The Autotransporter Esterase EstA of Pseudomonas aeruginosa Is Required for Rhamnolipid Production, Cell Motility, and Biofilm Formation

Abstract

Pseudomonas aeruginosa PAO1 produces the biodetergent rhamnolipid and secretes it into the extracellular environment. The role of rhamnolipids in the life cycle and pathogenicity of P. aeruginosa has not been completely understood, but they are known to affect outer membrane composition, cell motility, and biofilm formation. This report is focused on the influence of the outer membrane-bound esterase EstA of P. aeruginosa PAO1 on rhamnolipid production. EstA is an autotransporter protein which exposes its catalytically active esterase domain on the cell surface. Here we report that the overexpression of EstA in the wild-type background of P. aeruginosa PAO1 results in an increased production of rhamnolipids whereas an estA deletion mutant produced only marginal amounts of rhamnolipids. Also the known rhamnolipid-dependent cellular motility and biofilm formation were affected. Although only a dependence of swarming motility on rhamnolipids has been known so far, the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation. In order to demonstrate that EstA enzyme activity is responsible for these effects, inactive variant EstA* was constructed by replacement of the active serine by alanine. None of the mutant phenotypes could be complemented by expression of EstA*, demonstrating that the phenotypes affected by the estA mutation depend on the enzymatically active protein.Pseudomonas aeruginosa is an increasingly prevalent opportunistic gram-negative pathogen, which can infect humans, but also plants, nematodes, insects, amoebae, and animals (14,45,47). In humans, it causes infections in immunocompromised hosts such as patients suffering from cystic fibrosis, burns, or cancer and represents a major problem in intensive care units. P. aeruginosa produces and secretes an arsenal of virulence factors, which are all involved in the initiation or establishment of infection processes. These extracellular virulence determinants include not only proteases, lipases, and phospholipases but also nonprotein compounds like iron scavenger siderophores, the polysaccharide alginate, and the biosurfactant rhamnolipid, which is also known as heat-stable hemolysin (46).Many of these factors appear to have functions in complex physiological processes related to virulence like adhesion to different surfaces, cellular surface motility, and biofilm formation. P. aeruginosa is capable of performing three different types of cell motility: flagellum-mediated "swimming," type IV pilus-dependent "twitching," and a complex coordinated multicellular migration called "swarming" (16,39,48,54). In contrast to that of many other bacteria, swarming of P. aeruginosa is not dependent only on flagella but also on type IV pili (32). Furthermore, rhamnolipids seem to be required for swarming motility by acting as a surface-modifying agent (32).The rhamnolipids produced by P. aeruginosa are composed of mono-or dirhamnose linked to 3-hydroxy fatty acids of various chain lengths. The most abunda...

Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa

“…Less is known about the influence of extracellular enzymes on biofilm formation and architecture. The first evidence for the influence of the esterase EstA on the biofilm structure of non-mucoid P. aeruginosa PAO1 was reported by Wilhelm et al (2007a).…”

“…For example, this technique has been applied to study the functions of LasB and EstA in the nonmucoid wild-type P. aeruginosa PAO1 (Kamath et al, 1998;Wilhelm et al 2007a). Although the construction of geneknockout mutants is a common strategy to investigate the function of enzymes and their role in the physiological processes of the cell, the phenotypic characterization of overexpression strains is also an accepted strategy to investigate specific questions (e.g.…”

“…Furthermore, rhamnolipids are required for swarming motility by acting as biosurfactants (Köhler et al, 2000;Deziel et al, 2003). Recently, a correlation between expression of the extracellular esterase EstA of P. aeruginosa and rhamnolipid production was described, indicating an important role for EstA in the biosynthesis of rhamnolipids (Wilhelm et al, 2007a).…”

“…Furthermore, rhamnolipids are required for swarming motility by acting as biosurfactants (Köhler et al, 2000;Deziel et al, 2003). Recently, a correlation between expression of the extracellular esterase EstA of P. aeruginosa and rhamnolipid production was described, indicating an important role for EstA in the biosynthesis of rhamnolipids (Wilhelm et al, 2007a).In the case of mucoid variants of P. aeruginosa, the EPS contain a high proportion of the exopolysaccharide alginate, which is a high-molecular-mass unbranched copolymer consisting of the (1A4)-linked uronic acid residues of b-D-mannuronate and a-L-guluronate (Jain & Ohman, 2004). In addition to this polysaccharide there is a significant fraction of proteins in the EPS of mucoid P. aeruginosa (Wingender et al, 2001).…”

EprS, an autotransporter serine protease, plays an important role in various pathogenic phenotypes of Pseudomonas aeruginosa

“…Disruption of eprS decreases the production of surfactants in P. aeruginosa P. aeruginosa is known to produce surfactants that are involved in its swarming motility behaviour, such as rhamnolipids and their precursors 3-(3-hydroxyalkanoyloxy)-alkanoic acids (Déziel et al, 2003;Wilhelm et al, 2007). We then performed drop collapse assays using spent supernatant for each P. aeruginosa strain to assess semiquantitatively the production of surfactants in P. aeruginosa.…”