Arne Spiessens 218039856
This saprotrophic, Gram-negative bacterium commonly infects the respiratory tract of immunocompromised individuals causing infection.
The size of this rod-shaped (bacillus) bacterium ranges from 0.5-1.0 µm by 1.5-5.0 µm, and produces a blue-green fluorescent Pigment which can be very bright under UV.
ABSTRACT
Pyoverdines are fluorescent siderophores of pseudomonads that play important roles for growth under iron-limiting conditions. The production of pyoverdines by fluorescent pseudomonads permits their colonization of hosts ranging from humans to plants. Prominent examples include pathogenic or non-pathogenic species such as Pseudomonas aeruginosa, P. putida, P. syringae, or P. fluorescens...
The bio-synthesis of the pyoverdine siderophore (PVD) in Pseudomonas aeruginosa involves multiple enzymatic steps including the action of Non-Ribosomal Peptide Synthetases (NRPS). One hallmark of NRPS is their ability to make usage of non-proteinogenic amino-acids synthesised by co-expressed accessory enzymes. It is generally accepted that different enzymes of a secondary metabolic pathway must organise into macro-molecular complexes. However, evidence for complexes like siderosomes in the cellular context are missing.
Here, we used in vitro single-molecule tracking and FRET–FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to explore the spatial partitioning of the ornithine hydroxylase PvdA and its interactions with NRPS. We found PvdA was mostly diffusing bound to large complexes in the cytoplasm with a small exchangeable trapped fraction. FRET-FLIM clearly showed PvdA is physically interacting with PvdJ, PvdI, PvdL and PvdD, the four NRPS of the pyoverdine pathway. The binding modes of PvdA are strikingly different according to the NRPS it is interacting with suggesting that PvdA binding sites have co-evolved with the enzymatic active sites of NRPS.
Our data provide evidence for strongly organised multi-enzymatic complexes responsible for the bio-synthesis of PVD and suggest that finely controlled co-localisation of sequential enzymes seems to be required to promote metabolic efficiency.
Pseudomonas aeruginosa airway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist, P. aeruginosa depends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and the Pseudomonas heme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation of P. aeruginosa to the host environment. Here we investigated the within-host evolution of the transmissible P. aeruginosa DK2 lineage. We found positive selection for promoter mutations leading to increased expression of the phu system. By mimicking conditions of the CF airways in vitro, we experimentally demonstrate that increased expression of phuR confers a growth advantage in the presence of hemoglobin, thus suggesting that P. aeruginosa evolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additional P. aeruginosa lineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages, phuR promoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment of P. aeruginosa infections in CF patients.
Pyoverdine is a substance which is excreted by fluorescent pseudomonads in order to scavenge iron from their environment. Due to specific receptors of the bacterial cell wall, the iron loaded pyoverdine molecules are recognized and transported into the cell. This process can be exploited for developing efficient isolation and enrichment strategies for members of the Pseudomonas genus, which are capable of colonizing various environments and also include human pathogens like P. aeruginosa and the less virulent P. fluorescens. A significant advantage over antibody based systems is the fact that siderophores like pyoverdine can be considered as “immutable ligands,” since the probability for mutations within the siderophore uptake systems of bacteria is very low. While each species of Pseudomonas usually produces structurally unique pyoverdines, which can be utilized only by the producer strain, cross reactivity does occur. In order to achieve a reliable identification of the captured pathogens, further investigations of the isolated cells are necessary. In this proof of concept study, we combine the advantages of an isolation strategy relying on “immutable ligands” with the high specificity and speed of Raman microspectroscopy. In order to isolate the bacterial cells, pyoverdine was immobilized covalently on planar aluminum chip substrates. After capturing, single cell Raman spectra of the isolated species were acquired. Due to the specific spectroscopic fingerprint of each species, the bacteria can be identified. This approach allows a very rapid detection of potential pathogens, since time-consuming culturing steps are unnecessary. We could prove that pyoverdine based isolation of bacteria is fully Raman compatible and further investigated the capability of this approach by isolating and identifying P. aeruginosa and P. fluorescens from tap water samples, which are both opportunistic pathogens and can pose a threat for immunocompromised patients
Pseudomonas aeruginosa is a clinically important pathogen that causes a variety of infections, including urinary, respiratory, and other soft-tissue infections, particularly in hospitalized patients with immune defects, cystic fibrosis, or significant burns. Antimicrobial resistance is a substantial problem in P. aeruginosa treatment due to the inherent insensitivity of the pathogen to a wide variety of antimicrobial drugs and its rapid acquisition of additional resistance mechanisms. One strategy to circumvent this problem is the use of anti-virulent compounds to disrupt pathogenesis without directly compromising bacterial growth. One of the principle regulatory mechanisms for P. aeruginosa’s virulence is the iron-scavenging siderophore pyoverdine, as it governs in-host acquisition of iron, promotes expression of multiple virulence factors, and is directly toxic. Some combination of these activities renders pyoverdine indispensable for pathogenesis in mammalian models. Here we report identification of a panel of novel small molecules that disrupt pyoverdine function. These molecules directly act on pyoverdine, rather than affecting its biosynthesis. The compounds reduce the pathogenic effect of pyoverdine and improve the survival of Caenorhabditis elegans when challenged with P. aeruginosa by disrupting only this single virulence factor. Finally, these compounds can synergize with conventional antimicrobials, forming a more effective treatment. These compounds may help to...
