additionally, we explained its safety and bioavailability, which provides evidence for cepharanthine as a potential drug for the treatment of a variety of diseases. Finally, we further discuss the potential new clinical applications of cepharanthine and provide direction for its future development.

Discovery and Characterization of Rubrinodin Provide Clues into the Evolution of Lasso Peptides.

Lasso peptides are unique natural products that comprise a class of ribosomally synthesized and post-translationally modified peptides. Their defining three-dimensional structure is a lariat knot, in which the C-terminal tail is threaded through a macrolactam ring formed between the N-terminal amino group and an Asp or Glu side chain (i.e., an isopeptide bond). Recent genome mining strategies have revealed various types of lasso peptide biosynthetic gene clusters and have thus redefined the known chemical space of lasso peptides. To date, over 20 different types of these gene clusters have been discovered, including several different clades from Proteobacteria. Despite the diverse architectures of these gene clusters, which may or may not encode various tailoring enzymes, most currently known lasso peptides are synthesized by two discrete clades defined by the presence of an ATP-binding cassette transporter or its absence and (sometimes) concurrent appearance of an isopeptidase, raising questions about their evolutionary history. Herein, we discovered and characterized the lasso peptide rubrinodin, which is assembled by a gene cluster encoding both an ATP-binding cassette transporter and an isopeptidase. Our bioinformatics analyses of this and other representative cluster types provided new clues into the evolutionary history of lasso peptides. Furthermore, our structural and biochemical investigations of rubrinodin permitted the conversion of this thermolabile lasso peptide into a more thermostable scaffold.

Biological characterization and genomic analysis of Acinetobacter baumannii phage BUCT628.

Acinetobacter baumannii is an opportunistic pathogen that is resistant to the most commonly used antibiotics. In this study, the Acinetobacter phage BUCT628 was isolated from hospital wastewater. BLASTn analysis showed that the genome sequence of BUCT628 shared 89.76% identity with 66% query coverage with that of Acinetobacter phage Bphi-R2919. Genome sequencing showed that the BUCT628 genome is a 44,935-bp linear dsDNA molecule with 37.5% G+C content and 86 open reading frames (ORFs), and no tRNAs were identified.

Genomic analysis of Acinetobacter phage BUCT629, a newly isolated member of the genus Obolenskvirus.

A new virulent Acinetobacter phage, BUCT629 (GenBank no. MZ712044.1), was isolated from hospital sewage. Next-generation sequencing (NGS) results demonstrated that the double-stranded linear DNA genome of phage BUCT629 is 46,325 bp in length with a G+C content of 38%. The BLASTn analysis showed that the genome sequence shared similarity with Acinetobacter phage vB_AbaM_IME285, with 65% query coverage and 98.23% identity, suggesting that phage BUCT629 is a novel phage. The phage genome contains 89 putative protein-coding genes, and no rRNA or tRNA genes were identified. The results of this study may be helpful for discovering new antibacterial agents and for understanding the evolution and genetic diversity of Acinetobacter phages.

2'-Fucosyllactose Inhibits Coxsackievirus Class A Type 9 Infection by Blocking Virus Attachment and Internalisation.

Coxsackieviruses, a genus of enteroviruses in the small RNA virus family, cause fatal infectious diseases in humans. Thus far, there are no approved drugs to prevent these diseases. Human milk contains various biologically active components against pathogens. Currently, the potential activity of breast milk components against the coxsackievirus remains unclear. In our study, the inhibitory effect of 16 major human milk components was tested on coxsackievirus class A type 9 isolate (CV-A9), BUCT01; 2'-Fucosyllactose (2'-FL) was identified to be effective. Time-of-addition, attachment internalisation assays, and the addition of 2'-FL at different time points were applied to investigate its specific role in the viral life cycle. Molecular docking was used to predict 2'-FL's specific cellular targets. The initial screening revealed a significant inhibitory effect (99.97%) against CV-A9 with 10 mg/mL 2'-FL, with no cytotoxicity observed. Compared with the control group, 2'-FL blocked virus entry (85%) as well as inhibited viral attachment (48.4%) and internalisation (51.3%), minimising its infection in rhabdomyosarcoma (RD) cells. The cell pre-incubation with 2'-FL exhibited significant inhibition (73.2-99.9%). Extended incubation between cells with 2'-FL reduced CV-A9 infection (93.9%), suggesting that 2'-FL predominantly targets cells to block infection. Molecular docking results revealed that 2'-FL interacted with the attachment receptor αvß6 and the internalisation receptor FCGRT and ß2M with an affinity of -2.14, -1.87, and -5.43 kcal/mol, respectively. This study lays the foundation for using 2'-FL as a food additive against CV-A9 infections.

Antiviral Drugs Screening for Swine Acute Diarrhea Syndrome Coronavirus.

Coronaviruses as possible cross-species viruses have caused several epidemics. The ongoing emergency of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has posed severe threats to the global economy and public health, which has generated great concerns about zoonotic viruses. Swine acute diarrhea syndrome coronavirus (SADS-CoV), an alpha-coronavirus, was responsible for mass piglet deaths, resulting in unprecedented economic losses, and no approved drugs or vaccines are currently available for SADS-CoV infection. Given its potential ability to cause cross-species infection, it is essential to develop specific antiviral drugs and vaccines against SADS-CoV. Drug screening was performed on a total of 3523 compound-containing drug libraries as a strategy of existing medications repurposing. We identified five compounds (gemcitabine, mycophenolate mofetil, mycophenolic acid, methylene blue and cepharanthine) exhibiting inhibitory effects against SADS-CoV in a dose-dependent manner. Cepharanthine and methylene blue were confirmed to block viral entry, and gemcitabine, mycophenolate mofetil, mycophenolic acid and methylene blue could inhibit viral replication after SADS-CoV entry. This is the first report on SADS-CoV drug screening, and we found five compounds from drug libraries to be potential anti-SADS-CoV drugs, supporting the development of antiviral drugs for a possible outbreak of SADS-CoV in the future.

Characterization and Comparative Genomics Analysis of a New Bacteriophage BUCT610 against Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae.

The spread of multidrug-resistant Klebsiella pneumoniae (MDR-KP) has become an emerging threat as a result of the overuse of antibiotics. Bacteriophage (phage) therapy is considered to be a promising alternative treatment for MDR-KP infection compared with antibiotic therapy. In this research, a lytic phage BUCT610 was isolated from hospital sewage. The assembled genome of BUCT610 was 46,774 bp in length, with a GC content of 48%. A total of 83 open reading frames (ORFs) and no virulence or antimicrobial resistance genes were annotated in the BUCT610 genome. Comparative genomics and phylogenetic analyses showed that BUCT610 was most closely linked with the Vibrio phage pYD38-A and shared 69% homology. In addition, bacteriophage BUCT610 exhibited excellent thermal stability (4-75 °C) and broad pH tolerance (pH 3-12) in the stability test. In vivo investigation results showed that BUCT610 significantly increased the survival rate of Klebsiella pneumonia-infected Galleria mellonella larvae from 13.33% to 83.33% within 72 h. In conclusion, these findings indicate that phage BUCT610 holds great promise as an alternative agent with excellent stability for the treatment of MDR-KP infection.

The Coxiella burnetii QpH1 plasmid is a virulence factor for colonizing bone marrow-derived murine macrophages.

Coxiella burnetii strains carry one of four large, conserved, autonomously replicating plasmids (QpH1, QpRS, QpDV, and QpDG) or a QpRS-like chromosomally integrated sequence of unknown function. Here we report the characterization of the QpH1 plasmid of C. burnetii Nine Mile phase II by making QpH1-deficient strains. A shuttle vector pQGK containing the CBUA0036-0039a region (predicted as being required for the QpH1 maintenance) was constructed. The pQGK vector can be stably transformed into the Nine Mile II and maintained at a similar low copy like QpH1. Importantly, transformation with pQGK cured the endogenous QpH1 due to plasmid incompatibility. Compared to a Nine Mile II transformant of a RSF1010-ori based vector, the pQGK transformant shows a similar growth curve in both axenic media and Buffalo green monkey kidney cells, a variable growth defect in macrophage-like THP-1 cells depending on the origin of inoculum, and dramatically reduced ability of colonizing wild-type bone marrow-derived murine macrophages. Furthermore, we found CBUA0037-0039 ORFs are essential for plasmid maintenance, and CBUA0037-0038 ORFs account for plasmid compatibility. And plasmid-deficient C. burnetii can be isolated by using CBUA0037 or -0038 deletion vectors. Furthermore, QpH1-deficient C. burnetii strains caused a lesser extent of splenomegaly in SCID mice but, intriguingly, they had significant growth in SCID mouse-sourced macrophages. Taken together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine, not human, macrophages. This study suggests a critical role of QpH1 for C. burnetii persistence in rodents and expands the toolkit for the genetic studies in C. burnetii Author summary All C. burnetii isolates carry one of four large, conserved, autonomously replicating plasmids or a plasmid-like chromosomally integrated sequence. The plasmid is a candidate virulence factor of unknown function. Here we describe the construction of novel shuttle vectors that allow making plasmid-deficient C. burnetii mutants. With this plasmid-curing approach, we characterized the role of the QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the plasmid plays a critical role for C. burnetii growth in murine macrophages. Our work suggests an essential role of the QpH1 plasmid for the acquisition of colonizing capability in rodents by C. burnetii This study represents a major step toward unravelling the mystery of the C. burnetii cryptic plasmids.

Ubiquitin-Modified Proteome of SARS-CoV-2-Infected Host Cells Reveals Insights into Virus-Host Interaction and Pathogenesis.

The outbreak of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a serious threat to global public health. The mechanism of pathogenesis and the host immune response to SARS-CoV-2 infection are largely unknown. In the present study, we applied a quantitative proteomic technology to identify and quantify the ubiquitination changes that occur in both the virus and the Vero E6 cells during SARS-CoV-2 infection. By applying label-free, quantitative liquid chromatography with tandem mass spectrometry proteomics, 8943 lysine ubiquitination sites on 3086 proteins were identified, of which 138 sites on 104 proteins were quantified as significantly upregulated, while 828 sites on 447 proteins were downregulated at 72 h post-infection. Bioinformatics analysis suggested that SARS-CoV-2 infection might modulate host immune responses through the ubiquitination of important proteins, including USP5, IQGAP1, TRIM28, and Hsp90. Ubiquitination modification was also observed on 11 SAR-CoV-2 proteins, including proteins involved in virus replication and inhibition of the host innate immune response. Our study provides new insights into the interaction between SARS-CoV-2 and the host as well as potential targets for the prevention and treatment of COVID-19.

Characterization and genome sequence of the genetically unique Escherichia bacteriophage vB_EcoM_IME392.

In this study, a novel Escherichia coli-specific bacteriophage, vB_EcoM_IME392, was isolated from chicken farm sewage in Qingdao, China. The genome of IME392 was found by next-generation sequencing to be 116,460 base pairs in length with a G+C content of 45.4% (GenBank accession number MH719082). BLASTn results revealed that only 2% of the genome sequence of IME392 shows sequence similarity to known phage sequences in the GenBank database, which indicates that IME392 is a novel bacteriophage. Transmission electron microscopy showed that IME392 belongs to the family Myoviridae. The host range, the multiplicity of infection, and a one-step growth curve were also determined.