A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity

The opportunistic human pathogen, Pseudomonas aeruginosa, is a major cause of infections in chronic wounds, burns and the lungs of cystic fibrosis patients. The P. aeruginosa genome encodes at least three proteins exhibiting the characteristic three domain structure of autotransporters, but much remains to be understood about the functions of these three proteins and their role in pathogenicity. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and those characterised are virulence factors. Here, we demonstrate that the PA0328 autotransporter is a cell-surface tethered, arginine-specific aminopeptidase, and have defined its active site by site directed mutagenesis. Hence, we have assigned PA0328 with the name AaaA, for arginine-specific autotransporter of P. aeruginosa. We show that AaaA provides a fitness advantage in environments where the sole source of nitrogen is peptides with an aminoterminal arginine, and that this could be important for establishing an infection, as the lack of AaaA led to attenuation in a mouse chronic wound infection which correlated with lower levels of the cytokines TNFα, IL-1α, KC and COX-2. Consequently AaaA is an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections

Immune-Instructive Polymers Control Macrophage Phenotype and Modulate the Foreign Body Response In Vivo

© 2020 The Author(s) Implantation of medical devices can result in inflammation. A large library of polymers is screened, and a selection found to promote macrophage differentiation towards pro- or anti-inflammatory phenotypes. The bioinstructive properties of these materials are validated within a rodent model. By identifying novel materials with immune-instructive properties, the relationship between material-immune cell interactions could be investigated, and this offers exciting possibilities to design novel bioinstructive materials that can be used for numerous clinical applications including medical implants

Discovery of a polymer resistant to bacterial biofilm, swarming, and encrustation

Innovative approaches to prevent catheter-associated urinary tract infections (CAUTIs) are urgently required. Here, we describe the discovery of an acrylate copolymer capable of resisting single- and multispecies bacterial biofilm formation, swarming, encrustation, and host protein deposition, which are major challenges associated with preventing CAUTIs. After screening ~400 acrylate polymers, poly(tert-butyl cyclohexyl acrylate) was selected for its biofilm- and encrustation-resistant properties. When combined with the swarming inhibitory poly(2-hydroxy-3-phenoxypropyl acrylate), the copolymer retained the bioinstructive properties of the respective homopolymers when challenged with Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Urinary tract catheterization causes the release of host proteins that are exploited by pathogens to colonize catheters. After preconditioning the copolymer with urine collected from patients before and after catheterization, reduced host fibrinogen deposition was observed, and resistance to diverse uropathogens was maintained. These data highlight the potential of the copolymer as a urinary catheter coating for preventing CAUTIs

Translational pharmacology of an inhaled small molecule αvβ6 integrin inhibitor for idiopathic pulmonary fibrosis

The αvβ6 integrin plays a key role in the activation of transforming growth factor-β (TGFβ), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvβ6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and diseaserelated end points. Here we report, GSK3008348 binds to αvβ6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFβ signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvβ6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvβ6, induces prolonged inhibition of TGFβ signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy

The role of the Raf family in development

The role of the Raf-1 protein in mouse development was investigated by generating a targeted disruption of the raf-1 gene. The pTC4.Raf-1 targeting vector was constructed using raf-1 genomic DNA 5' and 3' to the kinase domain. This was designed such that homologous recombination with the wild type raf-1 gene resulted in the deletion of the kinase domain. Four ES clones carrying integrations of raf-1 were used to generate chimaeric mice and -/- raf-1 homozygotes were obtained. Embryos-/-for raf-1 were isolated up to day E10.5. Two phenotypes were observed: 1) Developmental arrest at day E8.5-E9, 2) Vascular and somatogesis irregularities with cranial defects. The -/- embryos were also smaller. Expression patterns of raf-1 and A-raf- were analysed in adult tissues and embryos by a combination of in situ hybridisation, immunohistochemistry and a reporter transgenic mouse for A-raf. Embryonic expression analysis of raf-1 showed a general level of expression with elevated levels in the developing embryo, vascular system, tail bud, otic pit and hindbrain. Comparison of the expression of A-raf and raf-1 showed that raf-1 was ubiquitously expressed but had a higher level of expression spatially in development. A-raf was more localised to cell types with higher metabolic requirements. Generation of an ES cell -/- for raf-1 by two rounds of gene targeting in in vitro demonstrated that Raf-1 was not required for ES cell 'self renewal'. The results indicate that there is a requirement for raf-1 protooncogene during development in the mouse

The passenger and β-barrel domains of AaaA remain connected and are tethered to the cell surface.

<p><i>E. coli</i> LEMO21 bearing the empty vector pET21a or pET21a::<i>aaaA</i> was grown to mid exponential phase in LB, and induced with 1 mM IPTG for 1 h. Following harvesting, washing and resuspension in PBS-Hepes, half of the cells were lysed by sonication. The whole and lysed cells were split into three aliquots and incubated with (T) or without (−) trypsin according to the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002854#s4" target="_blank">Materials and Methods</a>. Trypsin inhibitor was added at the same time as trypsin to one of the aliquots (T+I). Proteins were separated through a 9% SDS PAGE and stained with Coomassie Blue (Panel A) or subjected to immunoblotting with either α-AaaA (Panel B, top), or α-IscS (Panel B, bottom) antisera. A parallel experiment was performed with <i>P. aeruginosa</i> Δ<i>aaaA</i> bearing either pME6032 or pME6032::<i>aaaA</i>. LB overnight cultures were diluted 1∶100 in fresh LB, grown for 3 h at 37°C, and induced with 1 mM IPTG for 1 h. The immunoblot of the <i>P. aeruginosa</i> proteins is shown in Panel C, with the cytoplasmic control protein being detected with α-RpoS in the bottom panel. The sizes of molecular weight markers are shown in kDa on the left, and the position of AaaA is indicated. In Panels B and C, densitometry was used to estimate the quantity of the cytoplasmic protein and the full length AaaA (indicated with the asterisk) detected in the immunoblots using imageJ software. The fold change of AaaA, IscS and RpoS are shown below the images of the respective immunoblots. The images in Panels D and E were captured by confocal fluorescent microscopy. <i>P. aeruginosa</i> Δ<i>aaaA</i>(pME6032::<i>aaaA</i>) was grown and induced as described for Panel C, probed with FM1-43 and either α-AaaA (Panel E) or pre-immune serum (Panel D). Incubation with donkey α-rabbit alexa fluor 680-conjugated secondary antibody (red) was performed before images were captured at either the apex or cross section of individual cells (as indicated in the dotted lines of the cartoon). Green fluorescence from FM1-43 (top Panel, green circle in cartoon), red fluorescence from alexa fluor 680 (middle Panel, red stars in cartoon), merged 2D and merged 3D shadowed images are shown.</p

AaaA can remove arginine from <i>p</i>-nitroanilide.

<p><b>Panel A.</b> The <i>P. aeruginosa</i> Δ<i>aaaA</i> mutant alone (open triangles) or bearing either the empty plasmid pME6032 (open circles) or its derivative carrying <i>aaaA</i> (pME6032::<i>aaaA</i>: closed circles) were treated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002854#ppat.1002854.s002" target="_blank">Figure S2B</a> except arginine-<i>p</i>-nitroanilide was used as a substrate. WT PAO1 cells were treated similarly (closed triangles), and activities (measured as changes in A_{405 nm}) are compared against a growth media blank (crosses). <b>Panel B. </b><i>E. coli</i> DH5α bearing either the empty plasmid pME6032 (open circles) or its derivative carrying <i>aaaA</i> (pME6032::<i>aaaA</i>: closed circles) were grown in LB until exponential phase, induced with 1 mM IPTG, and then incubated with arginine-<i>p</i>-nitroanilide as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002854#ppat.1002854.s002" target="_blank">Figure S2B</a>. Activities are compared against a growth media blank (crosses). Error bars are+/−1 S.D. (n = 15). All measurements have been corrected for differential growth of bacteria by normalising to an initial OD_{600 nm} of 0.1.</p

The AaaA deficient mutant is less virulent in the chronic mouse wound model.

<p>Either the <i>P. aeruginosa</i> wild type PAO1 (black bars), the Δ<i>aaaA</i> mutant (white bars), or the complemented Δ<i>aaaA</i> mutant PAJL2 (grey bars) was inoculated (10⁴ CFU) into a chronic wound in each of 9 mice. After 2 (3 mice per group) or 8 (7 mice per group) days, wound tissue was removed and the bacterial load was estimated by calculating the colony forming units (<b>Panel A</b>). Chronically-wounded mice were euthanized at post infection day 2 (3 mice per group) or day 8 (7 mice per group), and wound tissue was harvested for qRT-PCR to detect the mRNA of the indicated cytokines and other host enzymes in the infected wound tissue as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002854#s4" target="_blank">materials and methods</a> (<b>Panel B and C</b>). Tissue from the wounds of the 2 day infected mice (<b>Panels D,G,J</b>) or 8 day infected mice (<b>Panels E,F,H,I,K,L</b>) was stained with H&E and is shown at 100× magnification. Images of the <i>P. aeruginosa</i> wild type PAO1 (<b>Panels D,E,F</b>), Δ<i>aaaA</i> mutant (<b>Panels G,H,I</b>), and the complemented Δ<i>aaaA</i> mutant PAJL2 (<b>Panels J,K,L</b>) are shown with infiltrating neutrophils indicated by white arrows, elongated fibroblasts with a red arrow, single bacterial cells with white arrow heads and clumps of bacteria with a white asterisk. <b>Panels D–E,G–H,J–K</b> are representative of the wound site and <b>Panels F,I,L</b> are representative of the site of infection below the wound.</p

The activity of AaaA enables <i>P. aeruginosa</i> to grow using the tripeptide arg-gly-asp as the sole source of carbon and nitrogen.

<p><i>P. aeruginosa</i> PAO1 (closed circles) and its derived <i>aaaA</i> deficient mutant (Δ<i>aaaA</i>, open circles) alone or bearing pME6032 (vector, open triangles) or pME6032::<i>aaaA</i> (complemented, closed triangles) were grown to mid-exponential phase before the induction of AaaA production by 1 mM IPTG. Cells were resuspended in MMP to OD₆₀₀ of 1, and subsequently 20 µl of this solution diluted into 200 µl of MMP containing arginine at 10 mM (<b>Panel A</b>), or 10 mM of the tripeptide arg-gly-asp (<b>Panel B</b>). The graph shows the subsequent growth in the Tecan monitored by observing the increase in OD₄₉₂ over time. The data is representative of 3 independent repetitions of this experiment.</p