In vitro and in vivo screening for novel essential cell-envelope proteins in Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa represents a prototype of multi-drug resistant opportunistic pathogens for which novel therapeutic options are urgently required. In order to identify new candidates as potential drug targets, we combined large-scale transposon mutagenesis data analysis and bioinformatics predictions to retrieve a set of putative essential genes which are conserved in P. aeruginosa and predicted to encode cell envelope or secreted proteins. By generating unmarked deletion or conditional mutants, we confirmed the in vitro essentiality of two periplasmic proteins, LptH and LolA, responsible for lipopolysaccharide and lipoproteins transport to the outer membrane respectively, and confirmed that they are important for cell envelope stability. LptH was also found to be essential for P. aeruginosa ability to cause infection in different animal models. Conversely, LolA-depleted cells appeared only partially impaired in pathogenicity, indicating that this protein likely plays a less relevant role during bacterial infection. Finally, we ruled out any involvement of the other six proteins under investigation in P. aeruginosa growth, cell envelope stability and virulence. Besides proposing LptH as a very promising drug target in P. aeruginosa, this study confirms the importance of in vitro and in vivo validation of potential essential genes identified through random transposon mutagenesis

Environmental Burkholderia cenocepacia Strain Enhances Fitness by Serial Passages during Long-Term Chronic Airways Infection in Mice

Burkholderia cenocepacia is an important opportunistic pathogen in cystic fibrosis (CF) patients, and has also been isolated from natural environments. In previous work, we explored the virulence and pathogenic potential of environmental B. cenocepacia strains and demonstrated that they do not differ from clinical strains in some pathogenic traits. Here, we investigated the ability of the environmental B. cenocepacia Mex1 strain, isolated from the maize rhizosphere, to persist and increase its virulence after serial passages in a mouse model of chronic infection. B. cenocepacia Mex1 strain, belonging to the recA lineage IIIA, was embedded in agar beads and challenged into the lung of C57Bl/6 mice. The mice were sacrificed after 28 days from infection and their lungs were tested for bacterial loads. Agar beads containing the pool of B. cenocepacia colonies from the four sequential passages were used to infect the mice. The environmental B. cenocepacia strain showed a low incidence of chronic infection after the first passage; after the second, third and fourth passages in mice, its ability to establish chronic infection increased significantly and progressively up to 100%. Colonial morphology analysis and genetic profiling of the Mex1-derived clones recovered after the fourth passage from infected mice revealed that they were indistinguishable from the challenged strain both at phenotypic and genetic level. By testing the virulence of single clones in the Galleria mellonella infection model, we found that two Mex1-derived clones significantly increased their pathogenicity compared to the parental Mex1 strain and behaved similarly to the clinical and epidemic B. cenocepacia LMG16656(T). Our findings suggest that serial passages of the environmental B. cenocepacia Mex1 strain in mice resulted in an increased ability to determine chronic lung infection and the appearance of clonal variants with increased virulence in non-vertebrate hosts

Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes

Abstract We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine and K + depletion) and of caveolae-mediated uptake (filipin and genistein) on internalization of FITC–poly-l-lysine-labeled DOTAP/DNA lipoplexes and PEI/DNA polyplexes by A549 pneumocytes and HeLa cells and on the transfection efficiencies of these complexes with the luciferase gene. Uptake of the complexes was assayed by fluorescence-activated cell sorting. Lipoplex internalization was inhibited by chlorpromazine and K + depletion but unaffected by filipin and genistein. In contrast, polyplex internalization was inhibited by all four inhibitors. We conclude that lipoplex uptake proceeds only by clathrin-mediated endocytosis, while polyplexes are taken up by two mechanisms, one involving caveolae and the other clathrin-coated pits. Transfection by lipoplexes was entirely abolished by blocking clathrin-mediated endocytosis, whereas inhibition of the caveolae pathway had no effect. By contrast, transfection mediated by polyplexes was completely blocked by genistein and filipin but was unaffected by inhibitors of clathrin-mediated endocytosis. Fluorescence colocalization studies with a lysosomal marker, AlexaFluor–dextran, revealed that polyplexes taken up by clathrin-mediated endocytosis are targeted to the lysosomal compartment for degradation, while the polyplexes internalized via caveolae escape this compartment, permitting efficient transfection

Dampening Host Sensing and Avoiding Recognition in Pseudomonas aeruginosa Pneumonia

Pseudomonas aeruginosa is an opportunistic pathogen and causes a wide range of acute and chronic infections. P. aeruginosa infections are kept in check by an effective immune surveillance in the healthy host, while any imbalance or defect in the normal immune response can manifest in disease. Invasive acute infection in the immunocompromised patients is mediated by potent extracellular and cell bound bacterial virulence factors. Life-threatening chronic infection in cystic fibrosis patients is maintained by pathogenic variants that contribute to evade detection and clearance by the immune system. Here, we reviewed the molecular basis of receptor-mediated recognition of P. aeruginosa and their role in initiating inflammation and the colonization. In addition, the consequence of the P. aeruginosa genetic adaptation for the antibacterial defence and the maintaining of chronic infection are discussed

Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections

Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies

Mapping genetic determinants of host susceptibility to Pseudomonas aeruginosa lung infection in mice.

Background: P. aeruginosa is one of the top three causes of opportunistic human bacterial infections. The remarkable variability in the clinical outcomes of this infection is thought to be associated with genetic predisposition. However, the genes underlying host susceptibility to P. aeruginosa infection are still largely unknown. Results: As a step towards mapping these genes, we applied a genome wide linkage analysis approach to a mouse model. A large F2 intercross population, obtained by mating P. aeruginosa-resistant C3H/HeOuJ, and susceptible A/J mice, was used for quantitative trait locus (QTL) mapping. The F2 progenies were challenged with a P. aeruginosa clinical strain and monitored for the survival time up to 7 days post-infection, as a disease phenotype associated trait. Selected phenotypic extremes of the F2 distribution were genotyped with high-density single nucleotide polymorphic (SNP) markers, and subsequently QTL analysis was performed. A significant locus was mapped on chromosome 6 and was named P. aeruginosa infection resistance locus 1 (Pairl1). The most promising candidate genes, including Dok1, Tacr1, Cd207, Clec4f, Gp9, Gata2, Foxp1, are related to pathogen sensing, neutrophils and macrophages recruitment and inflammatory processes. Conclusions: We propose a set of genes involved in the pathogenesis of P. aeruginosa infection that may be explored to complement human studie

Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Pseudomonas aeruginosa. Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in in vitro and in vivo models of MDR P. aeruginosa infection. Our results show that the in vitro treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR P. aeruginosa in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR P. aeruginosa significantly reduces NF-κB activation and the production of TNF-α, IL-1β, and IL-6, while increasing IL-10 and TGF-β levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR P. aeruginosa. The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF

IL-17A impairs host tolerance during airway chronic infection by Pseudomonas aeruginosa

Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a(-/-) or IL-17ra(-/-) mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-β1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection

Pseudomonas aeruginosa Exploits Lipid A and Muropeptides Modification as a Strategy to Lower Innate Immunity during Cystic Fibrosis Lung Infection

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1β cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF