Bacterivorous nematodes decipher microbial iron siderophores as prey cue in predator-prey interactions.

The minimal levels of biological-available iron in the environment impose growth limitation on all living organisms. Microbes often secrete high iron-binding-affinity siderophores at high concentrations for scavenging iron from the iron-limited habitats. However, the high prevalence of siderophores released by bacteria into the environment raises an intriguing question whether this chemical cue can be detected by bacterivorous predators. Here, we show that the bacterivorous Caenorhabditis elegans nematode could employ its chemosensory receptor Odr-10 to detect pyoverdine, an iron siderophore secreted by an environmental bacterium, Pseudomonas aeruginosa. This enabled the nematode predator to migrate toward the prey. Our soil microcosm study showed that the detection of pyoverdine and subsequent feeding of P. aeruginosa prey by C. elegans could lead to the expansion of its population. These results showed that siderophores are a prey chemical cue by predators, with key implications in predator-prey interactions.

Pharmaceutical Residues in Edible Oysters along the Coasts of the East and South China Seas and Associated Health Risks to Humans and Wildlife.

The investigation of pharmaceuticals as emerging contaminants in marine biota has been insufficient. In this study, we examined the presence of 51 pharmaceuticals in edible oysters along the coasts of the East and South China Seas. Only nine pharmaceuticals were detected. The mean concentrations of all measured pharmaceuticals in oysters per site ranged from 0.804 to 15.1 ng g-1 of dry weight, with antihistamines being the most common. Brompheniramine and promethazine were identified in biota samples for the first time. Although no significant health risks to humans were identified through consumption of oysters, 100-1000 times higher health risks were observed for wildlife like water birds, seasnails, and starfishes. Specifically, sea snails that primarily feed on oysters were found to be at risk of exposure to ciprofloxacin, brompheniramine, and promethazine. These high risks could be attributed to the monotonous diet habits and relatively limited food sources of these organisms. Furthermore, taking chirality into consideration, chlorpheniramine in the oysters was enriched by the S-enantiomer, with a relative potency 1.1-1.3 times higher when chlorpheniramine was considered as a racemate. Overall, this study highlights the prevalence of antihistamines in seafood and underscores the importance of studying enantioselectivities of pharmaceuticals in health risk assessments.

Biofilm Potentiates Cancer-Promoting Effects of Tumor-Associated Macrophages in a 3D Multi-Faceted Tumor Model.

Components of the tumor microenvironment (TME), such as tumor-associated macrophages (TAMs), influence tumor progression. The specific polarization and phenotypic transition of TAMs in the tumor microenvironment lead to two-pronged impacts that can promote or hinder cancer development and treatment. Here, a novel microfluidic multi-faceted bladder tumor model (TAMPIEB ) is developed incorporating TAMs and cancer cells to evaluate the impact of bacterial distribution on immunomodulation within the tumor microenvironment in vivo. It is demonstrated for the first time that biofilm-induced inflammatory conditions within tumors promote the transition of macrophages from a pro-inflammatory M1-like to an anti-inflammatory/pro-tumor M2-like state. Consequently, multiple roles and mechanisms by which biofilms promote cancer by inducing pro-tumor phenotypic switch of TAMs are identified, including cancer hallmarks such as reducing susceptibility to apoptosis, enhancing cell viability, and promoting epithelial-mesenchymal transition and metastasis. Furthermore, biofilms formed by extratumoral bacteria can shield tumors from immune attack by TAMs, which can be visualized through various imaging assays in situ. The study sheds light on the underlying mechanism of biofilm-mediated inflammation on tumor progression and provides new insights into combined anti-biofilm therapy and immunotherapy strategies in clinical trials.

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.

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a ΔwspF mutant strain (high c-di-GMP levels), and a plac-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo ΔwspF mutant compared to the in vivo plac-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The ΔwspF mutant was less susceptible to host clearance than the plac-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A ΔwspF mutant with a defective T3SS was more susceptible to host clearance than a ΔwspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Biofilm dispersal induced by mechanical cutting leads to heightened foodborne pathogen dissemination.

The biofilm life cycle where bacteria alternate between biofilm and planktonic lifestyles poses major implications in food spoilage and gastrointestinal infections. Recent studies had shown that freshly biofilm-dispersed cells have a unique physiology from planktonic cells, raising the fundamental question if biofilm-dispersed cells and planktonic cells disseminate differently across food surfaces. Mechanical dislodging via cutting can cause biofilm dispersal and eventual food cross-contamination. Here, we showed that biofilm-dispersed bacteria from various foodborne pathogens were transferred from freshly cut surface at a higher rate to the cutting material than that of planktonic bacteria. When the cutting tool was used to cut a fresh surface, more biofilm-dispersed bacteria were disseminated from the cutting tool to the newly cut surface than planktonic bacteria. Our observations were applicable to cutting tools of various materials and cut surfaces, where polystyrene and surfaces with high water content were most susceptible to biofilm transfer, respectively. Simple washing with detergent and mechanical wiping could aid bacterial removal from cutting tools. Our work revealed that biofilm-dispersed cells were transferred at a higher rate than planktonic cells and cutting tool was an important medium for pathogen cross-contamination, thus providing insights in maintaining their cleanliness in food processing industries.

Demolishing the great wall of biofilms in Gram-negative bacteria: To disrupt or disperse?

Bacterial infections lead to high morbidity and mortality globally. While current therapies against bacteria often employ antibiotics, most bacterial pathogens can form biofilms and prevent effective treatment of infections. Biofilm cells can aggregate and encased themselves in a self-secreted protective exopolymeric matrix, to reduce the penetration by antibiotics. Biofilm formation is mediated by c-di-GMP signaling, the ubiquitous secondary messenger in bacteria. Synthesis of c-di-GMP by diguanylate cyclases leads to biofilm formation via the loss of motility, increased surface attachment, and production of biofilm matrix, whereas c-di-GMP degradation by phosphodiesterases causes biofilm dispersal to new sites via increased bacterial motility and matrix breakdown. The highly variable nature of biofilm development and antimicrobial tolerance imposes tremendous challenges in conventional antimicrobial therapies, indicating an imperative need to develop anti-biofilm drugs against biofilm infections. In this review, we focus on two main emergent approaches-active dispersal and disruption. While both approaches aim to demolish biofilms, we will discuss their fundamental differences and associated methods. Active dispersal of biofilms involves signaling the bacterial cells to leave the biofilm, where resident cells ditch their sessile lifestyle, gain motility and self-degrade their matrix. Biofilm disruption leads to direct matrix degradation that forcibly releases embedded biofilm cells. Without the protection of biofilm matrix, released bacterial cells are highly exposed to antimicrobials, leading to their eradication in biofilm infections. Understanding the advantages and disadvantages of both approaches will allow optimized utility with antimicrobials in clinical settings.

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention.

BACKGROUND: Emergence of drug-resistant cancer phenotypes is a challenge for anti-cancer therapy. Cancer stem cells are identified as one of the ways by which chemoresistance develops. METHOD: We investigated the anti-inflammatory combinatorial treatment (DA) of doxorubicin and aspirin using a preclinical microfluidic model on cancer cell lines and patient-derived circulating tumour cell clusters. The model had been previously demonstrated to predict patient overall prognosis. RESULTS: We demonstrated that low-dose aspirin with a sub-optimal dose of doxorubicin for 72 h could generate higher killing efficacy and enhanced apoptosis. Seven days of DA treatment significantly reduced the proportion of cancer stem cells and colony-forming ability. DA treatment delayed the inhibition of interleukin-6 secretion, which is mediated by both COX-dependent and independent pathways. The response of patients varied due to clinical heterogeneity, with 62.5% and 64.7% of samples demonstrating higher killing efficacy or reduction in cancer stem cell (CSC) proportions after DA treatment, respectively. These results highlight the importance of using patient-derived models for drug discovery. CONCLUSIONS: This preclinical proof of concept seeks to reduce the onset of CSCs generated post treatment by stressful stimuli. Our study will promote a better understanding of anti-inflammatory treatments for cancer and reduce the risk of relapse in patients.

Repurposing the anticancer drug cisplatin with the aim of developing novel Pseudomonas aeruginosa infection control agents.

Antibiotic resistance threatens effective treatment of microbial infections globally. This situation has spurred the hunt for new antimicrobial compounds in both academia and the pharmaceutical industry. Here, we report how the widely used antitumor drug cisplatin may be repurposed as an effective antimicrobial against the nosocomial pathogen Pseudomonas aeruginosa. Cisplatin was found to effectively kill strains of P. aeruginosa. In such experiments, transcriptomic profiling showed upregulation of the recA gene, which is known to be important for DNA repair, implicating that cisplatin could interfere with DNA replication in P. aeruginosa. Cisplatin treatment significantly repressed the type III secretion system (T3SS), which is important for the secretion of exotoxins. Furthermore, cisplatin was also demonstrated to eradicate in vitro biofilms and in vivo biofilms in a murine keratitis model. This showed that cisplatin could be effectively used to eradicate biofilm infections which were otherwise difficult to be treated by conventional antibiotics. Although cisplatin is highly toxic for humans upon systemic exposure, a low toxicity was demonstrated with topical treatment. This indicated that higher-than-minimal inhibitory concentration (MIC) doses of cisplatin could be topically applied to treat persistent and recalcitrant P. aeruginosa infections.

In Vitro and In Vivo Efficacy of an LpxC Inhibitor, CHIR-090, Alone or Combined with Colistin against Pseudomonas aeruginosa Biofilm.

With the rapid spread of antimicrobial resistance in Gram-negative pathogens, biofilm-associated infections are increasingly harder to treat and combination therapy with colistin has become one of the most efficient therapeutic strategies. Our study aimed to evaluate the potential for the synergy of colistin combined with CHIR-090, a potent LpxC inhibitor, against in vitro and in vivoPseudomonas aeruginosa biofilms. Four P. aeruginosa isolates with various colistin susceptibilities were chosen for evaluation. The tested isolates of P. aeruginosa exhibited MIC values ranging from 1 to 64 and 0.0625 to 0.5 µg/ml for colistin and CHIR-090, respectively. Against 24-h static biofilms, minimum biofilm eradication concentration values ranged from 256 to 512 and 8 to >128 µg/ml for colistin and CHIR-090, respectively. Interestingly, subinhibitory concentrations of CHIR-090 contributed to the eradication of subpopulations of P. aeruginosa with the highest colistin MIC values. The combination of colistin and CHIR-090 at subinhibitory concentrations demonstrated synergistic activity both in vivo and in vitro and prevented the formation of colistin-tolerant subpopulations in in vitro biofilms. In summary, our study highlights the in vivo and in vitro synergistic activity of the colistin and CHIR-090 combination against both colistin-susceptible and -nonsusceptible P. aeruginosa biofilms. Further studies are warranted to investigate the clinical relevance of the combination of these two antimicrobials and outline the underlying mechanism for their synergistic action.

RpoN Regulates Virulence Factors of Pseudomonas aeruginosa via Modulating the PqsR Quorum Sensing Regulator.

The alternative sigma factor RpoN regulates many cell functions, such as motility, quorum sensing, and virulence in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa often evolves rpoN-negative variants during the chronic infection in cystic fibrosis patients. It is unclear how RpoN interacts with other regulatory mechanisms to control virulence of P. aeruginosa. In this study, we show that RpoN modulates the function of PqsR, a quorum sensing receptor regulating production of virulence factors including the phenazine pyocyanin. The ∆rpoN mutant is able to synthesize 4-quinolone signal molecule HHQ but unable to activate PqsR and Pseudomonas quinolone signal (pqs) quorum sensing. The ∆rpoN mutant produces minimal level of pyocyanin and is unable to produce the anti-staphylococcal agents. Providing pqsR in trans in the ∆rpoN mutant restores its pqs quorum sensing and virulence factor production to the wild-type level. Our study provides evidence that RpoN has a regulatory effect on P. aeruginosa virulence through modulating the function of the PqsR quorum sensing regulator.

Comparative systems biology analysis to study the mode of action of the isothiocyanate compound Iberin on Pseudomonas aeruginosa.

Food is now recognized as a natural resource of novel antimicrobial agents, including those that target the virulence mechanisms of bacterial pathogens. Iberin, an isothiocyanate compound from horseradish, was recently identified as a quorum-sensing inhibitor (QSI) of the bacterial pathogen Pseudomonas aeruginosa. In this study, we used a comparative systems biology approach to unravel the molecular mechanisms of the effects of iberin on QS and virulence factor expression of P. aeruginosa. Our study shows that the two systems biology methods used (i.e., RNA sequencing and proteomics) complement each other and provide a thorough overview of the impact of iberin on P. aeruginosa. RNA sequencing-based transcriptomics showed that iberin inhibits the expression of the GacA-dependent small regulatory RNAs RsmY and RsmZ; this was verified by using gfp-based transcriptional reporter fusions with the rsmY or rsmZ promoter regions. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomics showed that iberin reduces the abundance of the LadS protein, an activator of GacS. Taken together, the findings suggest that the mode of QS inhibition in iberin is through downregulation of the Gac/Rsm QS network, which in turn leads to the repression of QS-regulated virulence factors, such as pyoverdine, chitinase, and protease IV. Lastly, as expected from the observed repression of small regulatory RNA synthesis, we also show that iberin effectively reduces biofilm formation. This suggests that small regulatory RNAs might serve as potential targets in the future development of therapies against pathogens that use QS for controlling virulence factor expression and assume the biofilm mode of growth in the process of causing disease.

Synergistic microbial consortium for bioenergy generation from complex natural energy sources.

Microbial species have evolved diverse mechanisms for utilization of complex carbon sources. Proper combination of targeted species can affect bioenergy production from natural waste products. Here, we established a stable microbial consortium with Escherichia coli and Shewanella oneidensis in microbial fuel cells (MFCs) to produce bioenergy from an abundant natural energy source, in the form of the sarcocarp harvested from coconuts. This component is mostly discarded as waste. However, through its usage as a feedstock for MFCs to produce useful energy in this study, the sarcocarp can be utilized meaningfully. The monospecies S. oneidensis system was able to generate bioenergy in a short experimental time frame while the monospecies E. coli system generated significantly less bioenergy. A combination of E. coli and S. oneidensis in the ratio of 1:9 (v:v) significantly enhanced the experimental time frame and magnitude of bioenergy generation. The synergistic effect is suggested to arise from E. coli and S. oneidensis utilizing different nutrients as electron donors and effect of flavins secreted by S. oneidensis. Confocal images confirmed the presence of biofilms and point towards their importance in generating bioenergy in MFCs.

Oxidative stress induced by Etoposide anti-cancer chemotherapy drives the emergence of tumor-associated bacteria resistance to fluoroquinolones.

INTRODUCTION: Antibiotic-resistant bacterial infections, such as Pseudomonas aeruginosa and Staphylococcus aureus, are prevalent in lung cancer patients, resulting in poor clinical outcomes and high mortality. Etoposide (ETO) is an FDA-approved chemotherapy drug that kills cancer cells by damaging DNA through oxidative stress. However, it is unclear if ETO can cause unintentional side effects on tumor-associated microbial pathogens, such as inducing antibiotic resistance. OBJECTIVES: We aimed to show that prolonged ETO treatment could unintendedly confer fluoroquinolone antibiotic resistance to P. aeruginosa, and evaluate the effect of tumor-associated P. aeruginosa on tumor progression. METHODS: We employed experimental evolution assay to treat P. aeruginosa with prolonged ETO exposure, evaluated the ciprofloxacin resistance, and elucidated the gene mutations by DNA sequencing. We also established a lung tumor-P. aeruginosa bacterial model to study the role of ETO-evolved intra-tumoral bacteria in tumor progression using immunostaining and confocal microscopy. RESULTS: ETO could generate oxidative stress and lead to gene mutations in P. aeruginosa, especially the gyrase (gyrA) gene, resulting in acquired fluoroquinolone resistance. We further demonstrated using a microfluidic-based lung tumor-P. aeruginosa coculture model that bacteria can evolve ciprofloxacin (CIP) resistance in a tumor microenvironment. Moreover, ETO-induced CIP-resistant (EICR) mutants could form multicellular biofilms which protected tumor cells from ETO killing and enabled tumor progression. CONCLUSION: Overall, our preclinical proof-of-concept provides insights into how anti-cancer chemotherapy could inadvertently allow tumor-associated bacteria to acquire antibiotic resistance mutations and shed new light on the development of novel anti-cancer treatments based on anti-bacterial strategies.

Dual-species proteomics and targeted intervention of animal-pathogen interactions.

INTRODUCTION: Host-microbe interactions are important to human health and ecosystems globally, so elucidating the complex host-microbe interactions and associated protein expressions drives the need to develop sensitive and accurate biochemical techniques. Current proteomics techniques reveal information from the point of view of either the host or microbe, but do not provide data on the corresponding partner. Moreover, it remains challenging to simultaneously study host-microbe proteomes that reflect the direct competition between host and microbe. This raises the need to develop a dual-species proteomics method for host-microbe interactions. OBJECTIVES: We aim to establish a forward + reverse Stable Isotope Labeling with Amino acids in Cell culture (SILAC) proteomics approach to simultaneously label and quantify newly-expressed proteins of host and microbe without physical isolation, for investigating mechanisms in direct host-microbe interactions. METHODS: Using Caenorhabditis elegans-Pseudomonas aeruginosa infection model as proof-of-concept, we employed SILAC proteomics and molecular pathway analysis to characterize the differentially-expressed microbial and host proteins. We then used molecular docking and chemical characterization to identify chemical inhibitors that intercept host-microbe interactions and eliminate microbial infection. RESULTS: Based on our proteomics results, we studied the iron competition between pathogen iron scavenger and host iron uptake protein, where P. aeruginosa upregulated pyoverdine synthesis protein (PvdA) (fold-change of 5.2313) and secreted pyoverdine, and C. elegans expressed ferritin (FTN-2) (fold-change of 3.4057). Targeted intervention of iron competition was achieved using Galangin, a ginger-derived phytochemical that inhibited pyoverdine production and biofilm formation in P. aeruginosa. The Galangin-ciprofloxacin combinatorial therapy could eliminate P. aeruginosa biofilms in a fish wound infection model, and enabled animal survival. CONCLUSION: Our work provides a novel SILAC-based proteomics method that can simultaneously evaluate host and microbe proteomes, with future applications in higher host organisms and other microbial species. It also provides insights into the mechanisms dictating host-microbe interactions, offering novel strategies for anti-infective therapy.

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.

Bacterial Iron Siderophore Drives Tumor Survival and Ferroptosis Resistance in a Biofilm-Tumor Spheroid Coculture Model.

Interactions between tumoral cells and tumor-associated bacteria within the tumor microenvironment play a significant role in tumor survival and progression, potentially impacting cancer treatment outcomes. In lung cancer patients, the Gram-negative pathogen Pseudomonas aeruginosa raises questions about its role in tumor survival. Here, a microfluidic-based 3D-human lung tumor spheroid-P. aeruginosa model is developed to study the bacteria's impact on tumor survival. P. aeruginosa forms a tumor-associated biofilm by producing Psl exopolysaccharide and secreting iron-scavenging pyoverdine, which is critical for establishing a bacterial community in tumors. Consequently, pyoverdine promotes cancer progression by reducing susceptibility to iron-induced death (ferroptosis), enhancing cell viability, and facilitating several cancer hallmarks, including epithelial-mesenchymal transition and metastasis. A promising combinatorial therapy approach using antimicrobial tobramycin, ferroptosis-inducing thiostrepton, and anti-cancer doxorubicin could eradicate biofilms and tumors. This work unveils a novel phenomenon of cross-kingdom cooperation, where bacteria protect tumors from death, and it paves the way for future research in developing antibiofilm cancer therapies. Understanding these interactions offers potential new strategies for combatting cancer and enhancing treatment efficacy.

Identification of five structurally unrelated quorum-sensing inhibitors of Pseudomonas aeruginosa from a natural-derivative database.

Bacteria communicate by means of small signal molecules in a process termed quorum sensing (QS). QS enables bacteria to organize their activities at the population level, including the coordinated secretion of virulence factors. Certain small-molecule compounds, known as quorum-sensing inhibitors (QSIs), have been shown to effectively block QS and subsequently attenuate the virulence of Pseudomonas aeruginosa, as well as increasing its susceptibility to both antibiotics and the immune system. In this study, a structure-based virtual screening (SB-VS) approach was used for the discovery of novel QSI candidates. Three-dimensional structures of 3,040 natural compounds and their derivatives were obtained, after which molecular docking was performed using the QS receptor LasR as a target. Based on docking scores and molecular masses, 22 compounds were purchased to determine their efficacies as quorum-sensing inhibitors. Using a live reporter assay for quorum sensing, 5 compounds were found to be able to inhibit QS-regulated gene expression in P. aeruginosa in a dose-dependent manner. The most promising compound, G1, was evaluated by isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis, and it was found to significantly affect the abundance of 46 proteins (19 were upregulated; 27 were downregulated) in P. aeruginosa PAO1. It specifically reduced the expression of several quorum-sensing-regulated virulence factors, such as protease IV, chitinase, and pyoverdine synthetases. G1 was also able to reduce extracellular DNA release and inhibited the secretion of the virulence factor, elastase, whose expression is regulated by LasR. These results demonstrate the utility of SB-VS for the discovery of target-specific QSIs.

Bis-(3'-5')-cyclic dimeric GMP regulates antimicrobial peptide resistance in Pseudomonas aeruginosa.

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that controls the lifestyles of many bacteria. A high intracellular level of c-di-GMP induces a biofilm lifestyle, whereas a low intracellular level of c-di-GMP stimulates dispersal of biofilms and promotes a planktonic lifestyle. Here, we used the expression of different reporters to show that planktonic cells, biofilm cells, and cells dispersed from biofilms (DCells) had distinct intracellular c-di-GMP levels. Proteomics analysis showed that the low intracellular c-di-GMP level of DCells induced the expression of proteins required for the virulence and development of antimicrobial peptide resistance in Pseudomonas aeruginosa. In accordance with this, P. aeruginosa cells with low c-di-GMP levels were found to be more resistant to colistin than P. aeruginosa cells with high c-di-GMP levels. This finding contradicts the current dogma stating that dispersed cells are inevitably more susceptible to antibiotics than their sessile counterparts.

Biofilms exacerbate atherogenesis through macrophage-induced inflammatory responses in a fibrous plaque microsystem model.

BACKGROUND: Microbes have been implicated in atherosclerosis development and progression, but the impact of bacterial-based biofilms on fibrous plaque rupture remains poorly understood. RESULTS: Here, we developed a comprehensive atherosclerotic model to reflect the progression of fibrous plaque under biofilm-induced inflammation (FP-I). High expressions of biofilm-specific biomarkers algD, pelA and pslB validated the presence of biofilms. Biofilm promotes the polarization of macrophages towards a pro-inflammatory (M1) phenotype, as demonstrated by an increase in M1 macrophage-specific marker CD80 expression in CD68+ macrophages. The increase in the number of intracellular lipid droplets (LDs) and foam cell percentage highlighted the potential role of biofilms on lipid synthesis or metabolic pathways in macrophage-derived foam cells. In addition, collagen I production by myofibroblasts associated with the fibrous cap was significantly reduced along with the promotion of apoptosis of myofibroblasts, indicating that biofilms affect the structural integrity of the fibrous cap and potentially undermine its strength. CONCLUSION: We validated the unique role of biofilm-based inflammation in exacerbating fibrous plaque damage in the FP-I model, increasing fibrous plaque instability and risk of thrombosis. Our results lay the foundation for mechanistic studies of the role of biofilms in fibrous plaques, allowing the evaluation of preclinical combination strategies for drug therapy. STATEMENT OF SIGNIFICANCE: A microsystem-based model was developed to reveal interactions in fibrous plaque during biofilm-induced inflammation (FP-I). Real-time assessment of biofilm formation and its role in fibrous plaque progression was achieved. The presence of biofilms enhanced the expression of pro-inflammatory (M1) specific marker CD80, lipid droplets, and foam cells and reduced anti-inflammatory (M2) specific marker CD206 expression. Fibrous plaque exposure to biofilm-based inflammation reduced collagen I expression and increased apoptosis marker Caspase-3 expression significantly. Overall, we demonstrate the unique role of biofilm-based inflammation in exacerbating fibrous plaque damage in the FP-I model, promoting fibrous plaque instability and enhanced thrombosis risk. Our findings lay the groundwork for mechanistic studies, facilitating the evaluation of preclinical drug combination strategies.