Identification and characterization of biogenic silencers of Pseudomonas aeruginosa quorum sensing

Nemogućnost kontrole pojave i širenja rezistencije na antibiotike usmerila je istraživanja u poslednje dve dekade ka iznalaženju novih terapeutskih opcija, sa ciljem tretmana infekcija izazvanih patogenim bakterijama rezistentnim na veći broj kliniĉki znaĉajnih antibiotika. Uzevši u obzir ĉinjenicu da je antibiotska rezistencija kompleksan, multifaktorijalni fenomen, rešenje ovog problema ukljuĉuje niz pristupa usmerenih na kontrolu faktora koji olakšavaju nastanak i širenje rezistencije. Jedan od tih pristupa sastoji se u razvoju novih terapeutika koji bi delovali mehanizmima razliĉitim od trenutno dostupnih antibiotika. U tom pogledu, antivirulentna terapija zamišljena je kao obećavajuća alternativa sa ciljem kontrole virulencije specifiĉne za odreĊene patogene, bez vršenja snažnog selektivnog pritiska na bakterijske ćelije. Imajući u vidu da je Pseudomonas aeruginosa jedan od vodećih uzroĉnika unutarbolniĉkih infekcija širom sveta, leĉenje infekcija izazvanih ovim patogenom predstavlja veliki terapeutski izazov. P. aeruginosa svoj patogeni potencijal ostvaruje zahvaljujući mnogobrojnim uroĊenim, steĉenim i adaptivnim mehanizmima rezistencije. Pored toga, posedovanje sistema meĊućelijske komunikacije (eng. quorum sensing, QS) ovom patogenu omogućava fleksibilnost u regulaciji ekspresije gena ukljuĉenih u virulenciju, formiranje biofilma, produkciju sekundarnih metabolita i faktora koji imaju ulogu u zaštiti od imunskog sistema domaćina. Stoga bi primena antivirulentne terapije zasnovane na utišavanju meĊućelijske komunikacije bakterija mogla poslužiti kao obećavajuće oruĊe u kontroli infekcija izazvanih predstavnicima P. aeruginosa za koje ne postoji adekvatna terapija trenutno dostupnim antibioticima. U skladu sa time, predmet prouĉavanja ove teze bila je potraga za novim utišivaĉima meĊućelijske komunikacije bakterija (eng. quorum quenching, QQ) produkovanih od strane kliniĉkih izolata koji tokom infekcija dele istu ekološku nišu sa P. aeruginosa. Analizirana je kolekcija Gramnegativnih kliniĉkih izolata Laboratorije za molekularnu mikrobiologiju, Instituta za molekularnu genetiku i genetiĉko inženjerstvo, Univerziteta u Beogradu sa ciljem pronalaženja sojeva koji produkuju QQ molekule. Nakon odabira sojeva nosioca QQ fenotipa, usledila je njihova identifikacija kao i funkcionalna karakterizacija na model sistemu kliniĉkog izolata P. aeruginosa MMA83. Upotrebom Chromobacterium violaceum CV026 biosenzora, selektovano je 19 sojeva (od ukupno 633 analiziranih izolata) koji su ispoljavali QQ fenotip, od kojih su dva izolata Delftia sp. 11304 i Burkholderia sp. BCC4135, na osnovu najznaĉajnije QQ aktivnosti izdvojena za detaljniju karakterizaciju. UtvrĊeno je da Delftia sp. 11304 soj pripada Delftia tsuruhatensis vrsti, dok Burkholderia sp. BCC4135 pripada Burkholderia cepacia vrsti sa novim sekvencnim tipom ST1485. In silico analizom genomskih sekvenci ustanovljeno je da oba odabrana soja poseduju izuzetan potencijal virulencije i rezistencije na antimikrobna jedinjenja. Priroda QQ molekula koje produkuju ovi sojevi bila je razliĉita; konstatovano je da soj 11304 produkuje male molekule neproteinske prirode (QS inhibitore, QSI), dok BCC4135 produkuje QQ enzime. Pored toga, kod BCC4135 izolata utvrĊeno je prisustvo i QS fenomena. Ukupni etil-acetatni ekstrakt supernatanta soja D. tsuruhatensis 11304 (QSI ekstrakt) ostvario je znaĉajan antivirulentni potencijal na model sistemu kliniĉkog izolata P. aeruginosa MMA83...finding novel therapeutic options to combat multidrug-resistant pathogenic bacteria during the last two decades. Given the fact that antimicrobial resistance is a complex, multifactorial phenomenon, the solution to this problem comprises a range of approaches focused on monitoring the factors that facilitate the emergence and spread of resistance. One of proposed strategies consists of developing novel therapeutics that operate under different principles to the currently available antibiotics. In this respect, antivirulence therapy has been conceived as a promising alternative to control virulence in a pathogen-specific manner, without exerting strong selective pressure on the bacterial cells. Having in mind that Pseudomonas aeruginosa has been considered as a leading cause of nosocomial infections worldwide, the treatment of infections caused by this pathogen represents a major therapeutic challenge. The pathogenic potential of P. aeruginosa has been accomplished due to a numerous innate, acquired, and adaptive resistance mechanisms. In addition, the presence of cell-to-cell communication system (quorum sensing, QS) allows this pathogen the flexibility in the regulation of virulence gene expression responsible for biofilm development, production of secondary metabolites, and immuneevasive factors. Therefore, the use of antivirulence therapy based on the silencing of bacterial communication could serve as a promising tool in the control of infections caused by P. aeruginosa for which there is no adequate therapy with currently available antibiotics. Accordingly, the subject of this thesis was discovery and characterization of novel quenchers of bacterial cell-to-cell communication produced by clinical isolates that share the same ecological niche with P. aeruginosa during infections. A collection of Gram-negative clinical isolates from the Laboratory for Molecular Microbiology Institute of Molecular Genetics and Genetic Engineering University of Belgrade was analyzed in order to select the strains that produce quorum quenching (QQ) molecules. After the selection of strains carrying QQ phenotype, the identification of QQ molecules as well as their functional characterization on the model system of P. aeruginosa MMA83 clinical isolate was performed. Using Chromobacterium violaceum CV026 biosensor, 19 strains (out of a total of 633 analyzed isolates) with QQ phenotype were discovered, among which two clinical isolates Delftia sp. 11304 and Burkholderia sp. BCC4135 have been selected as strongest producers of QQ molecules for detailed characterization. According to the genomic sequence analysis, Delftia sp. 11304 was identified as a Delftia tsuruhatensis species while Burkholderia sp. BCC4135 was unveiled as Burkholderia cepacia with novel sequence type ST1485. In silico analysis of genomic sequences indicated that both selected strains possess significant antimicrobial resistance and virulence potential. The nature of the QQ molecules produced by these strains was determined as different; strain 11304 was found to produce small molecules of non-proteinaceous nature (QS inhibitors, QSI), while BCC4135 produced QQ enzymes. Besides, the presence of the QS phenomenon was determined in BCC4135 isolate. The total ethyl acetate extract of the D. tsuruhatensis 11304 culture supernatant (QSI extract) showed significant antivirulence potential on the model system of clinical isolate P. aeruginosa MMA83..

LACTONASE MEDIATED QUORUM QUENCHING OF PSEUDOMONAS AERUGINOSA VIRULENCE

Solving the problem of the antimicrobial resistance crisis is one of the primary challenges currently confronting the healthcare system. One of the most promising new strategies to combat antimicrobial resistance is the antivirulence therapy, based on silencing bacterial cell-to-cell communication (quorum quenching - QQ). QQ enzymes lactonases represent a diverse group of enzymes capable of inactivating signaling molecules of bacterial communication – N-acyl homoserine lactones (AHLs), resulting in alterations ofbacterial virulence. The numerous virulence factors and resistance to most conventional antibiotics have led to Pseudomonas aeruginosa being listed as one of the top-priority pathogens on the ESCAPE pathogen list, highlighting the urgent need for the development of new therapies to combat this pathogen. P. aeruginosauses cell-to-cell communication known as quorum sensing (QS) that allows bacteria to monitor their own population density via signal molecules and subsequently control bacterial pathogenesis. Our hypothesis was that bacterial pathogens which share the same ecological niche with P. aeruginosa during infection have developed a system to disrupt its QS system, in order to survive in polymicrobial communities alongside this successful pathogen. In our research we identified QQ enzymes lactonases originating from two Gramnegative bacterial pathogens Burkholderiacepacia and Stenotrophomonas maltophilia. The genes encoding for the enzymes were cloned and expressed in pQE30 expression vector. B. cepacia BCC4135 synthesizes two lactonases YtnP and Y2-aiiA, that have the different cellular localization, but also different substrate specificity, which could imply the difference in their biological roles. S. maltophilia 6960 YtnP lactonase has several advantageous biotechnological properties, such as high thermostability, activity in a wide pH range, and no cytotoxic microscopy analysis showed a strong effect of analyzed lactonases on preventing biofilm formationand initiating the decomposition of the preformed biofilm of P. aeruginosa MMA83. Functional assays showed that lactonases have the ability to significantly reduce extracellular virulence factors production – elastase, pyocyanin and rhamnolipid. Additionally, the results obtained by real-time quantitative PCR showed that analyzed recombinant enzymes significantly downregulated all three analyzed P. aeruginosa QS networks at the transcriptional level. Finally, S. maltophilia 6960 YtnP lactonase significantly prolonged survival of Caenorhabditis elegans by reducing virulence of P. aeruginosa using fastkilling liquid assay. The described properties make B. cepacia and S. maltophilia lactonases the promising therapeutic candidates for the development of nextgeneration antivirulence agents.Book of abstracts and conference proceedings / 3rd International Conference Antimicrobial Resistance - Current State and Perspectives, 16-18. May 2024, Novi Sad, Serbia

Biogenic silencers of Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa jedan je od najznačajnijih uzročnika unutarbolničkih infekcija čiji je terapijski tretman konvencionalnim antibioticima sve češće neefikasan usled rezistencije na antibiotike. Inovativni vidovi kontrole infekcija, poput utišavanja međućelijske komunikacije bakterija, a time i onemogućavanja virulencije i inhibicije patogenog fenotipa su stoga od izuzetnog značaja. U ovom radu biće predstavljena istraživanja koja su bazirana na prirodnom svojstvu bakterija koje dele ekološke niše da sarađuju, ali i kompetiraju, na osnovu čega su analizirane Delftia tsuruhatensis i Burkholderia cepacia koje tokom infekcija kolokalizuju sa P. aeruginosa. Pokazano je da D. tsuruhatensis 11304 produkuje C18-HSL koji inhibira virulenciju P. aeruginosa i rekonstituiše osetljivost na antibiotike, a takođe je po prvi put u literaturi opisano prisustvo dihidroksi- C18-HSL u biološkim uzorcima. Opisane su i laktonaze vrste B. cepacia BCC4135 koje degraduju autoinducere komunikacije P. aeruginosa i inhibiraju ekspresiju faktora virulencije. Utvrđena je njihova supstratna specifičnost i ukazano na različitu biološku funkciju u zavisnosti od lokalizacije.Pseudomonas aeruginosa is a leading cause of nosocomial infections, whose therapeutic treatment with conventional antibiotics is increasingly ineffective due to antibiotic resistance. Inovative approaches of infection control, such as silencing the bacterial quorum sensing system and thus virulence and pathogenic phenotype inhibition are of great importance. In this study, there will be presented research based on natural feature of bacteria that share the same ecological niche to coordinate, but also to compete, based on which Delftia tsuruhatensis and Burkholderia cepacia that colocalize with P. aeruginosa during infections were analysed. D. tsuruhatensis 11304 has been shown to produce C18-HSL which inhibits P. aeruginosa virulence and reconstitutes antibiotic susceptibility, and the presence of dihydroxy-C18-HSL in biological samples has also been described for the first time in the literature. B. cepacia BCC4135 lactonases that degrade autoinducers of P. aeruginosa quorum sensing system and inhibit virulence factor expression have also been reported. Their substrate specificity was determined and different biological function depending on their localization was indicated.Jedan deo ovog rada realizovan je na Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Napulj, Italija pod rukovodstvom prof dr Antonio Molinaro i dr Flaviana Di Lorenzo, kojima se ovom prilikom zahvaljujem. Hvala dr sci med Zorici Vasiljević sa Instituta za zdravstvenu zaštitu majke i deteta Srbije ‘’Dr Vukan Čupić‘’ koja je obezbedila kliničke izolate korišćene u ovom radu, kao i dr Milanu Kojiću i drugim saradnicima Laboratorije za molekularnu mikrobiologiju Instituta za molekularnu genetiku i genetičko inženjerstvo za izuzetnu pomoć tokom izrade eksperimenata

A novel thermostable YtnP lactonase inhibits biofilm formation and induces decomposition of preformed Pseudomonas aeruginosa biofilms

Biofilm-associated infections are the main cause of biomaterial implant failure today. The increasing prevalence of antibiotic-resistant pathogens often results in the only solution of implant movement, with serious consequences for patients. Recently, various antimicrobial agents have been recognized as a promising strategy to prevent biofilm formation on implant surfaces. Quorum sensing (QS) plays a central role in the control of bacterial virulence and biofilm formation. The use of quorum quenching (QQ) enzymes to target QS is therefore a promising innovative approach for the development of enzyme-based antivirulence therapeutics, which represent a potential solution to combat infections caused by multidrug-resistant pathogens. This study aimed to characterize the novel YtnP lactonase from the clinical isolate Stenotrophomonas maltophilia 6960 and to investigate its potential to combat the virulence of multidrug-resistant (MDR) Pseudomonas aeruginosa MMA83

RclS Sensor Kinase Modulates Virulence of Pseudomonas capeferrum

Signal transduction systems are the key players of bacterial adaptation and survival. The orthodox two-component signal transduction systems perceive diverse environmental stimuli and their regulatory response leads to cellular changes. Although rarely described, the unorthodox three-component systems are also implemented in the regulation of major bacterial behavior such as the virulence of clinically relevant pathogen P. aeruginosa. Previously, we described a novel three-component system in P. capeferrum WCS358 (RclSAR) where the sensor kinase RclS stimulates the intI1 transcription in stationary growth phase. In this study, using rclS knock-out mutant, we identified RclSAR regulon in P. capeferrum WCS358. The RNA sequencing revealed that activity of RclSAR signal transduction system is growth phase dependent with more pronounced regulatory potential in early stages of growth. Transcriptional analysis emphasized the role of RclSAR in global regulation and indicated the involvement of this system in regulation of diverse cellular activities such as RNA binding and metabolic and biocontrol processes. Importantly, phenotypic comparison of WCS358 wild type and Delta rclS mutant showed that RclS sensor kinase contributes to modulation of antibiotic resistance, production of AHLs and siderophore as well as host cell adherence and cytotoxicity. Finally, we proposed the improved model of interplay between RclSAR, RpoS and LasIR regulatory systems in P. capeferrum WCS358

Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU

Introduction: Lactolisterin BU (LBU) is a potent bacteriocin derived from Lactococcuslactis subsp. lactis bv. diacetylactis BGBU1-4. It exhibits antimicrobial properties against Gram-positive food spoilage and foodborne pathogens. This research aimed to explore the impact of amino acid substitution in LBU on its antimicrobial activity by utilizing in silico prediction of LBU’ssecondary structure and amino acid substitutions. Methods: The secondary structure of LBU was predicted using Phyre2 software. Five variants of LBU were selected and chemically synthesized, along with unaltered LBU and BHT-B,serving as controls. Peptides were twofold diluted in distilled water, resulting in final concentrations ranging from 1000 µg/ml to 0.5 µg/ml. An agarspot test, employing 5 µl of the dilution, was conducted on three indicatorstrains: Lactococcus lactis BGMN1-596, Listeria monocytogenes ATCC19111, and Staphylococcus aureus ATCC25923. The presence of inhibition zones was analyzed after overnight incubation at 37°C (S. aureus) and 30°C (L. lactis and L. monocytogenes). Results: Phyre2 analysis unveiled the presence of two α-helices in LBU’s structure. The majority of LBU variants displayed altered antimicrobial activity, with some changes being genusspecific, potentially attributable to variances in cell wall composition. Some variants completely lost their activity, underscoring the significance of native amino acids or their physicochemical properties in the corresponding positions within LBU’s structure. Furthermore, it was confirmed that chemically synthesized LBU effectively retains its antimicrobial activity. Conclusion: Changesin amino acid composition give insight on structure-function relationship of LBU

Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU

Introduction: Lactolisterin BU (LBU) is a potent bacteriocin derived from Lactococcuslactis subsp. lactisbv. diacetylactis BGBU1-4. It exhibits antimicrobial properties against Gram-positive food spoilage andfoodborne pathogens. This research aimed to explore the impact of amino acid substitution in LBU onits antimicrobial activity by utilizing in silico prediction of LBU’ssecondary structure and amino acid substitutions.Methods: The secondary structure of LBU was predicted using Phyre2 software. Five variants of LBUwere selected and chemically synthesized, along with unaltered LBU and BHT-B,serving as controls. Peptides were twofold diluted in distilled water, resulting in final concentrations ranging from 1000 µg/mlto 0.5 µg/ml. An agarspot test, employing 5 µl of the dilution, was conducted on three indicatorstrains:Lactococcus lactis BGMN1-596, Listeria monocytogenes ATCC19111, and Staphylococcus aureusATCC25923. The presence of inhibition zones was analyzed after overnight incubation at 37°C (S. aureus)and 30°C (L. lactis and L. monocytogenes).Results: Phyre2 analysis unveiled the presence of two α-helices in LBU’s structure. The majority of LBUvariants displayed altered antimicrobial activity, with some changes being genusspecific, potentially attributable to variances in cell wall composition. Some variants completely lost their activity, underscoring the significance of native amino acids or their physicochemical properties in the correspondingpositions within LBU’s structure. Furthermore, it was confirmed that chemically synthesized LBU effectively retains its antimicrobial activity.Conclusion: Changesin amino acid composition give insight on structure-function relationship of LBU

A novel thermostable YtnP lactonase from Stenotrophomonas maltophilia inhibits Pseudomonas aeruginosa virulence in vitro and in vivo

Infections caused by multidrug-resistant pathogens are one of the biggest challenges facing the healthcare system today. Quorum quenching (QQ) enzymes have the potential to be used as innovative enzyme-based antivirulence therapeutics to combat infections caused by multidrug-resistant pathogens. The main objective of this research was to describe the novel YtnP lactonase derived from the clinical isolate Stenotrophomonas maltophilia and to investigate its antivirulence potential against multidrug-resistant Pseudomonas aeruginosa MMA83. YtnP lactonase, the QQ enzyme, belongs to the family of metallo-β-lactamases. The recombinant enzyme has several advantageous biotechnological properties, such as high thermostability, activity in a wide pH range, and no cytotoxic effect. High-performance liquid chromatography analysis revealed the activity of recombinant YtnP lactonase toward a wide range of N-acyl-homoserine lactones (AHLs), quorum sensing signaling molecules, with a higher preference for long-chain AHLs. Recombinant YtnP lactonase was shown to inhibit P. aeruginosa MMA83 biofilm formation, induce biofilm decomposition, and reduce extracellular virulence factors production. Moreover, the lifespan of MMA83-infected Caenorhabditis elegans was prolonged with YtnP lactonase treatment. YtnP lactonase showed synergistic inhibitory activity in combination with gentamicin and acted additively with meropenem against MMA83. The described properties make YtnP lactonase a promising therapeutic candidate for the development of next-generation antivirulence agents

Polyphenols as Inhibitors of Antibiotic Resistant Bacteria-Mechanisms Underlying Rutin Interference with Bacterial Virulence

The rising incidence of antibiotic resistant microorganisms urges novel antimicrobials development with polyphenols as appealing potential therapeutics. We aimed to reveal the most promising polyphenols among hesperetin, hesperidin, naringenin, naringin, taxifolin, rutin, isoquercitrin, morin, chlorogenic acid, ferulic acid, p-coumaric acid, and gallic acid based on antimicrobial capacity, antibiofilm potential, and lack of cytotoxicity towards HaCaT, and to further test its antivirulence mechanisms. Although the majority of studied polyphenols were able to inhibit bacterial growth and biofilm formation, the most promising activities were observed for rutin. Further investigation proved rutin's ability to prevent/eradicate Pseudomonas aeruginosa and MRSA urinary catheter biofilms. Besides reduction of biofilm biomass, rutin antibiofilm mechanisms included reduction of cell viability, exopolysaccharide, and extracellular DNA levels. Moderate reduction of bacterial adhesion to human keratinocytes upon treatment was observed. Rutin antivirulence mechanisms included an impact on P. aeruginosa protease, pyocyanin, rhamnolipid, and elastase production and the downregulation of the lasI, lasR, rhlI, rhlR, pqsA and mvfR genes. Rutin also interfered with membrane permeability. Polyphenols could repress antibiotic resistant bacteria. Rutin has shown wide antimicrobial and antibiofilm capacity employing a range of mechanisms that might be used for the development of novel antimicrobials

Virulence traits associated with Burkholderia cenocepacia ST856 epidemic strain isolated from cystic fibrosis patients

Background: Burkholderia cenocepacia is considered one of the most problematic cystic fibrosis (CF) pathogens. Colonization prevalence in the Serbian CF population is high and virtually exclusively limited to a single highly transmissible clone of B. cenocepacia ST856 which is positive for both the B. cepacia epidemic strain marker (BCESM) and cable pilin, and is closely related to the epidemic strain CZ1 (ST32). Methods: Biofilm formation for 182 isolates, and adhesion to components of the host extracellular matrix, proteolytic activity, mucoidy and motility of selected ST856 representatives, as well as B. cenocepacia ST858 and ST859, and B. stabilis ST857, novel STs isolated from Serbian CF patients, were investigated in this study. The presence of the cepI, cepR, fliG, llpE, wbiI, and bcscV genes was analyzed. Results: Biofilm-formation ability of analyzed strains was poor under standard laboratory conditions, but changed in stress conditions (cold stress) and conditions that mimic CF milieu (increased CO2). All strains expressed ability to bind to collagen and fibronectin albeit with different intensity. Representatives of ST856 exhibited gelatinase activity. ST858, ST859 and 9/11 of ST856 genotypes were positive for swimming and twitching motility whereas ST857 was non-motile. Mucoidy was demonstrated in all ST856 genotypes, ST857 was semi-mucoid, and ST858 and ST859 were non-mucoid. Molecular analysis for major virulence factors revealed that ST856 and ST857 carried the six analyzed genes, while ST858 and ST859 were negative for the llpE gene. Conclusion: Variations in virulence phenotypes in different genotypes of epidemic B. cenocepacia ST856 clone, in vitro, could be a consequence of diversification driven by pathoadaptation. Diversity of epidemic clone genotypes virulence, could be challenging for accurate diagnosis and treatment, as well as for infection control