The Exopolysaccharide Alginate Protects Pseudomonas aeruginosa Biofilm Bacteria from IFN-γ-Mediated Macrophage Killing

Abstract

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host’s immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-γ. Human leukocytes, in the presence of recombinant human IFN-γ, killed biofilm bacteria lacking alginate after a 4-h challenge at 37°C. Bacterial killing was dependent on the presence of IFN-γ. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-γ vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.

Caenorhabditis elegans NPR-1–mediated behaviors are suppressed in the presence of mucoid bacteria

“…Mucoid strains of P. aeruginosa appear to have increased resistance to antibiotic therapy and innate and adaptive immune responses, but the mechanisms are not well understood. In vitro studies of murine leukocyte killing have indicated impaired survival of P. aeruginosa mutants defective in alginate synthesis relative to wild-type P. aeruginosa (including PA14) and mucoid P. aeruginosa strains (31). These data were suggestive of impaired phagocyte-mediated killing stimulated by IFN-γ and do not indicate increased resistance of mucoid strains relative to nonmucoid strains, but suggest a functional role for alginate biosynthesis in the ability of P. aeruginosa to survive phagocytemediated killing.…”

Regulation of bacterial virulence gene expression by cell envelope stress responses

“…These include treatment with hydrogen peroxide, osmotic shock and oxygen limitation. 50,54,59,60,[63][64][65][66] This raises the intriguing question of whether or not the RpoE/AlgU regulon, and alginate production, are induced immediately after environmental P. aeruginosa enters the lung. Indeed, there is evidence to support this.…”

“…Alginate facilitates biofilm formation and promotes resistance to host defenses and antibiotics. 50 The alginate biosynthesis operon is positively controlled by RpoE, which is known as AlgU (or AlgT) in P. aeruginosa. 51 A proteolytic cascade analogous to that in E. coli is triggered to degrade the antisigma factor RseA (called MucA in P. aeruginosa) when misfolded OMPs interact with its DegS homolog AlgW, and LPS interacts with it RseB homolog, MucB.…”

Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates

“…Biofilms of mucoid P. aeruginosa strains are more tolerant to antibiotics than wild-type biofilms (10), and mucoidy has been implicated in increased tolerance to host immune responses (11).…”