Surface Attachment Induces Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa infects every type of host that has been examined by deploying multiple virulence factors. Previous studies of virulence regulation have largely focused on chemical cues, but P. aeruginosa may also respond to mechanical cues. Using a rapid imaging-based virulence assay, we demonstrate that P. aeruginosa activates virulence in response to attachment to a range of chemically distinct surfaces, suggesting that this bacterial species responds to mechanical properties of its substrates. Surface-activated virulence requires quorum sensing, but activating quorum sensing does not induce virulence without surface attachment. The activation of virulence by surfaces also requires the surface-exposed protein PilY1, which has a domain homologous to a eukaryotic mechanosensor. Specific mutation of the putative PilY1 mechanosensory domain is sufficient to induce virulence in non-surface-attached cells, suggesting that PilY1 mediates surface mechanotransduction. Triggering virulence only when cells are both at high density and attached to a surface—two host-nonspecific cues—explains how P. aeruginosa precisely regulates virulence while maintaining broad host specificity

NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target.

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target

NirA is an alternative nitrite reductase from Pseudomonas aeruginosa with potential as an antivirulence target

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA. IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target

Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione- Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target

negative pathogen Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione) had anti-P. aeruginosa action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using in silico approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) lasB gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of Galleria mellonella larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag+ and Cu2+ complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the in vitro cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide, with Cu- phendione having the best inhibitory action (Ki = 90 nM). Treating living bacteria with a sub-inhibitory concentration (1/2 × MIC value) of the test compounds caused a significant reduction in the expression of the lasB gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective rg 1 August 2019 | Volume 10 | Article 1701 1,10-Phenanthroline-5,6-Dione-Based Compounds: As Anti-Virulence Drugs action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the in vivo model, increasing the survival time of G. mellonella larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against P. aeruginosa

NrsZ: a novel, processed, nitrogen-dependent, small non-coding RNA that regulates Pseudomonas aeruginosa PAO1 virulence.

The opportunistic pathogen Pseudomonas aeruginosa PAO1 has a remarkable capacity to adapt to various environments and to survive with limited nutrients. Here, we report the discovery and characterization of a novel small non-coding RNA: NrsZ (nitrogen-regulated sRNA). We show that under nitrogen limitation, NrsZ is induced by the NtrB/C two component system, an important regulator of nitrogen assimilation and P. aeruginosa's swarming motility, in concert with the alternative sigma factor RpoN. Furthermore, we demonstrate that NrsZ modulates P. aeruginosa motility by controlling the production of rhamnolipid surfactants, virulence factors notably needed for swarming motility. This regulation takes place through the post-transcriptional control of rhlA, a gene essential for rhamnolipids synthesis. Interestingly, we also observed that NrsZ is processed in three similar short modules, and that the first short module encompassing the first 60 nucleotides is sufficient for NrsZ regulatory functions

PvdQ Quorum Quenching Acylase Attenuates Pseudomonas aeruginosa Virulence in a Mouse Model of Pulmonary Infection

Pseudomonas aeruginosa is the predominant pathogen in pulmonary infections associated with cystic fibrosis. Quorum sensing (QS) systems regulate the production of virulence factors and play an important role in the establishment of successful P. aeruginosa infections. Inhibition of the QS system (termed quorum quenching) renders the bacteria avirulent thus serving as an alternative approach in the development of novel antibiotics. Quorum quenching in Gram negative bacteria can be achieved by preventing the accumulation of N-acyl homoserine lactone (AHL) signaling molecule via enzymatic degradation. Previous work by us has shown that PvdQ acylase hydrolyzes AHL signaling molecules irreversibly, thereby inhibiting QS in P. aeruginosa in vitro and in a Caenorhabditis elegans model of P. aeruginosa infection. The aim of the present study is to assess the therapeutic efficacy of intranasally instilled PvdQ acylase in a mouse model of pulmonary P. aeruginosa infection. First, we evaluated the deposition pattern of intranasally administered fluorochrome-tagged PvdQ (PvdQ-VT) in mice at different stages of pulmonary infection by in vivo imaging studies. Following intranasal instillation, PvdQ-VT could be traced in all lung lobes with 42 ± 7.5% of the delivered dose being deposited at 0 h post-bacterial-infection, and 34 ± 5.2% at 72 h post bacterial-infection. We then treated mice with PvdQ during lethal P. aeruginosa pulmonary infection and that resulted in a 5-fold reduction of lung bacterial load and a prolonged survival of the infected animals with the median survival time of 57 hin comparison to 42 h for the PBS-treated group. In a sublethal P. aeruginosa pulmonary infection, PvdQ treatment resulted in less lung inflammation as well as decrease of CXCL2 and TNF-α levels at 24 h post-bacterial-infection by 15 and 20%, respectively. In conclusion, our study has shown therapeutic efficacy of PvdQ acylase as a quorum quenching agent during P. aeruginosa infection