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, Sadenosyl-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. Q uorum sensing (QS) is an intercellular signaling process that mediates certain behaviors of bacteria (including bioluminescence, biofilm formation, motility, and virulence factor production) in response to the bacterial cell population density (1-3). In Gram-negative bacteria, QS is often mediated by Nacyl-L-homoserine lactones (acyl-HSLs), which are synthesized by the LuxI family of acyl-HSL synthases from S-adenosyl-Lmethionine (SAM) and acylated acyl-carrier protein (acyl-ACP), with the release of holo-ACP and 5′-methylthioadenosine (MTA) as byproducts (SI Appendix, Fig. S1A) (4, 5). Compounds of the acyl-HSL class share a homoserine lactone ring moiety, but the acyl chains conjugated to the ring via an amide bond vary in length, oxidation state at C3, and amount of saturation (SI Appendix, Fig. S1A). The recent finding that p-coumarate is an alternative substrate for acyl-ACP has extended the known range of possible acyl-HSL substrates (6). On the other hand, the acyl-HSL receptor is a transcriptional regulator that controls the expression of target genes in response to acyl-HSL binding (1-3).Among the hundreds of genes regulated by QS, the most widely studied genes are those related to virulence; these genes are of particular interest because QS disruption is being investigated as a strategy for controlling virulent pathogens (7-9). QS inhibitors can act by suppressing a...

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.

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).

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