Dataset Information

Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

ABSTRACT: Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

SUBMITTER: Ma L

PROVIDER: S-EPMC2654510 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

Ma Luyan L Conover Matthew M Lu Haiping H Parsek Matthew R MR Bayles Kenneth K Wozniak Daniel J DJ

PLoS pathogens 20090327 3

Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism f ...[more]

PMID: 19325879

Similar Datasets

Project description:The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.

Project description:Pseudomonas aeruginosa is an opportunistic pathogen that is widely known for infecting patients with underlying conditions. This species often survives antibiotic therapy by forming biofilms, in which the cells produce a protective extracellular matrix. P. aeruginosa also produces virulence factors that enhance its ability to cause disease. One signaling pathway that influences virulence is the nitrogen-related phosphotransferase system (Nitro-PTS), which consists of an initial phosphotransferase, PtsP, a phosphocarrier, PtsO, and a terminal phosphate receptor, PtsN. The physiological role of the Nitro-PTS in P. aeruginosa is poorly understood. However, PtsN, when deprived of its upstream phosphotransfer proteins, has an antagonistic effect on biofilm formation. We thus conducted a transposon mutagenesis screen in an unphosphorylated-PtsN (i.e., ∆ptsP) background to identify downstream proteins with unacknowledged roles in PtsN-mediated biofilm suppression. We found an unstudied gene, PA14_04030, whose disruption restored biofilm production. This gene encodes a predicted phospholipase with signature alpha/beta hydrolase folds and a lipase signature motif with an active-site Ser residue. Hence, we renamed the gene bipL, for biofilm-impacting phospholipase. Deletion of bipL in a ∆ptsP background increased biofilm formation, supporting the idea that BipL is responsible for reducing biofilm formation in strains with unphosphorylated PtsN. Moreover, substituting the putative catalytic Ser for Ala phenocopied bipL deletion, indicating that this residue is important for the biofilm-suppressive activity of BipL in vivo. As our preliminary data suggest that BipL is a lipase, we performed lipidomics to detect changes in the lipid profile due to bipL deletion and found changes in some lipid species. IMPORTANCE Biofilm formation by bacteria occurs when cells secrete an extracellular matrix that holds them together and shields them from environmental insults. Biofilms of bacterial opportunistic human pathogens such as Pseudomonas aeruginosa pose a substantial challenge to clinical antimicrobial therapy. Hence, a more complete knowledge about the bacterial factors that influence and regulate production of the biofilm matrix is one key to formulate more effective therapeutic strategies. In this study, we screen for factors that are important for reducing biofilm matrix production in certain genetic backgrounds. We unexpectedly found a gene encoding a putative lipase enzyme and showed that its predicted catalytic site is important for its ability to reduce biofilm formation. Our findings suggest that lipase enzymes have previously uncharacterized functions in biofilm matrix regulation.

Project description:Biofilms are commonly recalcitrant to antibiotics, through incompletely elucidated mechanisms such as tolerance and persistence. We aimed at investigating how a Pseudomonas aeruginosa biofilm escapes ciprofloxacin treatment. P. aeruginosa PA14 in vitro mature biofilms were challenged with supra-MIC ciprofloxacin concentrations. Cell viability was quantified by fluorescein diacetate assay. Population dynamics were determined by counts of surviving culturable cells. Biofilms were analyzed using confocal laser scanning microscopy (CLSM), and the expression of genes involved in stringent response, toxin-antitoxin HigB/HigA, and type 3 secretion system (T3SS) was quantified by RT-qPCR in untreated and treated biofilms. Ciprofloxacin exposure resulted in an initial reduction of bacterial counts following a biphasic time-kill curve. After 24 h of treatment, the overall cell activity and the density of culturable cells significantly decreased as compared to untreated biofilm. No resistant mutant was isolated among the <1% surviving cells. Phenotypic adaptation toward persistence appeared to start after only 1 h of antibiotic exposure, by an overexpression of the genes involved in stringent response and in the toxin-antitoxin system, whereas the expression of genes encoding for the T3SS remained unchanged. After 4 h of ciprofloxacin exposure, stringent response genes returned to their basal level of expression. After a prolonged ciprofloxacin exposure, a deep alteration in the matrix structure that became thinner and lost mushroom-like aggregates was observed, in relation with reduced biovolumes of exopolysaccharides and extracellular DNA. These results support that ciprofloxacin might first induce the bacterial killing of most bacterial cells, but simultaneously activate stringent response mechanisms contributing to the switch of a subpopulation toward a persister phenotype. Once the persister phenotype is expressed, and despite an unexpected alteration of the biofilm matrix, ciprofloxacin fails to eradicate biofilm.

Dataset Information

Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

Publications

Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets