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The opportunistic bacterial pathogen Pseudomonas aeruginosa has a layered acyl-homoserine lactone (AHL) quorum-sensing (QS) system, which controls production of a variety of extracellular metabolites and enzymes. The LasRI system activates genes including those coding for the extracellular protease elastase and for the second AHL QS system, RhlRI. Growth of P. aeruginosa on casein requires elastase production and LasR-mutant social cheats emerge in populations growing on casein. P. aeruginosa colonizes the lungs of individuals with the genetic disease cystic fibrosis (CF), and LasR mutants can be isolated from the colonized lungs; however, unlike laboratory-generated LasR mutants, many of these CF isolates have functioning RhlR-RhlI systems. We show that one such mutant can use the RhlR-RhlI system to activate expression of elastase and grow on casein. We carried out social-evolution experiments by growing this isolate on caseinate and, as with wild-type P. aeruginosa, elastase-negative mutants emerge as cheats, but these are not RhlR mutants; rather, they are mutants that do not produce the non-AHL Pseudomonas quinolone signal (PQS). Furthermore,we generated a RhlRImutant and showed it had a fitness defect when growing together with the parent. Apparently, RhlR QS and PQS collude to support growth on caseinate in the absence of a functional LasR. Our findings provide a plausible explanation as to why P. aeruginosa LasR mutants, but not RhlR mutants, are common in CF lungs.

Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.

In recent years, the effectiveness of antimicrobials in the treatment of Pseudomonas aeruginosainfections has gradually decreased. This pathogen can be observed in several clinical cases, such as pneumonia, urinary tract infections, sepsis, in immunocompromised hosts, such as neutropenic cancer, burns, and AIDS patients. Furthermore, Pseudomonas aeruginosa causes diseases in both livestock and pets. The highly flexible and versatile genome of P. aeruginosa allows it to have a high rate of pathogenicity. The numerous secreted virulence factors, resulting from its numerous secretion systems, the multi-resistance to different classes of antibiotics, and the ability to produce biofilms are pathogenicity factors that cause numerous problems in the fight against P. aeruginosa infections and that must be better understood for an effective treatment. Infections by P. aeruginosa represent, therefore, a major health problem and, as resistance genes can be disseminated between the microbiotas associated with humans, animals, and the environment, this issue needs be addressed on the basis of an One Health approach. This review intends to bring together and describe in detail the molecular and metabolic pathways in P. aeruginosa’s pathogenesis, to contribute for the development of a more targeted therapy against this pathogen

Pseudomonas aeruginosa is an important opportunistic human pathogen that can establish bacterial communication by synchronizing the behavior of individual cells in a molecular phenomenon known as \"quorum sensing\". Through an elusive mechanism involving gene products of the pqs operon, the PqsE enzyme is absolutely required for the synthesis of extracellular phenazines, including the toxic blue pigment pyocyanin, effectively allowing cells to achieve full-fledged virulence. Despite several functional and structural attempts at deciphering the role of this relevant enzymatic drug target, no molecular function has yet been ascribed to PqsE. In the present study, we report a series of alanine scanning experiments aimed at altering the biological function of PqsE, allowing us to uncover key amino acid positions involved in the molecular function of this enzyme. We use sequence analysis and structural overlays with members of homologous folds to pinpoint critical positions located in the vicinity of the ligand binding cleft and surrounding environment, revealing the importance of a unique C-terminal α-helical motif in the molecular function of PqsE. Our results suggest that the active site of the enzyme involves residues that extend further into the hydrophobic core of the protein, advocating for a lid-like movement of the two terminal helices. This information should help design virtual libraries of PqsE inhibitors, providing means to counter P. aeruginosa virulence acquisition and helping to reduce nosocomial infections.

The main pathology involved in chronic idiopathic urticaria (CIU) is immunological dysfunction which mainly adapts to the immune system of body. Pseudomonas aeruginosa-mannose-sensitive hemagglutinin (PA) is an inactivated Pseudomonas aeruginosa biological product which displays a broad immune regulatory effect. The current study was designed to explore the protective nature of PA as an immune regulator in CIU. The participants were randomly divided into CIU + PA, CIU, control + PA and control group. lg E, anti FcεRI, anti IgE antibody, IL-4, IL-17, TGF-β1 and interferon-γ in the sera were assayed by ELISA. Then CD4 + T cells and CD19 + B cells were isolated from peripheral blood of patients with CIU ( n  = 10) and healthy control ( n  = 10). CD4 + T cells and CD19 + B proliferation and apoptosis were analyzed through CCK-8 and flow cytometry respectively. T helper cells differentiations were assessed by real-time PCR. The results revealed that compared with the control group, the curative effect of CIU + PA group was more effective than that of the CIU control group. There was a hyper proliferation of CD19 + B cells in the CIU patients. Moreover, it was also discovered that presence of Th1 decreased while Th2 cells increased in CIU patients. PA significantly inhibited the proliferation of CD19 + B and Th2 cells but at the same time promoted the proliferation of Th1 compared to healthy control. The conclusion arrived at from this study is that the PA displayed a remarkable regulatory effect in CD4 + T cells and CD19 + B cells function by promoting Th1 but inhibited Th2 and the hyperfunction of B cells of CIU patients.

Abstract Pseudomonas aeruginosa is a Gram-negative bacterium belonging to the γ-proteobacteria. Like other members of the Pseudomonas genus, it is known for its metabolic versatility and its ability to colonize a wide range of ecological niches, such as rhizosphere, water environments and animal hosts, including humans where it can cause severe infections. Another particularity of P. aeruginosa is its high intrinsic resistance to antiseptics and antibiotics, which is partly due to its low outer membrane permeability. In contrast to Enterobacteria, pseudomonads do not possess general diffusion porins in their outer membrane, but rather express specific channel proteins for the uptake of different nutrients. The major outer membrane ‘porin’, OprF, has been extensively investigated, and displays structural, adhesion and signaling functions while its role in the diffusion of nutrients is still under discussion. Other porins include OprB and OprB2 for the diffusion of glucose, the two small outer membrane proteins OprG and OprH, and the two porins involved in phosphate/pyrophosphate uptake, OprP and OprO. The remaining nineteen porins belong to the so-called OprD (Occ) family, which is further split into two subfamilies termed OccD (8 members) and OccK (11 members). In the past years, a large amount of information concerning the structure, function and regulation of these porins has been published, justifying why an updated review is timely. Porins of Pseudomonas aeruginosa play numerous important functions and their expression seems to be highly regulated, reflecting their involvement in the bacterial adaptability to evolving environmental conditions.

Laboratory experiments have uncovered many basic aspects of bacterial physiology and behavior. After the past century of mostly in vitro experiments, we now have detailed knowledge of bacterial behavior in standard laboratory conditions, but only a superficial understanding of bacterial functions and behaviors during human infection. It is well-known that the growth and behavior of bacteria are largely dictated by their environment, but how bacterial physiology differs in laboratory models compared with human infections is not known. To address this question, we compared the transcriptome of Pseudomonas aeruginosa during human infection to that of P. aeruginosa in a variety of laboratory conditions. Several pathways, including the bacterium’s primary quorum sensing system, had significantly lower expression in human infections than in many laboratory conditions. On the other hand, multiple genes known to confer antibiotic resistance had substantially higher expression in human infection than in laboratory conditions, potentially explaining why antibiotic resistance assays in the clinical laboratory frequently underestimate resistance in patients. Using a standard machine learning technique known as support vector machines, we identified a set of genes whose expression reliably distinguished in vitro conditions from human infections. Finally, we used these support vector machines with binary classification to force P. aeruginosa mouse infection transcriptomes to be classified as human or in vitro. Determining what differentiates our current models from clinical infections is important to better understand bacterial infections and will be necessary to create model systems that more accurately capture the biology of infection.

Polyurethane diol (PUR-diol), a synthetic polymer, is widely used as a modifier for water-soluble resins and emulsions in wood appliances and auto coatings. Non-biodegradability of polyurethanes (PUR) and PUR-based materials poses a threat to environment that has led scientists to isolate microbes capable of degrading PUR. However, the bio-degradation of PUR-diol has not yet been reported. In this study, we report isolation of a soil bacterium that can survive using PUR-diol as sole carbon source. PUR-diol degradation by the organism was confirmed by thin layer chromatographic analysis of the conditioned medium obtained after the growth wherein a significant reduction of PUR-diol was observed compared to non-inoculated medium. To quantify the PUR-diol degradation, a sensitive assay based on High Performance Thin Layer Chromatography has been developed that showed 32% degradation of PUR-diol by the organism in 10 days. Degradation kinetics showed the maximal depletion of PUR-diol during logarithmic growth of the organism indicating a direct relation between the growth and PUR-diol degradation. Mutagenic study and GC-MS analysis revealed that esterase activity is involved in this degradation event. The ribotyping and metabolic fingerprinting analysis showed that this organism is a strain of Pseudomonous aeruginosa (P. aeruginosa). It has also been observed that this strain is able to degrade Impranil DLN™, a variety of commercially available PUR. Therefore this study identifies a new bacterium from soil that has the potential to reduce PUR-related waste burden and adds a new facet to diverse functional activities of P. aeruginosa.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa , which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes. Antibiotics are life-saving medicines, but many bacteria now have the ability to resist their effects. For some infections, all frontline antibiotics are now ineffective. To treat infections caused by these highly resistant bacteria, clinicians must use so-called ‘antibiotics of last resort’. These antibiotics include a drug called colistin, which is moderately effective, but often fails to eradicate the infection. One of the challenges to making colistin more effective is that its mechanism is poorly understood. Bacteria have two layers of protection against the outside world: an outer cell membrane and an inner cell membrane. To kill them, colistin must punch holes in both. First, it disrupts the outer membrane by interacting with molecules called lipopolysaccharides. But how it disrupts the inner membrane was unclear. Bacteria have evolved several different mechanisms that make them resistant to the effects of colistin. Sabnis et al. reasoned that understanding how these mechanisms protected bacteria could reveal how the antibiotic works to damage the inner cell membrane. Sabnis et al. examined the effects of colistin on Escherichia coli bacteria with and without resistance to the antibiotic. Exposing these bacteria to colistin revealed that the antibiotic damages both layers of the cell surface in the same way, targeting lipopolysaccharide in the inner membrane as well as the outer membrane. Next, Sabnis et al. used this new information to make colistin work better. They found that the effects of colistin were magnified when it was combined with the experimental antibiotic murepavadin, which caused lipopolysaccharide to build up at the inner membrane. This allowed colistin to punch more holes through the inner membrane, making colistin more effective at killing bacteria. To find out whether this combination of colistin and murepavadin could work as a clinical treatment, Sabnis et al. tested it on mice with Pseudomonas aeruginosa infections in their lungs. Colistin was much better at killing Pseudomonas aeruginosa and treating infections when combined with murepavadin than it was on its own. Pseudomonas aeruginosa bacteria can cause infections in the lungs of people with cystic fibrosis. At the moment, patients receive colistin in an inhaled form to treat these infections, but it is not always successful. The second drug used in this study, murepavadin, is about to enter clinical trials as an inhaled treatment for lung infections too. If the trial is successful, it may be possible to use both drugs in combination to treat lung infections in people with cystic fibrosis.

Exopolysaccharides and extracellular DNA are important structural components that contribute to the self-assembly of large aggregates or microcolonies that are characteristic of biofilms. Pseudomonas aeruginosa is capable of producing multiple exopolysaccharides, including alginate, Psl, and Pel. At present, little is known about Pel’s chemical structure and its role in microcolony formation. Our results demonstrate that Pel is composed of cationic amino sugars. Using this knowledge, we have developed a Pel-specific lectin stain to directly visualize Pel in biofilms. We show that the positive charge on Pel facilitates its binding to extracellular DNA in the biofilm stalk, and that Pel can compensate for lack of Psl in the biofilm periphery. Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa , Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N -acetylgalactosamine and N -acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.