Anti-Biofilm Extracts and Molecules from the Marine Environment.

Pathogenic bacteria and their biofilms are involved in many diseases and represent a major public health problem, including the development of antibiotic resistance. These biofilms are known to cause chronic infections for which conventional antibiotic treatments are often ineffective. The search for new molecules and innovative solutions to combat these pathogens and their biofilms has therefore become an urgent need. The use of molecules with anti-biofilm activity would be a potential solution to these problems. The marine world is rich in micro- and macro-organisms capable of producing secondary metabolites with original skeletons. An interest in the chemical strategies used by some of these organisms to regulate and/or protect themselves against pathogenic bacteria and their biofilms could lead to the development of bioinspired, eco-responsible solutions. Through this original review, we listed and sorted the various molecules and extracts from marine organisms that have been described in the literature as having strictly anti-biofilm activity, without bactericidal activity.

Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system.

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.

Pseudoalteromonas ostreae sp. nov., a new bacterial species harboured by the flat oyster Ostrea edulis.

Three bacterial strains, named hOe-66T, hOe-124 and hOe-125, were isolated from the haemolymph of different specimens of the flat oyster Ostrea edulis collected in Concarneau bay (Finistère, France). These strains were characterized by a polyphasic approach, including (i) whole genome analyses with 16S rRNA gene sequence alignment and pangenome analysis, determination of the G+C content, average nucleotide identity (ANI), and in silico DNA-DNA hybridization (isDDH), and (ii) fatty acid methyl ester and other phenotypic analyses. Strains hOe-66T, hOe-124 and hOe-125 were closely related to both type strains Pseudoalteromonas rhizosphaerae RA15T and Pseudoalteromonas neustonica PAMC 28425T with less than 93.3% ANI and 52.3% isDDH values. Regarding their phenotypic traits, the three strains were Gram-negative, 1-2 µm rod-shaped, aerobic, motile and non-spore-forming bacteria. Cells grew optimally at 25 °C in 2.5% NaCl and at 7-8 pH. The most abundant fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0 and C17:1 ω8c. The strains carried a genome average size of 4.64 Mb and a G+C content of 40.28 mol%. The genetic and phenotypic results suggested that strains hOe-66T, hOe-124 and hOe-125 belong to a new species of the genus Pseudoalteromonas. In this context, we propose the name Pseudoalteromonas ostreae sp. nov. The type strain is hOe-66T (=CECT 30303T=CIP 111911T).

Antibiofilm activity in the culture supernatant of a marine Pseudomonas sp. bacterium.

In the marine environment, most solid surfaces are covered by microbial biofilms, mainly composed of bacteria and diatoms. The negative effects of biofilms on materials and equipment are numerous and pose a major problem for industry and human activities. Since marine micro-organisms are an important source of bioactive metabolites, it is possible that they synthesize natural ecofriendly molecules that inhibit the adhesion of organisms. In this work, the antibiofilm potential of marine bacteria was investigated using Flavobacterium sp. II2003 as a target. This strain is potentially a pioneer strain of bacteria that was previously selected from marine biofilms for its strong biofilm-forming ability. The culture supernatants of 86 marine heterotrophic bacteria were tested for their ability to inhibit Flavobacterium sp. II2003 biofilm formation and the Pseudomonas sp. IV2006 strain was identified as producing a strong antibiofilm activity. The Pseudomonas sp. IV2006 culture supernatant (SNIV2006) inhibited Flavobacterium sp. II2003 adhesion without killing the bacteria or inhibiting its growth. Moreover, SNIV2006 had no effect on the Flavobacterium sp. II2003 cell surface hydrophilic/hydrophobic and general Lewis acid-base characteristics, but modified the surface properties of glass, making it on the whole more hydrophilic and more alkaline and significantly reducing bacterial cell adhesion. The glass-coating molecules produced by Pseudomonas sp. IV2006 were found to probably be polysaccharides, whereas the antibiofilm molecules contained in SNIV2006 and acting during the 2 h adhesion step on glass and polystyrene surfaces would be proteinaceous. Finally, SNIV2006 exhibited a broad spectrum of antibiofilm activity on other marine bacteria such as Flavobacterium species that are pathogenic for fish, and human pathogens in both the medical environment, such as Staphylococcus aureus and Pseudomonas aeruginosa, and in the food industry, such as Yersinia enterocolitica. Thus, a wide range of applications could be envisaged for the SNIV2006 compounds, both in aquaculture and human health.

Alterocin, an Antibiofilm Protein Secreted by Pseudoalteromonas sp. Strain 3J6.

We sought to identify and study the antibiofilm protein secreted by the marine bacterium Pseudoalteromonas sp. strain 3J6. The latter is active against marine and terrestrial bacteria, including Pseudomonas aeruginosa clinical strains forming different biofilm types. Several amino acid sequences were obtained from the partially purified antibiofilm protein, named alterocin. The Pseudoalteromonas sp. 3J6 genome was sequenced, and a candidate alt gene was identified by comparing the genome-encoded proteins to the sequences from purified alterocin. Expressing the alt gene in another nonactive Pseudoalteromonas sp. strain, 3J3, demonstrated that it is responsible for the antibiofilm activity. Alterocin is a 139-residue protein that includes a predicted 20-residue signal sequence, which would be cleaved off upon export by the general secretion system. No sequence homology was found between alterocin and proteins of known functions. The alt gene is not part of an operon and adjacent genes do not seem related to alterocin production, immunity, or regulation, suggesting that these functions are not fulfilled by devoted proteins. During growth in liquid medium, the alt mRNA level peaked during the stationary phase. A single promoter was experimentally identified, and several inverted repeats could be binding sites for regulators. alt genes were found in about 30% of the Pseudoalteromonas genomes and in only a few instances of other marine bacteria of the Hahella and Paraglaciecola genera. Comparative genomics yielded the hypothesis that alt gene losses occurred within the Pseudoalteromonas genus. Overall, alterocin is a novel kind of antibiofilm protein of ecological and biotechnological interest.IMPORTANCE Biofilms are microbial communities that develop on solid surfaces or interfaces and are detrimental in a number of fields, including for example food industry, aquaculture, and medicine. In the latter, antibiotics are insufficient to clear biofilm infections, leading to chronic infections such as in the case of infection by Pseudomonas aeruginosa of the lungs of cystic fibrosis patients. Antibiofilm molecules are thus urgently needed to be used in conjunction with conventional antibiotics, as well as in other fields of application, especially if they are environmentally friendly molecules. Here, we describe alterocin, a novel antibiofilm protein secreted by a marine bacterium belonging to the Pseudoalteromonas genus, and its gene. Alterocin homologs were found in about 30% of Pseudoalteromonas strains, indicating that this new family of antibiofilm proteins likely plays an important albeit nonessential function in the biology of these bacteria. This study opens up the possibility of a variety of applications.

Complete genome sequence of the halophilic PHA-producing bacterium Halomonas sp. SF2003: insights into its biotechnological potential.

A halophilic Gram-negative eubacterium was isolated from the Iroise Sea and identified as an efficient producer of polyhydroxyalkanoates (PHA). The strain, designated SF2003, was found to belong to the Halomonas genus on the basis of 16S rRNA gene sequence similarity. Previous biochemical tests indicated that the Halomonas sp. strain SF2003 is capable of supporting various culture conditions which sometimes can be constraining for marine strains. This versatility could be of great interest for biotechnological applications. Therefore, a complete bacterial genome sequencing and de novo assembly were performed using a PacBio RSII sequencer and Hierarchical Genome Assembly Process software in order to predict Halomonas sp. SF2003 metabolisms, and to identify genes involved in PHA production and stress tolerance. This study demonstrates the complete genome sequence of Halomonas sp. SF2003 which contains a circular 4,36 Mbp chromosome, and replaces the strain in a phylogenetic tree. Genes related to PHA metabolism, carbohydrate metabolism, fatty acid metabolism and stress tolerance were identified and a comparison was made with metabolisms of relative species. Genes annotation highlighted the presence of typical genes involved in PHA biosynthesis such as phaA, phaB and phaC and enabled a preliminary analysis of their organization and characteristics. Several genes of carbohydrates and fatty acid metabolisms were also identified which provided helpful insights into both a better knowledge of the intricacies of PHA biosynthetic pathways and of production purposes. Results show the strong versatility of Halomonas sp. SF2003 to adapt to various temperatures and salinity which can subsequently be exploited for industrial applications such as PHA production.

Anti-Bacterial Adhesion Activity of Tropical Microalgae Extracts.

The evolution of regulations concerning biocidal products aimed towards an increased protection of the environment (e.g., EU Regulation No 528/2012) requires the development of new non-toxic anti-fouling (AF) systems. As the marine environment is an important source of inspiration, such AF systems inhibiting the adhesion of organisms without any toxicity could be based on molecules of natural origin. In this context, the antibiofilm potential of tropical microalgal extracts was investigated. The tropics are particularly interesting in terms of solar energy and temperatures which provide a wide marine diversity and a high production of microalgae. Twenty microalgal strains isolated from the Indian Ocean were studied. Their extracts were characterized in terms of global chemical composition by high resolution magic angle spinning (HR-MAS) and nuclear magnetic resonance (NMR) spectroscopy, toxicity against marine bacteria (viability and growth) and anti-adhesion effect. The different observations made by confocal laser scanning microscopy (CLSM) showed a significant activity of three extracts from Dinoflagellate strains against the settlement of selected marine bacteria without any toxicity at a concentration of 50 µg/mL. The Symbiodinium sp. (P-78) extract inhibited the adhesion of Bacillus sp. 4J6 (Atlantic Ocean), Shewanella sp. MVV1 (Indian Ocean) and Pseudoalteromonas lipolytica TC8 (Mediterranean Ocean) at 60, 76 and 52%, respectively. These results underlined the potential of using microalgal extracts to repel fouling organisms.

Marine bacteria from the French Atlantic coast displaying high forming-biofilm abilities and different biofilm 3D architectures.

BACKGROUND: Few studies have reported the species composition of bacterial communities in marine biofilms formed on natural or on man-made existing structures. In particular, the roles and surface specificities of primary colonizers are largely unknown for most surface types. The aim of this study was to obtain potentially pioneering bacterial strains with high forming-biofilm abilities from two kinds of marine biofilms, collected from two different surfaces of the French Atlantic coast: an intertidal mudflat which plays a central role in aquaculture and a carbon steel structure of a harbour, where biofilms may cause important damages. RESULTS: A collection of 156 marine heterotrophic aerobic bacteria isolated from both biofilms was screened for their ability to form biofilms on polystyrene 96-well microtiter plates. Out of 25 strains able to build a biofilm in these conditions, only four bacteria also formed a thick and stable biofilm on glass surfaces under dynamic conditions. These strains developed biofilms with four different three-dimensional architectures when observed by confocal laser scanning microscopy: Flavobacterium sp. II2003 biofilms harboured mushroom-like structures, Roseobacter sp. IV3009 biofilms were quite homogeneous, Shewanella sp. IV3014 displayed hairy biofilms with horizontal fibres, whereas Roseovarius sp. VA014 developed heterogeneous and tousled biofilms. CONCLUSIONS: This work led for the first time to the obtaining of four marine bacterial strains, potentially pioneering bacteria in marine biofilms, able to adhere to at least two different surfaces (polystyrene and glass) and to build specific 3D biofilms. The four selected strains are appropriate models for a better understanding of the colonization of a surface as well as the interactions that can occur between bacteria in a marine biofilm, which are crucial events for the initiation of biofouling.

The Pseudomonas aeruginosa rhlG and rhlAB genes are inversely regulated and RhlG is not required for rhamnolipid synthesis.

BACKGROUND: Pseudomonas aeruginosa produces rhamnolipid biosurfactants involved in numerous phenomena including virulence. The transcriptional study of the rhlAB operon encoding two key enzymes for rhamnolipid synthesis led to the discovery of the quorum sensing system RhlRI. The latter positively controls the transcription of rhlAB, as well as of rhlC, which is required for di-rhamnolipid synthesis. The rhlG gene encodes an NADPH-dependent ß-ketoacyl reductase. Although it was reported to be required for the biosynthesis of the fatty acid part of rhamnolipids, its function in rhamnolipid synthesis was later questioned. The rhlG transcription and its role in rhamnolipid production were investigated here. RESULTS: Using 5'-RACE PCR, a luxCDABE-based transcriptional fusion, and quantitative reverse transcription-PCR, we confirmed two previously identified σ70- and σ54-dependent promoters and we identified a third promoter recognized by the extra-cytoplasmic function sigma factor AlgU. rhlG was inversely regulated compared to rhlAB and rhlC: the rhlG transcription was down-regulated in response to N-butyryl-l-homoserine lactone, the communication molecule of the RhlRI system, and was induced by hyperosmotic stress in an AlgU-dependent manner. Consistently with this transcriptional pattern, the single or double deletions of rhlG and PA3388, which forms an operon with rhlG, did not dramatically impair rhamnolipid synthesis. CONCLUSION: This first detailed study of rhlG transcription reveals a complex regulation involving three sigma factors and N-butyryl-l-homoserine lactone. We furthermore present evidences that RhlG does not play a key role in rhamnolipid synthesis.

Genetic Insights into Biofilm Formation by a Pathogenic Strain of Vibrio harveyi.

The Vibrio genus includes bacteria widely distributed in aquatic habitats and the infections caused by these bacteria can affect a wide range of hosts. They are able to adhere to numerous surfaces, which can result in biofilm formation that helps maintain them in the environment. The involvement of the biofilm lifestyle in the virulence of Vibrio pathogens of aquatic organisms remains to be investigated. Vibrio harveyi ORM4 is a pathogen responsible for an outbreak in European abalone Haliotis tuberculata populations. In the present study, we used a dynamic biofilm culture technique coupled with laser scanning microscopy to characterize the biofilm formed by V. harveyi ORM4. We furthermore used RNA-seq analysis to examine the global changes in gene expression in biofilm cells compared to planktonic bacteria, and to identify biofilm- and virulence-related genes showing altered expression. A total of 1565 genes were differentially expressed, including genes associated with motility, polysaccharide synthesis, and quorum sensing. The up-regulation of 18 genes associated with the synthesis of the type III secretion system suggests that this virulence factor is induced in V. harveyi ORM4 biofilms, providing indirect evidence of a relationship between biofilm and virulence.

Antibiofilm Activity of the Marine Probiotic Bacillus subtilis C3 Against the Aquaculture-Relevant Pathogen Vibrio harveyi.

V. harveyi is a well-known pathogen-inducing vibriosis, especially for shrimp, fish, and invertebrates. Its virulence is related to biofilm formation and this negatively impacts the aquaculture industry. Therapeutic strategies such as the utilization of probiotic bacteria may slow down Vibrio infections. In this study, we investigated the potential antibiofilm activity of the probiotic Bacillus subtilis C3 for aquaculture. First, B. subtilis C3 biofilm was characterized by confocal laser scanning microscopy (CLSM) before testing its bioactivities. We demonstrated antibiofilm activity of B. subtilis C3 culture supernatant, which is mainly composed-among other molecules-of lipopeptidic surfactants belonging to the surfactin family as identified by ultra-high-performance liquid chromatography (UHPLC)-MS/MS. Their antibiofilm activity was confirmed on V. harveyi ORM4 (pFD086) biofilm by CLSM. These findings suggest that the marine probiotic B. subtilis C3 might inhibit or reduce Vibrio colonization and thus decrease the associated animal mortalities.

Extracts from Wallis Sponges Inhibit Vibrio harveyi Biofilm Formation.

Pathogenic bacteria and their biofilms are involved in many human and animal diseases and are a major public health problem with, among other things, the development of antibiotic resistance. These biofilms are known to induce chronic infections for which classical treatments using antibiotic therapy are often ineffective. Sponges are sessile filter-feeding marine organisms known for their dynamic symbiotic partnerships with diverse microorganisms and their production of numerous metabolites of interest. In this study, we investigated the antibiofilm efficacy of different extracts from sponges, isolated in Wallis, without biocidal activity. Out of the 47 tested extracts, from 28 different genera, 11 showed a strong activity against Vibrio harveyi biofilm formation. Moreover, one of these extracts also inhibited two quorum-sensing pathways of V. harveyi.

Sensitivity of Legionella pneumophila to phthalates and their substitutes.

Phthalates constitute a family of anthropogenic chemicals developed to be used in the manufacture of plastics, solvents, and personal care products. Their dispersion and accumulation in many environments can occur at all stages of their use (from synthesis to recycling). However, many phthalates together with other accumulated engineered chemicals have been shown to interfere with hormone activities. These compounds are also in close contact with microorganisms that are free-living, in biofilms or in microbiota, within multicellular organisms. Herein, the activity of several phthalates and their substitutes were investigated on the opportunistic pathogen Legionella pneumophila, an aquatic microbe that can infect humans. Beside showing the toxicity of some phthalates, data suggested that Acetyl tributyl citrate (ATBC) and DBP (Di-n-butyl phthalate) at environmental doses (i.e. 10-6 M and 10-8 M) can modulate Legionella behavior in terms of motility, biofilm formation and response to antibiotics. A dose of 10-6 M mostly induced adverse effects for the bacteria, in contrast to a dose of 10-8 M. No perturbation of virulence towards Acanthamoeba castellanii was recorded. These behavioral alterations suggest that L. pneumophila is able to sense ATBC and DBP, in a cross-talk that either mimics the response to a native ligand, or dysregulates its physiology.

Transcription of the oprF gene of Pseudomonas aeruginosa is dependent mainly on the SigX sigma factor and is sucrose induced.

The OprF porin is the major outer membrane protein of Pseudomonas aeruginosa. OprF is involved in several crucial functions, including cell structure, outer membrane permeability, environmental sensing, and virulence. The oprF gene is preceded by the sigX gene, which encodes the poorly studied extracytoplasmic function (ECF) sigma factor SigX. Three oprF promoters were previously identified. Two intertwined promoters dependent on σ(70) and SigX are located in the sigX-oprF intergenic region, whereas a promoter dependent on the ECF AlgU lies within the sigX gene. An additional promoter was found in the cmpX-sigX intergenic region. In this study, we dissected the contribution of each promoter region and of each sigma factor to oprF transcription using transcriptional fusions. In Luria-Bertani (LB) medium, the oprF-proximal region (sigX-oprF intergenic region) accounted for about 80% of the oprF transcription, whereas the AlgU-dependent promoter had marginal activity. Using the sigX mutant PAOSX, we observed that the SigX-dependent promoter was largely predominant over the σ(70)-dependent promoter. oprF transcription was increased in response to low NaCl or high sucrose concentrations, and this induced transcription was strongly impaired in the absence of SigX. The lack of OprF itself increased oprF transcription. Since these conditions led to cell wall alterations, oprF transcription could be activated by signals triggered by perturbation of the cell envelope.

Proteomic studies highlight outer-membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41.

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer-membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer-membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB-dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT-PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.

Influence of exudates of the kelp Laminaria digitata on biofilm formation of associated and exogenous bacterial epiphytes.

Wild populations of brown marine algae (Phaeophyta) provide extensive surfaces to bacteria and epiphytic eukaryotes for colonization. On one hand, various strategies allow kelps prevent frond surface fouling which would retard growth by reducing photosynthesis and increasing pathogenesis. On the other hand, production and release of organic exudates of high energy value, sometimes in association with more or less selective control of settlement of epiphytic strains, allow bacteria to establish surface consortia not leading to macrofouling. Here, we present the analysis of adhesion and biofilm formation of bacterial isolates from the kelp Laminaria digitata and of characterized and referenced marine isolates. When they were grown in flow cell under standard nutrient regimes, all used bacteria, except one, were able to adhere on glass and then develop as biofilms, with different architecture. Then, we evaluated the effect of extracts from undisturbed young Laminaria thalli and from young thalli subjected to oxidative stress elicitation; this latter condition induced the production of defense molecules. We observed increasing or decreasing adhesion depending on the referenced strains, but no effects were observed against strains isolated from L. digitata. Such effects were less observed on biofilms. Our results suggested that L. digitata is able to modulate its bacterial colonization. Finally, mannitol, a regular surface active component of Laminaria exudates was tested individually, and it showed a pronounced increased on one biofilm strain. Results of these experiments are original and can be usefully linked to what we already know on the oxidative halogen metabolism peculiar to Laminaria. Hopefully, we will be able to understand more about the unique relationship that bacteria have been sharing with Laminaria for an estimated one billion years.

Alterins, a new family of marine antibacterial cyclolipopeptides.

Five strains of Pseudoalteromonas, isolated from oyster haemolymph, have exhibited antibacterial activity against several Gram-negative bacteria. Bioactive compounds have been identified in their cell-free supernatant and characterised as alterins, which are cyclolipopeptides comprising a heptapeptidic ring connected to a fatty acid chain. Using ultra-performance liquid chromatography-high-resolution mass spectrometry, this paper describes 37 structural analogues differing from each other by one or more amino acid residue, the length of the fatty acid chain, its hydroxylation and the presence of unsaturation.

Effect of Phthalates and Their Substitutes on the Physiology of Pseudomonas aeruginosa.

Phthalates are used in a variety of applications-for example, as plasticizers in polyvinylchloride products to improve their flexibility-and can be easily released into the environment. In addition to being major persistent organic environmental pollutants, some phthalates are responsible for the carcinogenicity, teratogenicity, and endocrine disruption that are notably affecting steroidogenesis in mammals. Numerous studies have thus focused on deciphering their effects on mammals and eukaryotic cells. While multicellular organisms such as humans are known to display various microbiota, including all of the microorganisms that may be commensal, symbiotic, or pathogenic, few studies have aimed at investigating the relationships between phthalates and bacteria, notably regarding their effects on opportunistic pathogens and the severity of the associated pathologies. Herein, the effects of phthalates and their substitutes were investigated on the human pathogen, Pseudomonas aeruginosa, in terms of physiology, virulence, susceptibility to antibiotics, and ability to form biofilms. We show in particular that most of these compounds increased biofilm formation, while some of them enhanced the bacterial membrane fluidity and altered the bacterial morphology.

Full virulence of Pseudomonas aeruginosa requires OprF.

OprF is a general outer membrane porin of Pseudomonas aeruginosa, a well-known human opportunistic pathogen associated with severe hospital-acquired sepsis and chronic lung infections of cystic fibrosis patients. A multiphenotypic approach, based on the comparative study of a wild-type strain of P. aeruginosa, its isogenic oprF mutant, and an oprF-complemented strain, showed that OprF is required for P. aeruginosa virulence. The absence of OprF results in impaired adhesion to animal cells, secretion of ExoT and ExoS toxins through the type III secretion system (T3SS), and production of the quorum-sensing-dependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. Accordingly, in the oprF mutant, production of the signal molecules N-(3-oxododecanoyl)-l-homoserine lactone and N-butanoyl-l-homoserine lactone was found to be reduced and delayed, respectively. Pseudomonas quinolone signal (PQS) production was decreased, while its precursor, 4-hydroxy-2-heptylquinoline (HHQ), accumulated in the cells. Taken together, these results show the involvement of OprF in P. aeruginosa virulence, at least partly through modulation of the quorum-sensing network. This is the first study showing a link between OprF, PQS synthesis, T3SS, and virulence factor production, providing novel insights into virulence expression.