Production Of Virulence Factors Research Articles

3-Hydroxypyridin-4(1H)-one Derivatives as pqs Quorum Sensing Inhibitors Attenuate Virulence and Reduce Antibiotic Resistance in Pseudomonas aeruginosa.

The development of quorum sensing inhibitors capable of decreasing the production of virulence factors is an effective strategy to overcome resistance in Pseudomonas aeruginosa due to the less selective pressure exerted on bacteria. In this study, a series of 3-hydroxypyridin-4(1H)-one derivatives bearing a 4-aminomethyl-1,2,3-triazole linker were designed and synthesized as antivirulence agents against P. aeruginosa. The most potent derivative 16e was identified as a selective inhibitor of the pqs system (IC50 = 3.7 μM) and its related virulence factor pyocyanin (IC50 = 2.7 μM). In addition, 16e exhibited moderate biofilm inhibition and significant inhibition of P. aeruginosa motility phenotypes with low cytotoxicity. Compound 16e showed an obvious antibacterial synergistic effect in combination with antibiotics such as ciprofloxacin and tobramycin in in vitro and in vivo Caenorhabditis elegans infection models. Overall, the excellent antivirulence properties of compound 16e make it a potential antibiotic adjuvant for the treatment of P. aeruginosa infections that may be advanced into preclinical development in the future.

Methyl gallate isolated from partridge tea (Mallotus oblongifolius (Miq.) Müll.Arg.) inhibits the biofilms and virulence factors of Burkholderia thailandensis

Ethnopharmcological relevanceThe formation of biofilms is a factor leading to chronic infection and drug resistance in melioidosis. The production of biofilm formation and many virulence factors are regulated by quorum sensing (QS). Therefore, the discovery of QS inhibitors to reduce antibiotic abuse has attracted a lot of attention. In this case, the methanol extract of a unique ethnic medicinal plant partridge tea (Mallotus oblongifolius (Miq.) Müll.Arg.) and its isolated active compound were used as biofilms and QS inhibitors against Burkholderia thailandensis. Aim of the studyThe purpose of this study is to investigate the anti-biofilm and anti-QS effect of the ethnic medicinal plant partridge tea and its active compounds against B. thailandensis. MethodsActive compound was isolated using classical phytochemical separation techniques under activity tracking. The biofilm and virulence factors (Proteases, lipases, rhamnolipids, and motility) of B. thailandensis were used to evaluate the activity of crude extracts and isolated compounds. ResultsIn this study, the extract of partridge tea and MG had good QS inhibitors activity against B. thailandensis E264. MG was investigated to inhibit QS-related virulence factors and the biofilm formation against B. thailandensis E264. The lipase activity of B. thailandensis E264 decreased by 49.41% at 150 μg/mL. At 75 μg/mL and 150 μg/mL, the erasion of mature biofilms reached 28.18% and 70.87%, respectively. Correspondingly, 150 μg/mL MG could significantly decrease btaR1 and btaR3 by 55.78% and 56.24%, respectively. Contradictorily, the rhamnolipid production of B. thailandensis E264 was 1.67 folds that of the control group at 150 μg/mL MG. ConclusionThrough molecular docking analysis and biological phenotype data, we speculate that MG may inhibit the biofilms and virulence factors of B. thailandensis E264 by interfering two QS systems, BtaI1/R1 and BtaI3/R3. Therefore, MG should be one potential QSI for the treatment of Burkholderia pathogens.

The secondary metabolite hydrogen cyanide protects Pseudomonas aeruginosa against sodium hypochlorite-induced oxidative stress.

The high pathogenicity of Pseudomonas aeruginosa is attributed to the production of many virulence factors and its resistance to several antimicrobials. Among them, sodium hypochlorite (NaOCl) is a widely used disinfectant due to its strong antimicrobial effect. However, bacteria develop many mechanisms to survive the damage caused by this agent. Therefore, this study aimed to identify novel mechanisms employed by P. aeruginosa to resist oxidative stress induced by the strong oxidizing agent NaOCl. We analyzed the growth of the P. aeruginosa mutants ΔkatA, ΔkatE, ΔahpC, ΔahpF, ΔmsrA at 1 μg/mL NaOCl, and showed that these known H2O2 resistance mechanisms are also important for the survival of P. aeruginosa under NaOCl stress. We then conducted a screening of the P. aeruginosa PA14 transposon insertion mutant library and identified 48 mutants with increased susceptibility toward NaOCl. Among them were 10 mutants with a disrupted nrdJa, bvlR, hcnA, orn, sucC, cysZ, nuoJ, PA4166, opmQ, or thiC gene, which also exhibited a significant growth defect in the presence of NaOCl. We focussed our follow-up experiments (i.e., growth analyzes and kill-kinetics) on mutants with defect in the synthesis of the secondary metabolite hydrogen cyanide (HCN). We showed that HCN produced by P. aeruginosa contributes to its resistance toward NaOCl as it acts as a scavenger molecule, quenching the toxic effects of NaOCl.

Tyramine, one quorum sensing inhibitor, reduces pathogenicity and restores tetracycline susceptibility in Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic respiratory pathogen of particular relevance to patients with cystic fibrosis (CF), primarily regulating its biological functions and virulence factors through two quorum sensing (QS) systems (CepI/R and CciI/R). The highly persistent incidence of multidrug resistant Burkholderia cenocepacia poses a global threat to public health. In this study, we investigated the effects of tyramine, one biogenic amine, on the QS systems of Burkholderia cenocepacia. Genetic and biochemical analyses revealed that tyramine inhibited the production of N-hexanoyl-homoserine (AHL) signaling molecules (C8-HSL and C6-HSL) by blocking the CepI/R and CciI/R systems. As a result, the inhibition of QS systems leads to reduced production of various virulence factors, such as biofilm formation, extracellular polysaccharides, lipase, and swarming motility. Notably, as a potential quorum sensing inhibitor, tyramine exhibits low toxicity in vivo in Galleria mellonella larvae and is well characterized by Lipinski’s five rules. It also shows high gastrointestinal absorption and the ability to cross the blood–brain barrier according to SwissADME database and ProTox-II server. Additionally, tyramine was found to enhance the efficacy of tetracycline in reducing the infectivity of Burkholderia cenocepacia in Galleria mellonella larvae infection model. Therefore, tyramine could be a promising candidate for combination therapy with traditional antimicrobials to improve their effectiveness against Burkholderia cenocepacia.

Phyloproteomic study by MALDI-TOF MS in view of intraspecies variation in a significant homogenous phytopathogen Dickeya solani

Dickeya solani is an economically significant pectinolytic phytopathogen belonging to the Pectobacteriaceae family, which causes soft rot and blackleg diseases. Despite its notable impact on global potato production, there are no effective methods to control this pest. Here, we undertook a phyloproteomic study on 20 D. solani strains, of various origin and year of isolation, with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) supported by an in-depth characterization of the strains in terms of the virulence-associated phenotype. In spite of high homogeneity in this species, we herein revealed for the first time intraspecies variation in the MALDI-TOF MS protein profiles among the studied D. solani isolates. Finally, representative mass spectra for the four delineated clades are presented. A majority of the analysed D. solani strains showed high virulence potential, while two strains stood out in their growth dynamics, virulence factors production and ability to macerate plant tissue. Nonetheless, the metabolic profiles of D. solani strains turned out to be uniform, except for gelatinase activity. Given that all D. solani isolates distinctly grouped from the other Dickeya species in the MALDI-TOF MS analysis, there is strong evidence supporting the potential routine use of this method for fast and reliable to-species identification of D. solani isolates of environmental origin.

Trichoderma-derived emodin competes with ExpR and ExpI of Pectobacterium carotovorum subsp. carotovorum to biocontrol bacterial soft rot.

Quorum sensing inhibitors (QSIs) are an emerging control tool that inhibits the quorum sensing (QS) system of pathogenic bacteria. We aimed to screen for potential QSIs in the metabolites of Trichoderma and to explore their inhibitory mechanisms. We screened a strain of Trichoderma asperellum LN004, which demonstrated the ability to inhibit the color development of Chromobacterium subtsugae CV026, primarily attributed to the presence of emodin as its key QSI component. The quantitative polymerase chain reaction with reverse transcription results showed that after emodin treatment of Pectobacterium carotovorum subsp. carotovorum (Pcc), plant cell wall degrading enzyme-related synthetic genes were significantly downregulated, and the exogenous enzyme synthesis gene negative regulator (rsmA) was upregulated 3.5-fold. Docking simulations indicated that emodin could be a potential ligand for ExpI and ExpR proteins because it exhibited stronger competition than the natural ligands in Pcc. In addition, western blotting showed that emodin attenuated the degradation of n-acylhomoserine lactone on the ExpR protein and protected it. Different concentrations of emodin reduced the activity of pectinase, cellulase, and protease in Pcc by 20.81%-72.21%, 8.38%-52.73%, and 3.57%-47.50%. Lesion size in Chinese cabbages, carrots and cherry tomatoes following Pcc infestation was reduced by 10.02%-68.57%, 40.17%-88.56% and 11.36%-86.17%. Emodin from T. asperellum LN004 as a QSI can compete to bind both ExpI and ExpR proteins, interfering with the QS of Pcc and reducing the production of virulence factors. The first molecular mechanism reveals the ability of emodin as a QSI to competitively inhibit two QS proteins simultaneously. © 2023 Society of Chemical Industry.

Development of a dual fluorescent reporter system to identify inhibitors of Staphylococcus aureus virulence factors.

Staphylococcus aureus is a formidable pathogen responsible for a wide range of infections, and the emergence of antibiotic-resistant strains has posed significant challenges in treating these infections. In this study, we have established a novel dual reporter system capable of concurrently monitoring the activities of two critical virulence regulators in S. aureus. By incorporating both reporters into a single screening platform, we provide a time- and cost-efficient approach for assessing the activity of compounds against two distinct targets in a single screening round. This innovative dual reporter system presents a promising strategy for the identification of molecules capable of modulating virulence gene expression in S. aureus, potentially expediting the development of antivirulence therapies.

Modified Impedance Sensing System Determination of Virulence Characteristics of Pathogenic Bacteria Klebsiella Species.

An impedance sensing system is a family of biosensors that measure changes in electrical impedance to perform their functions. Physical and chemical changes in the impedance of the sensing element, such as changes in the concentration of a target analyte or changes in the physical properties of the sensing element, can result in changes in the impedance of the sensing element. Many impedance biosensors have been developed for the detection of pathogens in the past few decades. Several types of biosensors have been developed for the detection of infections, including transduction elements, biorecognition components, and electrochemical approaches. In this review, we discuss the characteristics and pathogenic factors associated with 2,3-butanediol-producing Klebsiella pneumoniae collected using impedance sensors. An impedance sensing system was introduced as a great method for monitoring the virulence factors of Klebsiella spp. in situ. Klebsiella pneumoniae produces virulence factors, including capsules, lipopolysaccharides, fimbriae, and siderophores, as part of its pathogenesis. It is possible to examine virulence factors' pathogenic characteristics in vitro and in vivo using real tissues or mouse models in order to conduct experiments. For the monitoring of virulence factors in situ, a novel alternative method has been developed to mimic the environment of real tissues. For the purpose of developing tissue-mimicking models, mucin and mannose were used to modify the surface of gold electrodes. These components are known to contribute to the adhesion of pathogens to epithelial cells in mammals.

Comparative Genomics, Phylogenetic and Functional Analysis of Yersinia enterocolitica, a Gastrointestinal Pathogen, with Other Soil-Borne Bacteria Causing Diseases

Yersinia enterocolitica is a harmful bacterium transmitted through contaminated food, causing gastrointestinal illness and lymph node inflammation. The rise of drug-resistant strains of Y. enterocolitica poses a serious public health threat, necessitating research on its ecology, related species, and unique genes linked to virulence and antibiotic resistance. This study identified eight microorganisms similar to Y. enterocolitica and conducted a pan-genomic analysis, revealing specific genes exclusive to Y. enterocolitica. Enrichment analysis of these genes unveiled their involvement in antibiotic synthesis pathways, such as siderophore production, osmoregulated periplasmic glucan activation, and antibiotic resistance. These pathways, including biofilm formation and increased antibiotic tolerance, are vital for Yersinia’s virulence. Furthermore, specific genes related to glutamate metabolism, nitrogen regulation, motility, purine, and pyrimidine synthesis may contribute to Y. enterocolitica’s pathogenicity, growth, and virulence factor production. Phylogenetic analysis demonstrated the evolutionary relationship between Y. enterocolitica and similar species like Escherichia coli, Campylobacter jejuni, and Salmonella enterica, stressing the need to monitor Y. enterocolitica in slaughterhouses due to animal carriers. The study’s findings shed light on the ecological factors and genetic mechanisms driving Y. enterocolitica’s pathogenicity and antibiotic resistance. Targeting genes involved in purine and pyrimidine synthesis, such as ushA, cpdB, and deoB, could be potential strategies for controlling pathogenicity and antimicrobial resistance. Understanding the relationships and genetic interactions between Y. enterocolitica and related microorganisms is crucial for developing effective surveillance and management approaches in the future.

Repurposing carvacrol, cinnamaldehyde, and eugenol as potential anti-quorum sensing agents against uropathogenic Escherichia coli isolates in Alexandria, Egypt

BackgroundUrinary tract infections represent one of the most frequent hospital and community-acquired infections with uropathogenic Escherichia coli (UPEC) being the main causative agent. The global increase in the emergence of multidrug-resistant (MDR) UPEC necessitates exploring novel approaches. Repurposing natural products as anti-quorum sensing (QS) agents to impede bacterial virulence is gaining momentum nowadays. Hence, this study investigates the anti-QS potentials of carvacrol, cinnamaldehyde, and eugenol against E. coli isolated from urine cultures of Egyptian patients.ResultsAntibiotic susceptibility testing was performed for 67 E. coli isolates and 94% of the isolates showed MDR phenotype. The usp gene was detected using PCR and accordingly, 45% of the isolates were categorized as UPEC. Phytochemicals, at their sub-inhibitory concentrations, inhibited the swimming and twitching motilities of UPEC isolates, with eugenol showing the highest inhibitory effect. The agents hindered the biofilm-forming ability of the tested isolates, at two temperature sets, 37 and 30 °C, where eugenol succeeded in significantly inhibiting the biofilm formation by > 50% at both investigated temperatures, as compared with untreated controls. The phytochemicals were shown to downregulate the expression of the QS gene (luxS) and critical genes related to motility, asserting their anti-QS potential. Further, the combinatory activity of the phytoproducts with five antibiotics was assessed by checkerboard assay. The addition of the phytoproducts significantly reduced the minimum inhibitory concentrations of the antibiotics and generated several synergistic or partially synergistic combinations, some of which have not been previously explored.ConclusionsOverall, carvacrol, cinnamaldehyde, and eugenol could be repurposed as potential anti-QS agents, which preferentially reduce the QS-based communication and attenuate the cascades of gene expression, thus decreasing the production of virulence factors in UPEC, and eventually, subsiding their pathogenicity. Furthermore, the synergistic combinations of these agents with antibiotics might provide a new perspective to circumvent the side effects brought about by high antibiotic doses, thereby paving the way for overcoming antibiotic resistance.

T3SS chaperone of the CesT family is required for secretion of the anti-sigma factor BtrA in Bordetella pertussis

ABSTRACT Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA. In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.

Bactericidal effect of Iberin combined with photodynamic antimicrobial chemotherapy against Pseudomonas aeruginosa biofilm cultured on ex vivo wound model

Wounds infected by Pseudomonas aeruginosa (P. aeruginosa) biofilms are characterized by poor healing and by being long lasting. Pyocyanin and pyoverdine are exotoxins that contribute to P. aeruginosa pathogenicity in wound infections and are known as virulence factors. Despite the usefulness of antimicrobial photodynamic therapy (PDT) in the management of wound infections, biofilms are hurdle for microbial photoinactivation. Quorum sensing (QS) is a cell density-dependent chemical signaling system P. aeruginosa uses to regulate biofilm formation and virulence factors production. In the current study, QS attenuation was used in combination with PDT against P. aeruginosa biofilm cultured on skin explant. Iberin is a QS inhibitor that attenuates P. aeruginosa virulence and affects biofilm integrity. The antibiofilm and QS inhibitory activities of iberin in combination with either riboflavin or 5,10,15,20-Tetrakis(1-methyl-4-pyridinio) porphyrin tetra p-toluenesulfonate (TMP) mediated PDT were investigated using viable count method and pyocyanin and pyoverdine assays, respectively.No bactericidal activity was reported when iberin was added to a mature biofilm (24 h) followed by PDT. When added to a growing biofilm at multiple time points (0 h, 24 h and 48 h), iberin inhibited P. aeruginosa biofilm QS signaling system. This inhibitory effect resulted in an observable decrease in the levels of the QS-regulated virulence factors, pyocyanin and pyoverdine, without any effect on the growth of the biofilm cultures. These changes in biofilm virulence were associated with a decrease in biofilm resistance to PDT and caused bactericidal effect upon photosensitizers treatment and irradiation. Iberin-treated-riboflavin-mediated PDT resulted in a significant 1.3 log reduction in biofilm population. Similarly, iberin-treated-TMP-mediated PDT caused a significant 1.8 log reduction in biofilm population. The combination of QS inhibitor with PDT is a promising alternative antimicrobial therapy for the management of biofilms.

The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans.

V-ATPase, which comprises 13-14 subunits, is essential for pH homeostasis in all eukaryotes, but its proper function requires a regulator to assemble its subunits. While RAVE (regulator of H+-ATPase of vacuolar and endosomal membranes) and Raboconnectin-3 complexes assemble V-ATPase subunits in Saccharomyces cerevisiae and humans, respectively, the function of the RAVE complex in fungal pathogens remains largely unknown. In this study, we identified two RAVE complex components, Rav1 and Wdr1, in the fungal meningitis pathogen Cryptococcus neoformans, and analyzed their roles. Rav1 and Wdr1 are orthologous to yeast RAVE and human Rabconnectin-3 counterparts, respectively, forming the hybrid RAVE (hRAVE) complex. Deletion of RAV1 caused severe defects in growth, cell cycle control, morphogenesis, sexual development, stress responses, and virulence factor production, while the deletion of WDR1 resulted in similar but modest changes, suggesting that Rav1 and Wdr1 play central and accessary roles, respectively. Proteomics analysis confirmed that Wdr1 was one of the Rav1-interacting proteins. Although the hRAVE complex generally has V-ATPase-dependent functions, it also has some V-ATPase-independent roles, suggesting a unique role beyond conventional intracellular pH regulation in C. neoformans. The hRAVE complex played a critical role in the pathogenicity of C. neoformans, and RAV1 deletion attenuated virulence and impaired blood-brain barrier crossing ability. This study provides comprehensive insights into the pathobiological roles of the fungal RAVE complex and suggests a novel therapeutic strategy for controlling cryptococcosis.

Dimethylpolysulfides production as the major mechanism behind wheat fungal pathogen biocontrol, by Arthrobacter and Microbacterium actinomycetes.

Volatile organic compounds (VOCs) produced by plant-associated microorganisms could serve as natural biocontrol compounds. In this work, we investigate the potential of wheat rhizosphere Microbacterium and Arthrobacter actinomycetes to inhibit the growth of major wheat phytopathogenic fungi, Fusarium graminearum and Zymoseptoria tritici via production of antifungal VOCs. A correlative analysis between fungal-growth inhibition versus bacterial volatilomic profiles identified four dimethylpolysulfide (DMPS) VOCs as the main active compounds behind their biocontrol potential. Subsequent inhibition assays reveal that DMTriS (dimethyltrisulfide) exhibits the strongest inhibition effects, then dimethyldisulfide (DMDiS). Further investigation focuses on the mechanisms behind F. graminearum-growth inhibition by the most active strain Microbacterium JM188 in dual culture. Surprisingly, nine interaction-induced VOCs, including two fungal sesquiterpenes, were exclusively detected in dual cultures, suggesting a complex interplay between microbial VOC production and sensing. More importantly, all JM188 VOCs, including antifungal DMPS, were less abundant in dual cultures, suggesting an uptake of bacterial VOCs by the fungus. Quantification of pure DMPS after confrontation with F. graminearum confirmed complete and partial uptake by the fungus of DMTriS and DMDiS VOCs, respectively, suggesting a potential link between the fungal uptake level of bacterial VOCs and their toxicity. Finally, we demonstrated that F. graminearum growth inhibition leads to a complete depletion of DON (deoxynivalenol) carcinogenic mycotoxins, highlighting a significant modulation of fungal metabolism. Collectively, these results pinpoint DMPS as broad-range fungal-inhibiting VOCs produced by rhizosphere Microbacterium and Arthrobacter, and emphasize the extensive VOC-mediated interplay between bacterial biocontrol agents and fungal pathogens.IMPORTANCEAs the management of wheat fungal diseases becomes increasingly challenging, the use of bacterial agents with biocontrol potential against the two major wheat phytopathogens, Fusarium graminearum and Zymoseptoria tritici, may prove to be an interesting alternative to conventional pest management. Here, we have shown that dimethylpolysulfide volatiles are ubiquitously and predominantly produced by wheat-associated Microbacterium and Arthrobacter actinomycetes, displaying antifungal activity against both pathogens. By limiting pathogen growth and DON virulence factor production, the use of such DMPS-producing strains as soil biocontrol inoculants could limit the supply of pathogen inocula in soil and plant residues, providing an attractive alternative to dimethyldisulfide fumigant, which has many non-targeted toxicities. Notably, this study demonstrates the importance of bacterial volatile organic compound uptake by inhibited F. graminearum, providing new insights for the study of volatiles-mediated toxicity mechanisms within bacteria-fungus signaling crosstalk.

Phenotypic and Genotypic Characterization of Recently Isolated Multidrug-Resistant Acinetobacter baumannii Clinical and Aquatic Strains and Demonstration of Silver Nanoparticle Potency.

This study aims to demonstrate the effectiveness of silver nanoparticles (Ag NPs) on multidrug-resistant (MDR) Acinetobacter baumannii (AB) strains isolated from the clinical and aquatic environment. Three types of Ag NPs were investigated for their antimicrobial, antibiofilm, and antivirulence properties on a total number of 132 AB strains isolated in the same temporal sequence from intra-hospital infections (IHIs), wastewater (WW), and surface water (SW) samples between 2019 and 2022 from different Romanian locations and characterized at the phenotypic and genotypic levels. The comparative analysis of the antimicrobial resistance (AR) profiles according to the isolation source and the geographical location demonstrated a decrease in MDR level in AB recovered from WW samples in 2022 from north-eastern/central/southern regions (N-E/C-W/analyzed strains S): 87.5/60/32.5%. The AB strains were lecithinase, caseinase, amylase, and lipase producers, had variable biofilm formation ability, and belonged to six genotypes associated with the presence of different virulence genes (ompA, csuE, bap, and bfmS). The Ag NPs synthesized with the solvothermal method exhibited an inhibitory effect on microbial growth, the adherence capacity to the inert substratum, and on the production of soluble virulence factors. We report here the first description of a powerful antibacterial agent against MDR AB strains circulating between hospitals and anthropically polluted water in Romania.

A putative lipase affects Pseudomonas aeruginosa biofilm matrix production.

Pseudomonas aeruginosa is an opportunistic pathogen that is widely known for infecting patients with underlying conditions. This species often survives antibiotic therapy by forming biofilms, in which the cells produce a protective extracellular matrix. P. aeruginosa also produces virulence factors that enhance its ability to cause disease. One signaling pathway that influences virulence is the nitrogen-related phosphotransferase system (Nitro-PTS), which consists of an initial phosphotransferase, PtsP, a phosphocarrier, PtsO, and a terminal phosphate receptor, PtsN. The physiological role of the Nitro-PTS in P. aeruginosa is poorly understood. However, PtsN, when deprived of its upstream phosphotransfer proteins, has an antagonistic effect on biofilm formation. We thus conducted a transposon mutagenesis screen in an unphosphorylated-PtsN (i.e., ∆ptsP) background to identify downstream proteins with unacknowledged roles in PtsN-mediated biofilm suppression. We found an unstudied gene, PA14_04030, whose disruption restored biofilm production. This gene encodes a predicted phospholipase with signature alpha/beta hydrolase folds and a lipase signature motif with an active-site Ser residue. Hence, we renamed the gene bipL, for biofilm-impacting phospholipase. Deletion of bipL in a ∆ptsP background increased biofilm formation, supporting the idea that BipL is responsible for reducing biofilm formation in strains with unphosphorylated PtsN. Moreover, substituting the putative catalytic Ser for Ala phenocopied bipL deletion, indicating that this residue is important for the biofilm-suppressive activity of BipL in vivo. As our preliminary data suggest that BipL is a lipase, we performed lipidomics to detect changes in the lipid profile due to bipL deletion and found changes in some lipid species. IMPORTANCE Biofilm formation by bacteria occurs when cells secrete an extracellular matrix that holds them together and shields them from environmental insults. Biofilms of bacterial opportunistic human pathogens such as Pseudomonas aeruginosa pose a substantial challenge to clinical antimicrobial therapy. Hence, a more complete knowledge about the bacterial factors that influence and regulate production of the biofilm matrix is one key to formulate more effective therapeutic strategies. In this study, we screen for factors that are important for reducing biofilm matrix production in certain genetic backgrounds. We unexpectedly found a gene encoding a putative lipase enzyme and showed that its predicted catalytic site is important for its ability to reduce biofilm formation. Our findings suggest that lipase enzymes have previously uncharacterized functions in biofilm matrix regulation.

S. aureus virulence factors decrease epithelial barrier function and increase susceptibility to viral infection.

Individuals with atopic dermatitis (AD) are highly colonized by Staphylococcus aureus and are more susceptible to severe viral complications. We hypothesized that S. aureus secreted virulence factors may alter keratinocyte biology to enhance viral susceptibility through disruption of the skin barrier, impaired keratinocyte differentiation, and/or inflammation. To address this hypothesis, human keratinocytes were exposed to conditioned media from multiple S. aureus strains that vary in virulence factor production (USA300, HG003, and RN4220) or select purified virulence factors. We have identified the S. aureus enterotoxin-like superantigen SElQ, as a virulence factor of interest, since it is highly produced by USA300 and was detected on the skin of 53% of AD subjects (n = 72) in a study conducted by our group. Treatment with USA300 conditioned media or purified SElQ resulted in a significant increase in keratinocyte susceptibility to infection with vaccinia virus, and also significantly decreased barrier function. Importantly, we have previously demonstrated that keratinocyte differentiation influences susceptibility to viral infection, and our qPCR observations indicated that USA300 S. aureus and SElQ alter differentiation in keratinocytes. CRISPR/Cas9 was used to knock out CD40, a potential enterotoxin receptor on epithelial cells. We found that CD40 expression on keratinocytes was not completely necessary for SElQ-mediated responses, as measured by proinflammatory cytokine expression and barrier function. Together, these findings support that select S. aureus virulence factors, particularly SElQ, enhance the susceptibility of epidermal cells to viral infection, which may contribute to the increased cutaneous infections observed in individuals with AD. IMPORTANCE Staphylococcus aureus skin colonization and infection are frequently observed in individuals with atopic dermatitis. Many S. aureus strains belong to the clonal group USA300, and these strains produce superantigens including the staphylococcal enterotoxin-like Q (SElQ). Our studies highlight that SElQ may play a key role by altering keratinocyte differentiation and reducing barrier function; collectively, this may explain the AD-specific enhanced infection risk to cutaneous viruses. It is unclear what receptor mediates SElQ's effects on keratinocytes. We have shown that one putative surface receptor, CD40, was not critical for its effects on proinflammatory cytokine production or barrier function.

Indole-3-acetic acid impacts biofilm formation and virulence production of Pseudomonas aeruginosa

Bacterial pathogenesis involves complex mechanisms contributing to virulence and persistence of infections. Understanding the multifactorial nature of bacterial infections is crucial for developing effective interventions. The present study investigated the efficacy of indole-3-acetic acid (IAA) against Pseudomonas aeruginosa with various end points including antibacterial activity, minimum inhibitory concentration (MIC), virulence factor production, biofilm inhibition, bacterial cell detachment, and viability assays. Results showed significant biofilm inhibition, bacterial cell detachment, and modest effects on bacterial viability. Microscopic analysis confirmed the disintegrated biofilm matrix, supporting the inhibitory effect of IAA. Additionally, molecular docking studies revealed potential mechanisms of action through active bond interactions between IAA and virulence proteins. These findings highlight IAA as an effective antibiofilm agent against P. aeruginosa.

Development of Quinazolinone Derivatives as Modulators of Virulence Factors of Pseudomonas aeruginosa Cystic Fibrosis Strains.

Pseudomonas aeruginosa (PA), one of the ESKAPE pathogens, is an opportunistic Gram-negative bacterium responsible for nosocomial infections in humans but also for infections in patients affected by AIDS, cancer, or cystic fibrosis (CF). Treatment of PA infections in CF patients is a global healthcare problem due to the ability of PA to gain antibiotic tolerance through biofilm formation. Anti-virulence compounds represent a promising approach as adjuvant therapy, which could reduce or eliminate the pathogenicity of PA without impacting its growth. Pyocyanin is one of the virulence factors whose production is modulated by the Pseudomonas quinolone signal (PQS) through its receptor PqsR. Different PqsR modulators have been synthesized over the years, highlighting this new powerful therapeutic strategy. Based on the promising structure of quinazolin-4(3H)-one, we developed compounds 7a-d, 8a,b, 9, 10, and 11a-f able to reduce biofilm formation and the production of virulence factors (pyocyanin and pyoverdine) at 50 µM in two PA strains responsible for CF acute and chronic infections. The developed compounds did not reduce the cell viability of IB3-1 bronchial CF cells, and computational studies confirmed the potential ability of novel compounds to act as potential Pqs system modulators.

MSphere of Influence: If virulence is energetically costly, how can it be maintained?

Kimberly Davis works in the field of bacterial pathogenesis and studies heterogeneity in bacterial populations within host tissues. In this mSphere of Influence article, she reflects on how the paper "Stabilization of cooperative virulence by the expression of an avirulent phenotype" by M. Diard et al. (M. Diard et al., Nature 494:353-6, 2013, DOI: 10.1038/nature11913) impacted the way she thinks about bacterial population dynamics and the costs and benefits of producing virulence factors during infection.