Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategy

Hard-to-heal wounds are typically infected with biofilm-producing microorganisms, such as Pseudomonas aeruginosa, which strongly contribute to delayed healing. Due to the global challenge of antimicrobial resistance, alternative treatment strategies are needed. Here, we investigated whether inhibition of quorum sensing (QS) by sodium salicylate in different P. aeruginosa strains (QS-competent, QS-mutant, and chronic wound strains) influences biofilm formation and tolerance to silver. Biofilm formation was evaluated in simulated serum-containing wound fluid in the presence or absence of sodium salicylate (NaSa). Biofilms were established using a 3D collagen-based biofilm model, collagen coated glass, and the Calgary biofilm device. Furthermore, the susceptibility of 48-h-old biofilms formed by laboratory and clinical strains in the presence or absence of NaSa towards silver was evaluated by assessing cell viability. Biofilms formed in the presence of NaSa were more susceptible to silver and contained reduced levels of virulence factors associated with biofilm development than those formed in the absence of NaSa. Biofilm aggregates formed by the wild-type but not the QS mutant strain, were smaller and less heterogenous in size when grown in cultures with NaSa compared to control. These data suggest that NaSa, via a reduction of cell aggregation in biofilms, allows the antiseptic to become more readily available to cells.

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Cell aggregation of Pseudomonas aeruginosa strain PAO1 as an energy-dependent stress response during growth with sodium dodecyl sulfate

Archives of Microbiology ◽

10.1007/s00203-006-0111-y ◽

2006 ◽

Vol 185 (6) ◽

pp. 417-427 ◽

Cited By ~ 42

Author(s):

Janosch Klebensberger ◽

Oliver Rui ◽

Eva Fritz ◽

Bernhard Schink ◽

Bodo Philipp

Keyword(s):

Pseudomonas Aeruginosa ◽

Stress Response ◽

Sodium Dodecyl Sulfate ◽

Sodium Dodecyl ◽

Cell Aggregation ◽

Strain Pao1 ◽

Dodecyl Sulfate ◽

Energy Dependent

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Targeted and untargeted quantification of quorum sensing signalling molecules in bacterial cultures and biological samples via HPLC-TQ MS techniques

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-020-03040-6 ◽

2020 ◽

Author(s):

Federica Dal Bello ◽

Michael Zorzi ◽

Riccardo Aigotti ◽

Davide Medica ◽

Vito Fanelli ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biological Samples ◽

Analytical Methods ◽

Neutral Loss ◽

Multiple Reaction Monitoring ◽

Cell Aggregation ◽

Liquid Chromatography Mass Spectrometry ◽

Signalling Molecules ◽

Bacterial Cultures

AbstractQuorum sensing (QS) is the ability of some bacteria to detect and to respond to population density through signalling molecules. QS molecules are involved in motility and cell aggregation mechanisms in diseases such as sepsis. Few biomarkers are currently available to diagnose sepsis, especially in high-risk conditions. The aim of this study was the development of new analytical methods based on liquid chromatography-mass spectrometry for the detection and quantification of QS signalling molecules, including N-acyl homoserine lactones (AHL) and hydroxyquinolones (HQ), in biofluids. Biological samples used in the study were Pseudomonas aeruginosa bacterial cultures and plasma from patients with sepsis. We developed two MS analytical methods, based on neutral loss (NL) and product ion (PI) experiments, to identify and characterize unknown AHL and HQ molecules. We then established a multiple-reaction-monitoring (MRM) method to quantify specific QS compounds. We validated the HPLC-MS-based approaches (MRM-NL-PI), and data were in accord with the validation guidelines. With the NL and PI MS-based methods, we identified and characterized 3 and 13 unknown AHL and HQ compounds, respectively, in biological samples. One of the newly found AHL molecules was C12-AHL, first quantified in Pseudomonas aeruginosa bacterial cultures. The MRM quantitation of analytes in plasma from patients with sepsis confirmed the analytical ability of MRM for the quantification of virulence factors during sepsis.

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Cell aggregation promotes pyoverdine-dependent iron uptake and virulence in Pseudomonas aeruginosa

Frontiers in Microbiology ◽

10.3389/fmicb.2015.00902 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 20

Author(s):

Daniela Visaggio ◽

Martina Pasqua ◽

Carlo Bonchi ◽

Volkhard Kaever ◽

Paolo Visca ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Iron Uptake ◽

Cell Aggregation

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Two-pronged survival strategy for the major cystic fibrosis pathogen, Pseudomonas aeruginosa, lacking the capacity to degrade nitric oxide during anaerobic respiration

The EMBO Journal ◽

10.1038/sj.emboj.7601787 ◽

2007 ◽

Vol 26 (15) ◽

pp. 3662-3672 ◽

Cited By ~ 48

Author(s):

Sang Sun Yoon ◽

Ahmet C Karabulut ◽

John D Lipscomb ◽

Robert F Hennigan ◽

Sergei V Lymar ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Anaerobic Respiration ◽

Survival Strategy

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Small-Colony Variant Selection as a Survival Strategy for Staphylococcus aureus in the Presence of Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.01211-09 ◽

2009 ◽

Vol 75 (21) ◽

pp. 6910-6912 ◽

Cited By ~ 82

Author(s):

Lalitha Biswas ◽

Raja Biswas ◽

Martin Schlag ◽

Ralph Bertram ◽

Friedrich Götz

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Electron Transport ◽

Electron Transport Chain ◽

Survival Strategy ◽

Variant Selection ◽

Small Colony Variant ◽

Transport Chain ◽

Small Colony ◽

Selection Of

ABSTRACT Previously it has been demonstrated that Staphylococcus aureus is sensitive toward Pseudomonas-secreted exotoxins, which preferentially target the electron transport chain in staphylococci. Here it is shown that a subpopulation of S. aureus survives these respiratory toxins of P seudomonas aeruginosa by selection of the small-colony variant (SCV) phenotype. Purified pyocyanin alone causes the same effect. A hem B mutant of S. aureus survives cocultivation with P. aeruginosa without a decrease in CFU.

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Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

Journal of Bacteriology ◽

10.1128/jb.00369-15 ◽

2015 ◽

Vol 198 (1) ◽

pp. 66-76 ◽

Cited By ~ 24

Author(s):

Richard B. Cooley ◽

T. Jarrod Smith ◽

Wilfred Leung ◽

Valerie Tierney ◽

Bradley R. Borlee ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Second Messenger ◽

Cell Aggregation ◽

Large Cell ◽

Opportunistic Pathogen ◽

Secretion System ◽

Signaling System ◽

Signaling Mechanism ◽

Content Type ◽

Environmental Bacterium

ABSTRACTWe previously identified a second-messenger-regulated signaling system in the environmental bacteriumPseudomonas fluorescenswhich controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogenPseudomonas aeruginosaharbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identifiedP. aeruginosaCdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen.IMPORTANCEHere, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogenPseudomonas aeruginosais revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger.

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Alginate Polymerization and Modification Are Linked in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00453-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 33

Author(s):

M. Fata Moradali ◽

Ivan Donati ◽

Ian M. Sims ◽

Shirin Ghods ◽

Bernd H. A. Rehm

Keyword(s):

Pseudomonas Aeruginosa ◽

Material Properties ◽

Molecular Mechanisms ◽

Cell Aggregation ◽

Activation Mechanism ◽

Cellulose Synthesis ◽

Multiprotein Complex ◽

Site Specific ◽

Content Type

ABSTRACTThe molecular mechanisms of alginate polymerization/modification/secretion by a proposed envelope-spanning multiprotein complex are unknown. Here, bacterial two-hybrid assays and pulldown experiments showed that the catalytic subunit Alg8 directly interacts with the proposed copolymerase Alg44 while embedded in the cytoplasmic membrane. Alg44 additionally interacts with the lipoprotein AlgK bridging the periplasmic space. Site-specific mutagenesis of Alg44 showed that protein-protein interactions and stability were independent of conserved amino acid residues R17 and R21, which are involved in c-di-GMP binding, the N-terminal PilZ domain, and the C-terminal 26 amino acids. Site-specific mutagenesis was employed to investigate the c-di-GMP-mediated activation of alginate polymerization by the PilZAlg44domain and Alg8. Activation was found to be different from the proposed activation mechanism for cellulose synthesis. The interactive role of Alg8, Alg44, AlgG (epimerase), and AlgX (acetyltransferase) on alginate polymerization and modification was studied by using site-specific deletion mutants, inactive variants, and overproduction of subunits. The compositions, molecular masses, and material properties of resulting novel alginates were analyzed. The molecular mass was reduced by epimerization, while it was increased by acetylation. Interestingly, when overproduced, Alg44, AlgG, and the nonepimerizing variant AlgG(D324A) increased the degree of acetylation, while epimerization was enhanced by AlgX and its nonacetylating variant AlgX(S269A). Biofilm architecture analysis showed that acetyl groups promoted cell aggregation while nonacetylated polymannuronate alginate promoted stigmergy. Overall, this study sheds new light on the arrangement of the multiprotein complex involved in alginate production. Furthermore, the activation mechanism and the interplay between polymerization and modification of alginate were elucidated.IMPORTANCEThis study provides new insights into the molecular mechanisms of the synthesis of the unique polysaccharide, alginate, which not only is an important virulence factor of the opportunistic human pathogenPseudomonas aeruginosabut also has, due to its material properties, many applications in medicine and industry. Unraveling the assembly and composition of the alginate-synthesizing and envelope-spanning multiprotein complex will be of tremendous significance for the scientific community. We identified a protein-protein interaction network inside the multiprotein complex and studied its relevance with respect to alginate polymerization/modification as well as the c-di-GMP-mediated activation mechanism. A relationship between alginate polymerization and modification was shown. Due to the role of alginate in pathogenesis as well as its unique material properties harnessed in numerous applications, results obtained in this study will aid the design and development of inhibitory drugs as well as the commercial bacterial production of tailor-made alginates.

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A Survival Strategy for Pseudomonas aeruginosa That Uses Exopolysaccharides To Sequester and Store Iron To Stimulate Psl-Dependent Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01307-16 ◽

2016 ◽

Vol 82 (21) ◽

pp. 6403-6413 ◽

Cited By ~ 20

Author(s):

Shan Yu ◽

Qing Wei ◽

Tianhu Zhao ◽

Yuan Guo ◽

Luyan Z. Ma

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Iron Concentration ◽

Survival Strategy ◽

Iron Storage ◽

Content Type ◽

High Level ◽

Biofilm Development ◽

Biofilm Matrix

ABSTRACTExopolysaccharide Psl is a critical biofilm matrix component inPseudomonas aeruginosa, which forms a fiber-like matrix to enmesh bacterial communities. Iron is important forP. aeruginosabiofilm development, yet it is not clearly understood how iron contributes to biofilm development. Here, we showed that iron promoted biofilm formation via elevating Psl production inP. aeruginosa. The high level of iron stimulated the synthesis of Psl by reducing rhamnolipid biosynthesis and inhibiting the expression of AmrZ, a repressor ofpslgenes. Iron-stimulated Psl biosynthesis and biofilm formation held true in mucoidP. aeruginosastrains. Subsequent experiments indicated that iron bound with Pslin vitroand in biofilms, which suggested that Psl fibers functioned as an iron storage channel inP. aeruginosabiofilms. Moreover, among three matrix exopolysaccharides ofP. aeruginosa, Psl is the only exopolysaccharide that can bind with both ferrous and ferric ion, yet with higher affinity for ferrous iron. Our data suggest a survival strategy ofP. aeruginosathat uses exopolysaccharide to sequester and store iron to stimulate Psl-dependent biofilm formation.IMPORTANCEPseudomonas aeruginosais an environmental microorganism which is also an opportunistic pathogen that can cause severe infections in immunocompromised individuals. It is the predominant airway pathogen causing morbidity and mortality in individuals affected by the genetic disease cystic fibrosis (CF). Increased airway iron and biofilm formation have been proposed to be the potential factors involved in the persistence ofP. aeruginosain CF patients. Here, we showed that a high level of iron enhanced the production of the key biofilm matrix exopolysaccharide Psl to stimulate Psl-dependent biofilm formation. Our results not only make the link between biofilm formation and iron concentration in CF, but also could guide the administration or use of iron chelators to interfere with biofilm formation inP. aeruginosain CF patients. Furthermore, our data also imply a survival strategy ofP. aeruginosaunder high-iron environmental conditions.

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