Development of a Lateral Flow Strip Membrane Assay for Rapid and Sensitive Detection of the SARS-CoV-2.

The infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) has threatened public health worldwide. The easy human-to-human transmission of this virus has rapidly evolved into a global pandemic. Therefore, to control the community spread of the virus, it is crucial to identify the infected individuals, including asymptomatic people. Hence, a specific and rapid assay is crucial for the early diagnosis and active monitoring of individuals potentially exposed to SARS-CoV-2 for controlling the COVID-19 outbreak. In this study, we have developed the novel lateral flow strip membrane (LFSM) assay that allows the simultaneous detection of RdRp, ORF3a, and N genes using the PCR product obtained by using the single-tube reverse transcription polymerase chain reaction (RT-PCR). The LFSM assay allows detection of SARS-CoV-2 in 30 min at 25 °C after the RT-PCR with the detection limit of 10 copies/test for each gene. The clinical performance of the LFSM assay for the detection of SARS-Cov-2 was evaluated using 162 clinical samples previously detected by using the commercial assay. The percent positive agreement, percent negative agreement, and overall percent agreement of the LFSM assay with the commercial assay were 100% (94.2-100%), 99.0% (94.6-100%), and 99.4% (96.6-100%), respectively. Therefore, the results of the LFSM assay showed significantly high concordance with the commercial assay for the detection of SARS-CoV-2 in clinical specimens. Therefore, we conclude that the developed LFSM assay can be used alone or complementary to the RT-PCR or other methods for the diagnosis and monitoring of the patients to curb community transmission and the pandemic.

The crystal structure of type III effector protein XopQ from Xanthomonas oryzae complexed with adenosine diphosphate ribose.

Effector proteins are virulence factors that promote pathogenesis by interfering with various cellular events and are delivered directly into host cells by the secretion systems of many Gram-negative bacteria. Type III effector protein XOO4466 from the plant pathogen Xanthomonas oryzae pv. oryzae (XopQ(Xoo)) and XopQ homologs from other phytopathogens have been predicted to be nucleoside hydrolases based on their sequence similarities. However, despite such similarities, recent structural and functional studies have revealed that XopQ(Xoo) does not exhibit the expected activity of a nucleoside hydrolase. On the basis of the conservation of a Ca(2+) coordination shell of a ribose-binding site and the spacious active site in XopQ(Xoo), we hypothesized that a novel compound containing a ribosyl moiety could serve as a substrate for XopQ(Xoo). Here, we report the crystal structure of XopQ(Xoo) in complex with adenosine diphosphate ribose (ADPR), which is involved in regulating cytoplasmic Ca(2+) concentrations in eukaryotic cells. ADPR is bound to the active site of XopQ(Xoo) with its ribosyl end tethered to the Ca(2+) coordination shell. The binding of ADPR is further stabilized by interactions mediated by hydrophobic residues that undergo ligand-induced conformational changes. These data showed that XopQ(Xoo) is capable of binding a novel chemical bearing a ribosyl moiety, thereby providing the first step toward understanding the functional role of XopQ(Xoo).

Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase.

Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

Crystal structure of the effector protein XOO4466 from Xanthomonas oryzae.

Many Gram-negative bacteria deliver their virulence factors into host cells through a secretion system. Those factors, called effector proteins, are involved in the pathogenicity in host cells by interfering with various cellular events. The phytopathogen Xanthomonas oryzae pv. oryzae uses a type III secretion system to inject its effectors, but the functional roles of these proteins remain largely uncharacterized. Here, we determined a crystal structure of XOO4466, an effector from X. oryzae pv. oryzae, and performed a functional analysis. We determined that XOO4466 is similar in sequence to Xanthomonas outer protein Q, a putative nucleoside hydrolase (NH). The overall structure of XOO4466 is homologous to that of NHs, including a metal-binding site, but differs in its oligomeric state and active site topology. Further analysis indicated that antiparallel ß-strands commonly found in NHs adjacent to the active site loop are replaced in XOO4466 with a short loop, causing the active site loop to adopt a conformation distinct from that of NHs. Thus, the catalytic residues emanating from the respective active site loop of NHs are absent in the putative active site of XOO4466. Consistent with these structural features, a functional assay indicated that XOO4466 does not exhibit NH activity and possibly catalyzes yet unknown reactions.