A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2.

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression to zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.

Curcumin Promotes the Clearance of Listeria monocytogenes both In Vitro and In Vivo by Reducing Listeriolysin O Oligomers.

The pore-forming toxin listeriolysin O (LLO), an essential virulence factor that is secreted by Listeria monocytogenes (L. monocytogenes), is responsible for bacterial breaching at the phagosomal membranes and subsequent release into the cytoplasm; it cannot be recognized by the host immune system. The vital role that LLO plays in bacterial pathogenicity and evading host immune clearance makes this virulence a promising target for addressing L. monocytogenes infection. In this study, we hypothesized that curcumin, a polyphenol derived from turmeric that could effectively inhibit LLO pore-forming activity, might be useful in the prevention or treatment of L. monocytogenes infection. Thus, the in vitro protective effects of curcumin against L. monocytogenes infection by targeting LLO were assessed via hemolytic activity assays, cytotoxicity tests, intracellular growth assays, and confocal microscopy. Our results revealed that treating infected macrophages with curcumin can lead to a decrease in LLO-mediated bacteria phagosomal escape and limit the intracellular growth of L. monocytogenes. Moreover, results from animal experiments show that this natural compound effectively increases protection against bacterial infection and helps the host to clear the invading pathogen completely from an animal model, establishing it as a potent antagonist of L. monocytogenes. The results from our molecular modeling and mutational analysis demonstrated that curcumin directly engages with domains 2 and 4 of LLO, thereby decreasing the hemolytic activity of LLO by influencing its oligomerization. Taken together, these results suggest that, as an antitoxin agent, curcumin can be further developed into a novel therapy against L. monocytogenes infections by targeting LLO.

Molecular Mechanism of the Flavonoid Natural Product Dryocrassin ABBA against Staphylococcus aureus Sortase A.

The intractability of bacterial resistance presents a dilemma for therapies against Staphylococcus aureus (S. aureus) infection. Effective anti-virulence strategies are urgently needed, reflecting the proliferation of resistant strains. Inhibitors of sortase A (SrtA), enzymes that anchor virulence-related surface proteins, are regarded as promising candidates for countermeasures against bacterial infections. In the present study, the inhibitory effect of dryocrassin ABBA (ABBA) against SrtA and its molecular basis has been examined. Fluorescence resonance energy transfer (FRET) assays were used to determine the inhibitory activity of ABBA against SrtA. To identify the mechanism underlying this activity, molecular dynamics simulations and mutagenesis assays were applied, and the results revealed that the direct engagement of SrtA via ABBA through binding to V166 and V168 significantly attenuated the catalytic activity of SrtA. Taken together, these findings indicated that ABBA is a potential novel antimicrobial agent for S. aureus infection via targeting SrtA.