Extracellular aminopeptidase modulates biofilm development of <i>Pseudomonas aeruginosa</i> by affecting matrix exopolysaccharide and bacterial cell death

2019

mBio

Biofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight into how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.

Section: Resultsmentioning

confidence: 99%

“…Recently, Zhao et al showed that PaAP can affect PAO1 P. aeruginosa biofilm pellicles formed in cultures using Jensen’s medium at 30°C (28). While the body of this work mainly focused on a late-stage phenotype, they also observed that PaAP deletion led to increased biomass in early biofilms.…”

Section: Resultsmentioning

confidence: 99%

Pseudomonas aeruginosa Leucine Aminopeptidase Influences Early Biofilm Composition and Structure via Vesicle-Associated Antibiofilm Activity

Esoda

2019

mBio

“…on the involvement of PslG in biofilm disruption, which was also, intriguingly, PaAP-dependent 531 33 . However, in this case, biofilm disruption was found to take place in late log-stage biofilms 532 under nutrient-limiting conditions such that the lack of PaAP lead to cell lysis, releasing the 533 periplasmic PslG enzyme.…”

Section: Discussion 504mentioning

confidence: 98%

“…Several factors from P. aeruginosa are known to facilitate biofilm detachment directly, including 483 those that can break apart the biofilm structure by enzymatic or biochemical means. We studied 484 the impact of three known disruption factors on this phenotype: rhamnolipids 35,36 , and the 485 exopolysaccharide hydrolases PslG 33,36 and AlgL 36 . Vesicles were isolated from cultures of 486 PA14 transposon mutants with a deletion in each potential target and evaluated for their ability 487 21 to reduce the biomass of 4.5 hpi S470∆PaAP coculture biofilms.…”

Section: Protease Inhibitors Decrease Biofilm Detachment Activity Of mentioning

confidence: 99%

Pseudomonas aeruginosaleucine aminopeptidase influences early biofilm composition and structure via vesicle-associated anti-biofilm activity

Esoda

2019

Preprint

1Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic 2 fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to 3 P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient 4 lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. 5We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly 6 expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during 7 infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), 8 structures known to contribute to virulence mechanisms in a variety of Gram-negative species 9 and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a 10 role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture 11 model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm 12 microcolony composition to increase cellular density, while decreasing matrix polysaccharide 13 content, and that OMVs from PaAP expressing strains but not PaAP alone or in combination 14with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally 15 found that OMVs from PaAP expressing strains could cause protease-mediated biofilm 16 detachment, leading to changes in matrix and colony composition. Finally, we showed that the 17OMVs could also mediate the detachment of biofilms formed by both non-self P. aeruginosa 18 strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel 19 roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture. 20 21 22 34 35 36 Introduction 37The Gram-negative bacterium Pseudomonas aeruginosa is a prominent opportunistic 38 pathogen capable of causing both acute and chronic disease in a variety of compromised hosts. 39 P. aeruginosa is most well known as a leading cause of morbidity and mortality in cystic fibrosis 40 (CF) patients 1 . In these individuals, the pathogen establishes chronic, biofilm-based infections 41 that may persist for decades in the unique, mucous-rich environment of the CF lung. In addition 42 to lung tissue, P. aeruginosa forms biofilms on a wide range of substrates relevant to human 43 infection, including corneal and skin tissue, as well as soil and water reservoirs and hospital 44 surfaces, which can contribute to infection initiation and spread 2 . 45 P. aeruginosa is considered a model organism for the study of biofilm formation, and 46 many of the cellular and matrix components contributing to this mode of growth have been 47 studied previously 3-5 . The process of forming biofilm communities has also been characterized, 48 revealing distinct growth phases. First the bacteria settle and attach onto a suitable host tissue 49 or abiotic surface, then they begin to form stationary microcolonies and secrete extrapolymeric 50 substance (EPS) to form a dense mat...

“…However, bacterial factors have been identified that can contribute to human disease in a counterintuitive fashion. Longitudinal studies that follow the progression of P. aeruginosa strains from early colonization through chronic infection have shown that the bacteria quickly evolve and adapt to the conditions found in the CF lung, and that many of the genetic changes seen in chronic P. aeruginosa strains function to decrease overall bacterial pathogenicity [32][33][34] . For example, mucA mutations, which downregulate the expression of virulence-associated type III secretion systems, are frequently observed 35,36 .…”

Section: Discussionmentioning

confidence: 99%

Vesicle-associatedPseudomonas aeruginosaleucine aminopeptidase modulates bacterial biofilms grown on host cellular substrates

2019

Preprint

Pseudomonas aeruginosa, known as one of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients, secretes a variety of virulence-associated proteases. These enzymes have been shown to contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. Our lab has previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is not only secreted solubly, it also associates with bacterial outer membrane vesicles (OMVs), structures known for their contribution to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. With this in mind, we hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background leads to increased early biofilm formation. We additionally found that only native vesicle-bound PaAP, as opposed to its soluble forms, could reconstitute the original PaAP-mediated inhibition phenotype, and that the PaAP-containing vesicles could disperse preformed biofilm microcolonies of Klebsiella pneumoniae, another lung pathogen. These data provide the basis for future work into the mechanism behind PaAP-OMV mediated bacterial microcolony dispersal and the application of these findings to clinical anti-biofilm research.