OprF Impacts Pseudomonas aeruginosa Biofilm Matrix eDNA Levels in a Nutrient-Dependent Manner

Cassin, Erin; Araujo-Hernandez, Sophia A.; Baughn, Dena S; Londono, Melissa C.; Rodriguez, Daniela Q; Al-Otaibi, Natalie S.; Picard, Aude; Bergeron, Julien R. C.; Tseng, Boo Shan

doi:10.1128/jb.00080-23

Cited by 5 publications

(3 citation statements)

References 34 publications

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“…Thus, the contributions of OmpT and OmpA to biofilm formation could be conditional and governed by environmental stress responses. In P. aeruginosa , the major OMP, OprF, is an abundant matrix protein and promotes or represses biofilm formation depending upon growth conditions ( 62 , 63 ). OprF interacts with surface-exposed lectin LecB, which binds P. aeruginosa exopolysaccharide Psl, thereby promoting biofilm matrix assembly and stability through increased retention of cells and exopolysaccharides in biofilms ( 64 ).…”

Section: Discussionmentioning

confidence: 99%

Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly

Potapova,

Garvey,

Dahl

et al. 2024

mBio

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Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae . Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae , which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae . IMPORTANCE Cholera remains a major public health concern. Vibrio cholerae , the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae . Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

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Section: Discussionmentioning

confidence: 99%

Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly

Potapova,

Garvey,

Dahl

et al. 2024

mBio

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“…Studies have shown that eDNA can form PsI-eDNA fiber webs with extracellular polysaccharide PsI ( Byrd et al., 2009 ), and in addition, eDNA can interact with Pel ( Jennings et al., 2015 ; Jennings et al., 2021 ) to protect eDNA from degradation by DNase I. Due to the important position of eDNA in biofilm EPS ( Kanampalliwar and Singh, 2020 ; Peng et al., 2020 ; Dawson et al., 2021 ; Cassin et al., 2023 ), more and more scholars have begun to pay attention to drug development targeting eDNA. Previous studies have reported the effects of natural compounds ( Meto et al., 2020 ), synthetic novel substances ( Patel et al., 2020 ) and amino acids ( Gnanadhas et al., 2015 ) on eDNA in P. aeruginosa biofilms.…”

Section: Discussionmentioning

confidence: 99%

In vitro inhibition of Pseudomonas aeruginosa PAO1 biofilm formation by DZ2002 through regulation of extracellular DNA and alginate production

Dai,

Luo,

et al. 2024

Front. Cell. Infect. Microbiol.

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IntroductionPseudomonas aeruginosa (P. aeruginosa) is a common pathogen associated with biofilm infections, which can lead to persistent infections. Therefore, there is an urgent need to develop new anti-biofilm drugs. DZ2002 is a reversible inhibitor that targets S-adenosylhomocysteine hydrolase and possesses anti-inflammatory and immune-regulatory activities. However, its anti-biofilm activity has not been reported yet.Methods and resultsTherefore, we investigated the effect of DZ2002 on P. aeruginosa PAO1 biofilm formation by crystal violet staining (CV), real-time quantitative polymerase chain reaction (RT-qPCR) and confocal laser scanning microscopy (CLSM). The results indicated that although DZ2002 didn’t affect the growth of planktonic PAO1, it could significantly inhibit the formation of mature biofilms. During the inhibition of biofilm formation by DZ2002, there was a parallel decrease in the synthesis of alginate and the expression level of alginate genes, along with a weakening of swarming motility. However, these results were unrelated to the expression of lasI, lasR, rhII, rhIR. Additionally, we also found that after treatment with DZ2002, the biofilms and extracellular DNA content of PAO1 were significantly reduced. Molecular docking results further confirmed that DZ2002 had a strong binding affinity with the active site of S-adenosylhomocysteine hydrolase (SahH) of PAO1.DiscussionIn summary, our results indicated that DZ2002 may interact with SahH in PAO1, inhibiting the formation of mature biofilms by downregulating alginate synthesis, extracellular DNA production and swarming motility. These findings demonstrate the potential value of DZ2002 in treating biofilm infections associated with P. aeruginosa.

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“…In a similar vein, OMVs have long been believed to interact with eDNA in the biofilm matrix (119)(120)(121). Though no direct interaction with OprF has been proposed, loss of the protein was shown to affect overall eDNA production under some conditions (122). Thus, the role of OprF in OMV-matrix interaction is an area ripe for deeper investigation, and a new understanding that OprF in OMVs adopts a different conformation than OprF in cells provides a new wrinkle to be further explored.…”

Section: Discussionmentioning

confidence: 99%

OprF functions as a latch to direct Outer Membrane Vesicle release inPseudomonas aeruginosa

Mathur,

Erickson,

Goldberg

et al. 2023

Preprint

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Bacterial Outer Membrane Vesicles (OMVs) contribute to virulence, competition, immune avoidance and communication. This has led to great interest in how they are formed. To date, investigation has focused almost exclusively on what controls the initiation of OMV biogenesis. Regardless of the mechanism of initiation, all species face a similar challenge before an OMV can be released: How does the OM detach from the underlying peptidoglycan (PG) in regions that will ultimately bulge and then vesiculate? The OmpA family of OM proteins (OprF inP. aeruginosa) is widely conserved and unusually abundant in OMVs across species considering their major role in PG attachment. OmpA homologs also have the interesting ability to adopt both PG-bound (two-domain) and PG-released (one-domain) conformations. Using targeted deletion of the PG-binding domain we showed that loss of cell wall association, and not general membrane destabilization, is responsible for hypervesiculation in OprF-modified strains. We therefore propose that OprF functions as a ‘latch’, capable of releasing PG in regions destined to become OMVs. To test this hypothesis, we developed a protocol to assess OprF conformation in live cells and purified OMVs. While >90% of OprF proteins exist in the two-domain conformation in the OM of cells, we show that the majority of OprF in OMVs is present in the one-domain conformation. With this work, we take some of the first steps in characterizing late-stage OMV biogenesis and identify a family of proteins whose critical role can be explained by their unique ability to fold into two distinct conformations.SignificanceVesicular transport is now recognized to operate in all domains of life. However, the study of OMV biogenesis has been challenging because genetic screens failed to identify proteins analogous to those involved in eukaryotic vesicular transport. With this work we identify the first protein whose direct action can both define the location and govern the mechanism of OMV release. Our latch model is consistent with previous observations linking OmpA family proteins to OMV biogenesis, but further describes a physical mechanism that has broad implications for vesicle production and function across species. The work presented here advances our understanding of a fundamental virulence-associated process in bacteria, while underscoring stark differences in how transport vesicles are formed in prokaryotesversuseukaryotes.

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OprF Impacts Pseudomonas aeruginosa Biofilm Matrix eDNA Levels in a Nutrient-Dependent Manner

Abstract: Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized.

Cited by 5 publications

References 34 publications

Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly

Outer membrane vesicles and the outer membrane protein OmpU govern Vibrio cholerae biofilm matrix assembly

In vitro inhibition of Pseudomonas aeruginosa PAO1 biofilm formation by DZ2002 through regulation of extracellular DNA and alginate production

OprF functions as a latch to direct Outer Membrane Vesicle release inPseudomonas aeruginosa

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