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.

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.

Several plant extracts exhibit anti-virulence properties due to the interruption of bacterial quorum sensing (QS). However, studies on their effects at the preclinical level are scarce. Here, we used a murine model of abscess/necrosis induced by Pseudomonas aeruginosa to evaluate the anti-pathogenic efficacy of 24 plant extracts at a sub-inhibitory concentration. We analyzed their ability to inhibit QS-regulated virulence factors such as swarming, pyocyanin production, and secretion of the ExoU toxin via the type III secretion system (T3SS). Five of the seven extracts with the best anti-pathogenic activity reduced ExoU secretion, and the extracts of Diphysa americana and Hibiscus sabdariffa were identified as the most active. Therefore, the abscess/necrosis model allows identification of plant extracts that have the capacity to reduce pathogenicity of P. aeruginosa. Furthermore, we evaluated the activity of the plant extracts on Chromobacterium violaceum. T3SS (ΔescU) and QS (ΔcviI) mutant strains were assessed in both the abscess/necrosis and sepsis models. Only the ΔescU strain had lower pathogenicity in the animal models, although no activity of plant extracts was observed. These results demonstrate differences between the anti-virulence activity recorded in vitro and pathogenicity in vivo and between the roles of QS and T3S systems as virulence determinants.

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

Options to combat bacterial infections are becoming scarce. We require innovative approaches to enhance the discovery of effective antimicrobials capable of combating bacteria resistant to multiple or all antibiotics. These methods should either directly eliminate resistant bacteria or indirectly influence their viability by inhibiting their virulence or reducing their resistance to antibiotics. One interesting approach is to analyze ancient remedies used to treat bacterial infections, formulate them, and test them against modern microbes. This field has recently been named "ancientbiotics." This approach allows us to leverage centuries of empirical knowledge accumulated, from traditional medicines across various ancient cultures worldwide. The strategy has already yielded promising formulations to combat the ESKAPE group of nosocomial pathogens. Additionally, molecular de-extinction, which involves genome analysis of extinct species to search for useful antimicrobials, such as peptides, offers another avenue. In this review, we compile the antimicrobial effects of ancient remedies and de-extinct molecules known to modern science and discuss possible new strategies to further harness the potential of past remedies and molecules to fight the rise of superbugs.