RESUMEN
Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1â4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.
Asunto(s)
Biopelículas , ADN Bacteriano/metabolismo , Polisacáridos Bacterianos/metabolismo , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cationes/química , ADN Bacteriano/química , ADN Bacteriano/genética , Matriz Extracelular/metabolismo , Espacio Extracelular/genética , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Immunoblotting , Microscopía Confocal , Mutación , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Polisacáridos Bacterianos/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Receptores N-Acetilglucosamina/química , Receptores N-Acetilglucosamina/metabolismo , Coloración y Etiquetado/métodosRESUMEN
BACKGROUND: Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A-L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the "scaffolding" polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A-L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated. PRINCIPAL FINDINGS: In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1-3)-α-D-QuiNAc-(1-3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic ß-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least â¼20% of dry weight) of LPS-like material. CONCLUSIONS: We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.