Juglone as a Natural Quorum Sensing Inhibitor against Pseudomonas aeruginosa pqs-Mediated Virulence and Biofilms.

Pseudomonas aeruginosa is a notorious opportunistic pathogen associated with chronic biofilm-related infections, posing a significant challenge to effective treatment strategies. Quorum sensing (QS) and biofilm formation are critical virulence factors employed by P. aeruginosa, contributing to its pathogenicity and antibiotic resistance. Other than the homoserine-based QS systems, P. aeruginosa also possesses the quinolone-based Pseudomonas quinolone signal (PQS) QS signaling. Synthesis of the PQS signaling molecule is achieved by the pqsABCDEH operon, whereas the PQS signaling response was mediated by the PqsR receptor. In this study, we report the discovery of a novel natural compound, Juglone, with potent inhibitory effects on pqs QS and biofilm formation in P. aeruginosa. Through an extensive screening of natural compounds from diverse sources, we identified Juglone, a natural compound from walnut, as a promising candidate. We showed that Juglone could inhibit PqsR and the molecular docking results revealed that Juglone could potentially bind to the PqsR active site. Furthermore, Juglone could inhibit pqs-regulated virulence factors, such as pyocyanin and the PQS QS signaling molecule. Juglone could also significantly reduce both the quantity and quality of P. aeruginosa biofilms. Notably, this compound exhibited minimal cytotoxicity toward mammalian cells, suggesting its potential safety for therapeutic applications. To explore the clinical relevance of Juglone, we investigated its combinatorial effects with colistin, a commonly used antibiotic against P. aeruginosa infections. The Juglone-colistin combinatorial treatment could eliminate biofilms formed by wild-type P. aeruginosa PAO1 and its clinical isolates collected from cystic fibrosis patients. The Juglone-colistin combinatorial therapy dramatically improved colistin efficacy and reduced inflammation in a wound infection model, indicating its potential for clinical utility. In conclusion, the discovery of Juglone provides insights into the development of innovative antivirulence therapeutic strategies to combat P. aeruginosa biofilm-associated infections.

Simultaneous Dissemination of Nanoplastics and Antibiotic Resistance by Nematode Couriers.

Nanoplastics (NPs) are increasingly recognized as a newly emerging pollutant in the environment. NPs can enable the colonization of microbial pathogens on their surfaces and adsorb toxic pollutants, such as heavy metals and residual antibiotics. Although the dissemination of plastic particles in water bodies and the atmosphere is widely studied, the dissemination of NPs and adsorbed pollutants on land, via biological means, is poorly understood. Since soil animals, such as the bacterivorous nematode Caenorhabditis elegans (C. elegans), are highly mobile, this raises the possibility that they play an active role in disseminating NPs and adsorbed pollutants. Here, we established that antibiotic-resistant bacteria could aggregate with antibiotic-adsorbed NPs to form antibiotic-adsorbed NP-antibiotic resistant bacteria (ANP-ARB) aggregates, using polymyxins (colistin) as a proof-of-concept. Colistin-resistant mcr-1 bearing Escherichia coli from a mixed population of resistant and sensitive bacteria selectively aggregate with colistin-ANPs. In the soil microcosm, C. elegans fed on ANP-ARB clusters, resulting in the rapid spread of ANP-ARB by the nematodes across the soil at a rate of 40-60 cm per day. Our work revealed insights into how NPs could still disseminate across the soil faster than previously thought by "hitching a ride" in soil animals and acting as agents of antibiotic-resistant pathogens and antibiotic contaminants. This poses direct risks to ecology, agricultural sustainability, and human health.

Biofilm matrix cloaks bacterial quorum sensing chemoattractants from predator detection.

Microbes often secrete high levels of quorum sensing (QS) autoinducers into the environment to coordinate gene expression and biofilm formation, but risk detection and subsequent predation by bacterivorous predators. With such prominent signaling molecules acting as chemoattractants that diffuse into the environment at alarmingly high concentrations, it is unclear if bacterial cells can mask their chemical trails from predator detection. Here, we describe a microbial-based anti-detection adaptation, termed as "biofilm cloak", where the biofilm prey produced biofilm matrix exopolysaccharides that "locked" and reduced the leaching of autoinducers into the milieu, thereby concealing their trails to the detection by the bacterivorous Caenorhabditis elegans nematode. The exopolysaccharides act as common good for the non-producers to hide their autoinducers from predator detection. Deficiency in chemosensory gene odr-10 in mutant animals abrogated their ability to detect autoinducers and migrate toward their prey in a directed manner, which led to lower population growth rate of animals. Hence, restriction of bacterial communication activities to the confinements of biofilms is a novel approach for predator evasion, which plays a fundamental role in shaping ecological dynamics of microbial communities and predator-prey interactions.

The effects of biofilms on tumor progression in a 3D cancer-biofilm microfluidic model.

Components within the tumor microenvironment, such as intratumoral bacteria (IB; within tumors), affect tumor progression. However, current experimental models have not explored the effects of extratumoral bacteria (EB; outside tumors) on cancer progression. Here, we developed a microfluidic platform to analyze the influence of bacterial distribution on bladder cancer progression under defined conditions, using uropathogenic Escherichia coli. This was achieved by establishing coating (CT) and colonizing (CL) models to simulate the different invasion and colonization modes of IB and EB in tumor tissues. We demonstrated that both EB and IB induced closer cell-cell contacts within the tumor cluster, but cancer cell viability was reduced only in the presence of IB. Interestingly, cancer stem cell counts increased significantly in the presence of EB. These outcomes were due to the formation of extracellular DNA-based biofilms by EB. Triple therapy of DNase (anti-biofilm agent), ciprofloxacin (antibiotic), and doxorubicin (anti-cancer drug) could effectively eradicate biofilms and tumors simultaneously. Our preclinical proof-of-concept provides insights on how bacteria can influence tumor progression and facilitate future research on anti-biofilm cancer management therapies.

Biofilm matrix disrupts nematode motility and predatory behavior.

In nature, bacteria form biofilms by producing exopolymeric matrix that encases its entire community. While it is widely known that biofilm matrix can prevent bacterivore predation and contain virulence factors for killing predators, it is unclear if they can alter predator motility. Here, we report a novel "quagmire" phenotype, where Pseudomonas aeruginosa biofilms could retard the motility of bacterivorous nematode Caenorhabditis elegans via the production of a specific exopolysaccharide, Psl. Psl could reduce the roaming ability of C. elegans by impeding the slithering velocity of C. elegans. Furthermore, the presence of Psl in biofilms could entrap C. elegans within the matrix, with dire consequences to the nematode. After being trapped in biofilms, C. elegans could neither escape effectively from aversive stimuli (noxious blue light), nor leave easily to graze on susceptible biofilm areas. Hence, this reduced the ability of C. elegans to roam and predate on biofilms. Taken together, our work reveals a new function of motility interference by specific biofilm matrix components, and emphasizes its importance in predator-prey interactions.

Vanillin inhibits PqsR-mediated virulence in Pseudomonas aeruginosa.

Reduced efficacy of antibiotics in bacterial diseases is a global concern in clinical settings. Development of anti-virulence compounds which disarm bacterial virulence is an attractive therapeutic agent for complementary antibiotics usage. One potential target for anti-virulence compounds is quorum sensing (QS), the intercellular communication system in most pathogens, such as Pseudomonas aeruginosa. QS inhibitors (QSIs) can inhibit QS effectively, attenuate QS-mediated virulence, and improve host clearance of infections. While studies focused on developing homoserine-based las QSI, few targeted the quinolone-based pqs QS, which implicated host cytotoxicity and biofilm formation. It is imperative to develop novel anti-pqs-QS therapeutics for combinatorial antibiotic treatment of microbial diseases. We employed a gfp-based transcriptional pqs biosensor to screen a natural compounds library and identify vanillin (4-hydroxy-3-methoxybenzaldehyde), the primary phenolic aldehyde of vanilla bean. The vanillin inhibited pqs expression and its associated phenotypes, namely pyocyanin production and twitching motility in P. aeruginosa. Molecular docking results revealed that vanillin binds to the active site of PqsR, the PQS-binding response regulator. Combinatorial treatment of vanillin with antimicrobial peptide (colistin) inhibited biofilm growth in vitro and improved treatment in the in vivo C. elegans acute infection model. We demonstrated that vanillin could dampen pqs QS and associated virulence, thus providing novel therapeutic strategies against P. aeruginosa infections.