| Listeria monocytogenes (L. monocytogenes) is a clinically significant bacterialzoonotic pathogen, which is the only causative agent of listeriosis in human beingsand animals, a disease which manifests primarily as bacteremia, nervous systeminfection (meningitis and meningoencephalitis), abortion and stillbirth with a highmortality (about25%). Recently, the increasing emergence of antibiotic resistant L.monocytogenes coupled with poor antibiotic penetration limits the treatment optionsfor this bacterial infection. Following bacterial internalization, the secretedlisteriolysin O (LLO) would bind to cholesterol on membrane of vacuole to formarc-shaped or ring-shaped pore structures, disrupting the vacuolar membranes andfacilitating the escape of trapped L. monocytogenes to cytoplasm, where the bacteriaare able to survive and replicate. Furthermore, LLO is necessary and sufficient toinduce immune cells and tissue cells appotosis during L. monocytogenes infection. Anindispensable role of LLO in the evading of immune system and the pathogenicity ofL. monocytogenes has been observed, as the inactivation the hly (the gene encodingLLO) results in the loss of hemolytic activity of bacterial cultures and the inability toescape from the bacterial vacuole, in which L. monocytogenes will be degraded, andthe mutant is absolutely nonvirulent in the mouse infection model. However, thecomplemented strain transformed with a plasmid carrying only hly displayed ahemolytic phenotype identical to that of the wild-type strain and is fully virulent.Therefore, LLO is one of the most promising targets for the development ofanti-virulence drugs against L. monocytogenes infection.In this study, the results from hemolysis and western blotting assays indicatedthat fisetin (FSN), myricetin, morin, baicalein, chrysin and naringenin could directlyneutralize the hemolytic activities caused by L. monocytogenes culture supernatants and purified LLO. In the co-culture system of J774cells and L. monocytogenes, theaddition of FSN could inhibit the escape of bacteria from vacuole to cytoplasm anddecrease the cytotoxicity caused by L. monocytogenes, facilitating J774cell-mediatedbacterial lysis. In the mouse model for infection with L. monocytogenes, the treatmentof FSN nanoemulsion effectively protects mice from L. monocytogenes infection.FSN nanoemulsion-treated mice survived significantly longer than untreated mice anda significantly lower bacterial burden was observed in the liver and spleen of thesemice. Furthermore, analyses of livers and spleens from infected mice treated with orwithout FSN nanoemulsion revealed a remarkable alleviation of pathological injury inboth organs in the FSN nanoemulsion-treated mice. Consistent with theseobservations, the levels of cytokines such as interleukin1β, interleukin6and tumornecrosis factor α in organ homogenates of FSN nanoemulsion-treated mice weresignificantly lower than those observed for control mice using ELISA assay. Resultsfrom our functional analyses strongly suggest direct targeting of LLO by FSN,hindering the pore-forming activity of LLO and, subsequently, inhibiting thevirulence of L. monocytogenes. Computational biology, site-directed mutagenesis andfluorescence quenching assays were further employed to determine the mechanism ofsuch inhibition. The results indicated that the engagement of FSN to Ile396in Loop2and Lys433in Loop3causes a significant shift for the conformation of the Loop1andLoop2binding cavity. Such shift caused a stretch between these two binding cavitiesand impaired the ability of LLO to interact with cholesterol, leading to loweroligomerization and lytic activity of LLO. The plasmids encoding LLOWT,LLOI396A and LLOK433A were constructed and the fluorescence quenchingconstants were determined for the reaction system of each of the recombinationprotein and FSN to verify the theoretical calculation results. The calculated bindingconstants of FSN and LLO and its mutants based on the fluorescence quenching assaydecreased in the following order: LLOWT-FSN> LLOK433A-FSN>LLOI396A-FSN. The results from cholesterol binding, LLO oligomerization andhemolysis assays indicated that FSN interferes with the cholesterol-binding activity of LLO and its oligomerization and hemolytic activity. However, the sensitivity of FSNagainst the LLO mutants in these assays is significantly lower than the wild type LLO.Although the other five natural compounds (myricetin, morin, baicalein, chrysinand naringenin) have similar structures, they possess different inhibitory effects on thehemolytic activity of LLO. The half effective inhibition concentrations of thesenatural compounds against LLO activity were in the range of0.46μg/ml to186.57μg/ml. Computational biology assays and mutagenesis assays were employed toinvestigate the mechanism by which the inhibitors attenuated the hemolytic activity ofLLO and the structure activity relationship of these natural compounds for suchdifferential inhibitory effect on LLO activity. The results suggested that themechanism of these natural compounds against LLO is consistent with FSN and asubstantial increase in anti-hemolytic activity was observed when the single bond(C1-C2). These theoretical calculation results have also been confirmed usingsite-directed mutagenesis and fluorescence quenching assays.Additionally, we have discovered that luteolin (LTN), a natural compound withneglect anti-L. monocytogenes activity, could inhibit the expression of LLO in culturesupernatants and bacteria via down-regulating the translation of hly, a gene encodingLLO, using hemolysis, western blotting, RT-PCR and β-LacZ translational reportergene fusions assays. Transcription kit and translation kit were further used todetermine the mechanism of LTN-mediated inhibition of LLO expression. The resultsindicated that LTN could specifically inhitbit the translation of hly. Moreover, thepotential target sequences (799-849nt) response for such inhibition has beenindentified via cell free protein synthesis system.Traditional Chinese Medicine is one of the quintessences of our country withmultiply biological activities, little side effects and low drug resistance characteristics,which is an important resource for development of novel drugs. In this study, we havescreened and identified some natural compounds from traditional Chinese medicine aspotential inhibitors of LLO. In summary, we have preliminarily elucidated themechanism by which LTN inhibits the translation of hly. Furthermore, the detailed mechanism of FSN and other flavanoids (myricetin, morin, baicalein, chrysin andnaringenin) against LLO activity has been clarified using hemolysis, computationalbiology and mutagenesis assays. The inhibitory effect of FSN on L. monocytogenespathogenicity has also been verified in the co-culture system of L. monocytogenes andmacrophages cell and the mouse model of L. monocytogenes infection. Taken together,these findings will provide an important experimental basis for the elucidation of themechanism of Traditional Chinese Medicine against bacterial infection and, also,leading compounds for the development of anti-virulence agents for L.monocytogenes specially targeting LLO. |
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