Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1Pseudomonas aeruginosabiofilms

Abstract

The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilmmode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of MucoidPseudomonas aeruginosaBiofilms

“…For FRD1 biofilms cultivated in the presence of added Ca 2ϩ in the medium, the relative abundances of the absorbance bands changed, with the alginate-associated bands becoming dominant. In particular, the band at 1,615 (carboxylate ion) increased and partially obscured the amide I (1,650) and amide was not a component of P. aeruginosa PAO1 biofilms (72). The FTIR spectra here verify those results.…”

“…Similar results were obtained using a mutant derivative of the nonmucoid strain P. aeruginosa PAO1 engineered to overproduce alginate (25,60). On the other hand, results using wild-type P. aeruginosa PAO1 and PA14 showed that alginate was not associated with biofilms (72) and that a second polysaccharide likely encoded by a separate polysaccharide biosynthetic gene cluster may provide the extracellular matrix material for those cells (18,19,29). In another report, using P. aeruginosa PAO1, DNA was suggested to be the extracellular matrix material, since the addition of DNase to biofilms dispersed the bacteria (64).…”

“…In nonmucoid strains, isolated from environmental sources other than chronic CF pulmonary infections, this sigma factor is sequestered in the membrane by the antisigma factor MucAB (40,43). Nonmucoid strains show no evidence of alginate production under any environmental conditions described, including in biofilms (72). Alginate-producing strains isolated from the pulmonary fluid of CF patients often have a mutation in mucA (40), causing release of e from membrane sequestration and subsequent expression of the e -controlled regulon, including the alginate biosynthetic gene cluster (43).…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…The surface-dependent induction of algC expression correlated with stability of attachment, with cells that did not undergo algC upregulation demonstrating reduced ability to remain attached to the surface relative to that of cells with activated expression (33). While these findings suggested increased alginate production upon attachment, with alginate production contributing to biofilm resistance and being inversely linked to both flagellum-driven motility and twitching motility (48,139,165), it is now apparent that alginate is neither the major matrix polysaccharide nor required for biofilm development by nonmucoid P. aeruginosa strains, which are the first to colonize CF patients (121,144,167). Instead, recent chemical and genetic studies have demonstrated that the major polysaccharides produced by P. aeruginosa strains PAO1 and PA14 are Psl and Pel, with the roles of these two polysaccharides differing with respect to attachment and biofilm formation in a strain-specific manner.…”

High-Resolution Visualization ofPseudomonas aeruginosaPAO1 Biofilms by Freeze-Substitution Transmission Electron Microscopy

“…A recent study describing the EPS of PAO1 biofilms identified the major carbohydrate moieties in this to include glucose, rhamnose, mannose, xylose, 3-deoxy-D-manno-oct-2-ulosonic acid (KDO),N-acetylgalactosamine, N-acetylfucosamine, and N-acetylglucosamine (70). More recently, two genetic loci (psl and pel) responsible for the production of mannose-rich (psl) and glucose-rich (pel) polymer components of P. aeruginosa EPS (18,19,52) were identified.…”