Investigation Of Virulence Inhibitors And The Quinolone Signal Mediated Regulatory System In Pseudomonas Aeruginosa

Conventional antibiotics target viability; such antiniotics exert as elective pressure on pathogenic bacteria and hence cause the rise of drug resistance. Antibiotic resistance in pathogens limits our abilities to treat infectious diseases and has become a serious threat to human health. Pseudomonas aeruginosa (PA) is a Gram-negative opportunistic human pathogen. It is the third most common infectious agent in hospitals, and it can cause various acute and chronic infections in patients, especially those who are immuno-compromised or suffered other chronic diseases. With decades of conventional antibiotic treatment, many P. aeruginosa stains have become a multi-drug resistant, which leads to enormous difficulties in clinical treatment.The ability of pathogens to cause disease depends on virulence factors, such as toxins, cytolysins or proteases which cause damage to host tissues. It is therefore plausible that bacterial infections can be treated or prevented by inhibitors of the bacterial virulence factors. Such inhibitors are a promising new kind of "antipathogenic"therapeutics.Chinese herbs have been effectively used to treat infectious diseases for thousands of years. They represent a rich resource for antibacterial compound exploration. In this study, we screened for P. aeruginosa virulence factor inhibitors from Chinese herbs and investigated the pathways and molecular mechanisms of a potent inhibitor baicalin isolated from Scutellariae Radix. We constructed an array of virulence factor reporters with the virulence related gene promoters fused with promoterless luxCDABE cluster. Chinese herbs that are known for the functions of Qing Re Jie Du (i.e. treating symptoms resembling infections) were screened using this reporter array. In addition, the target of baicalin, i.e. the Pseudomonas quinolone signal system (PQS), was also investigated. The regulation of virulence factors by the PQS system and the corelation between the signal molecules and the transcriptional regulator PqsR (cognate receptor of PQS) in virulence regulation were investigated. The results of our study revealed that the extract of medicinal plant Scutellariae Radix (the root of Scutellaria baicalensis Georgi) could inhibit the expression of Quorm sensing (QS) related genes including lasl, lasR, rhlI and rhlR, type Ⅲ secretion system (T3SS) effector gene exoS and flagella biosynthesis gene filC. We found Folium Artemisiae Argyi extract could inhibit the expression of QS lasR, rhlI, rhlR, exoS and filC in P. aeruginosa, while it had no influence on cell viability.Further investigation indicate that baicalin, an flavonoid isolated from Scutellariae Radix significantly inhibited a range of important virulence factors in P. aeruginosa such as the Type III secretion system (T3SS), swarming motility and the production of rhamnolipid and elastase, suggesting that baicalin was the active anti-virulence component in the medicinal plant Scutellariae Radix.Based on the observation that baicalin repressed the expression of T3SS effector gene exoS, we investigated the mechanism of baicalin inhibition on virulence exoS as a reporter. The exoS inhibition by baicalin was examined in the mutants of global regulatory system which are known controlling virulence factors in P. aeruginosa. The results demonstrated that PQS system is the target regulatory pathway through which baicalin excerted its inhibitory activity against virulence factors. Baicalin had no impact on exoS expression in the pqsH and pqsR mutants which are deficient in the PQS production and lack of regulatory protein PqsR (cognate receptor of PQS) respectively. The inhibition of exoS expression by baicalin was restored in the presence of PQS and PqsR, but not by the PQS precursor HHQ and PqsR or PqsR alone. The results demonstrated that the inhibition activity on the T3SS by baicalin is dependent on the PQS and its cognate receptor PqsR.The activity of baicalin on the pathogenicity of P. aeruginosa was further tested using in vitro EMT6cells, fruit fly infection model, and the rat lung alginate-bead infection model. The result demonstrated that baicalin reduced the cellular toxicity of PAO1on the mammalian cell and attenuated its in vivo pathogenicity in the fly infection model. Moreover, the results using the rat pulmonary infection model demonstrated that baicalin reduced the inflammatory response and enhanced bacterial clearance by the host. These results suggest that baicalin is apromissing antipathogenic drug candidate for treating P. aeruginosa infections. Study of the baicalin target-the PQS system showed that PQS stsyem is an important for P. aeruginosa motility and biofilm formation. We demonstrated that PQS molecules repressed the swarming motility of P. aeruginosa PAO1, and such regulation was independent on its cognate receptor PqsR but was not related to changes of the flagella, type IV pili or the production of the surface-wetting agent rhamnolipid surfactants. While PQS molecules did not affect twitching motility in PAO1, a pqsR deletion abolished twitching motility, indicating pqsR is required for twitching motility. The result for the first time revealed the distinct PqsR-dependent and PqsR-independent functions of PQS molecules. Our results also indicated that the enhancement of biofilm formation by PQS, at least partially, depended on the GacAS/Rsm regulatory pathway whereas without affecting the Las or Rhl QS systems. However, PQS still inhibited swarming motility in gacA mutant. It is likely that the effect of PQS on swarming motility and biofilm formation were exerted through different pathways.