Exploring the anti-virulence potential of plants used in traditional Mayan medicine to treat bacterial infections.

ETHNOPHARMACOLOGICAL RELEVANCE: While the antimicrobial activity of a number of plants used in traditional Mayan medicine against infectious diseases has been documented, their potential to inhibit quorum sensing (QS) as means of discovering novel anti-virulence agents remains unexplored. AIM OF THE STUDY: To evaluate the anti-virulence potential of plants used in traditional Mayan medicine by determining their inhibition of QS- regulated virulence factors in Pseudomonas aeruginosa. METHODS: A group of plants used in traditional Mayan medicine against infectious diseases was selected, and their methanolic extracts were evaluated at 10 mg/mL for their antibacterial and anti-virulence activity using the reference strain P. aeruginosa PA14WT. The broth microdilution method was used to determine antibacterial activity (MIC), while anti-virulence activity was evaluated by measuring the anti-biofilm effect and the inhibition of pyocyanin and protease activities. The most bioactive extract was fractionated using a liquid-liquid partition procedure and the semipurified fractions were evaluated at 5 mg/mL for antibacterial and anti-virulence activity. RESULTS: Seventeen Mayan medicinal plants traditionally used to treat infection-associated diseases were selected. None of the extracts exhibited antibacterial activity, whereas anti-virulence activity was detected in extracts of Bonellia flammea, Bursera simaruba, Capraria biflora, Ceiba aesculifolia, Cissampelos pareira and Colubrina yucatanensis. The most active extracts (74% and 69% inhibition) against biofilm formation were from C. aesculifolia (bark) and C. yucatanensis (root), respectively. Alternatively, the extracts of B. flammea (root), B. simaruba (bark), C. pareira (root), and C. biflora (root), reduced pyocyanin and protease production (50-84% and 30-58%, respectively). Fractionation of the bioactive root extract of C. yucatanensis allowed the identification of two semipurified fractions with anti-virulence activity. CONCLUSIONS: The anti-virulence activity detected in the crude extracts of B. flammea, B. simaruba, C. biflora, C. aesculifolia, C. pareira, and C. yucatanensis, confirms the efficacy and traditional use of these medicinal plants against infectious diseases. The activity of the extract and semipurified fractions of C. yucatanensis indicates the presence of hydrophilic metabolites capable of interfering with QS in P. aeruginosa. This study represents the first report of Mayan medicinal plants with anti-QS properties and suggests they represent an important source of novel anti-virulence agents.

A Brominated Furanone Inhibits Pseudomonas aeruginosa Quorum Sensing and Type III Secretion, Attenuating Its Virulence in a Murine Cutaneous Abscess Model.

Quorum sensing (QS) and type III secretion systems (T3SSs) are among the most attractive anti-virulence targets for combating multidrug-resistant pathogenic bacteria. Some halogenated furanones reduce QS-associated virulence, but their role in T3SS inhibition remains unclear. This study aimed to assess the inhibition of these two systems on Pseudomonas aeruginosa virulence. The halogenated furanones (Z)-4-bromo-5-(bromomethylene)-2(5H) (C-30) and 5-(dibromomethylene)-2(5H) (named hereafter GBr) were synthesized, and their ability to inhibit the secretion of type III exoenzymes and QS-controlled virulence factors was analyzed in P. aeruginosa PA14 and two clinical isolates. Furthermore, their ability to prevent bacterial establishment was determined in a murine cutaneous abscess model. The GBr furanone reduced pyocyanin production, biofilm formation, and swarming motility in the same manner or more effectively than C-30. Moreover, both furanones inhibited the secretion of ExoS, ExoT, or ExoU effectors in all tested strains. The administration of GBr (25 and 50 µM) to CD1 mice infected with the PA14 strain significantly decreased necrosis formation in the inoculation zone and the systemic spread of bacteria more efficiently than C-30 (50 µM). Molecular docking analysis suggested that the gem position of bromine in GBr increases its affinity for the active site of the QS LasR regulator. Overall, our findings showed that the GBr furanone displayed efficient multi-target properties that may favor the development of more effective anti-virulence therapies.

Anti-Pathogenic Properties of the Combination of a T3SS Inhibitory Halogenated Pyrrolidone with C-30 Furanone.

Antimicrobial resistance is one of the current public health challenges to be solved. The World Health Organization (WHO) has urgently called for the development of strategies to expand the increasingly limited antimicrobial arsenal. The development of anti-virulence therapies is a viable option to counteract bacterial infections with the possibility of reducing the generation of resistance. Here we report on the chemical structures of pyrrolidones DEXT 1-4 (previously identified as furan derivatives) and their anti-virulence activity on Pseudomonas aeruginosa strains. DEXT 1-4 were shown to inhibit biofilm formation, swarming motility, and secretion of ExoU and ExoT effector proteins. Also, the anti-pathogenic property of DEXT-3 alone or in combination with furanone C-30 (quorum sensing inhibitor) or MBX-1641 (type III secretion system inhibitor) was analyzed in a model of necrosis induced by P. aeruginosa PA14. All treatments reduced necrosis; however, only the combination of C-30 50 µM with DEXT-3 100 µM showed significant inhibition of bacterial growth in the inoculation area and systemic dispersion. In conclusion, pyrrolidones DEXT 1-4 are chemical structures capable of reducing the pathogenicity of P. aeruginosa and with the potential for the development of anti-virulence combination therapies.

Antivirulence Activity of a Dietary Phytochemical: Hibiscus Acid Isolated from Hibiscus sabdariffa L. Reduces the Virulence of Pseudomonas aeruginosa in a Mouse Infection Model.

Hibiscus sabdariffa L. (Hs) calyxes, rich in organic acids, are included in diets in different countries. In recent years, some phytochemicals have been shown to reduce bacterial virulence at sublethal concentrations by interfering with quorum sensing (QS) systems. Therefore, in this study the antivirulence properties of Hs calyxes and two γ-lactones (hibiscus acid [HA] and its methyl ester) in Pseudomonas aeruginosa were analyzed. Acetone and methanol extracts of Hs showed anti-QS activity by inhibiting violacein production (60% to 80% with 250 µg/mL). In molecular docking analysis, the γ-lactones registered a good binding score, which suggests strong interaction with the active site of LasR protein. To verify their effect in vitro, they were isolated from Hs and evaluated in six QS-regulated phenotypes, as well as in ExoU toxin that is released by the type III secretion system (T3SS). At 500 µg/mL they reduced alkaline protease (29-52%) and elastase (15-37%) activity, biofilm formation (∼75%), and swarming (50%), but there was no effect on pyocyanin production, hemolytic activity, or type III secretion. In a mouse abscess/necrosis model, HA at sublethal concentrations (15 and 31.2 µg/mL) affected infection establishment and prevented damage and systemic spread. In conclusion, HA is the first molecule identified with antivirulence properties in Hs with the potential to prevent infections caused by P. aeruginosa.