Mucin Promotes Rapid Surface Motility

An important environmental factor that determines the mode of motility adopted by Pseudomonas aeruginosa is the viscosity of the medium, often provided by adjusting agar concentrations in vitro. However, the viscous gel-like property of the mucus layer that overlays epithelial surfaces is largely due to the glycoprotein mucin. P. aeruginosa is known to swim within 0.3% (wt/vol) agar and swarm on the surface at 0.5% (wt/vol) agar with amino acids as a weak nitrogen source. When physiological concentrations or as little as 0.05% (wt/vol) mucin was added to the swimming agar, in addition to swimming, P. aeruginosa was observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular, with a bright green center surrounded by a thicker white edge. While intact flagella were required for the surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Replacement of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with mutants, the quorum-sensing systems (las and rhl) and the orphan autoinducer receptor QscR played important roles in this form of surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, we suggest that mucin may be promoting a new or highly modified form of surface motility, which we propose should be termed “surfing.” IMPORTANCE An important factor that dictates the mode of motility adopted by P. aeruginosa is the viscosity of the medium, often provided by adjusting agar concentrations in vitro. However, the gel-like properties of the mucous layers that overlay epithelial surfaces, such as those of the lung, a major site of Pseudomonas infection, are contributed mostly by the production of the glycoprotein mucin. In this study, we added mucin to swimming media and found that it promoted the ability of P. aeruginosa to exhibit rapid surface motility. These motility colonies appeared in a circular form, with a bright green center surrounded by a thicker white edge. Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella. Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility in P. aeruginosa. Received 13 March 2012 Accepted 9 April 2012 Published 1 May 2012 Citation Yeung ATY, Parayno A, and Hancock REW. 2012. Mucin promotes rapid surface motility in Pseudomonas aeruginosa.mBio 3(3):e00073-12. doi:10.1128/mBio.00073-12. Editor Karen Bush, Indiana University Copyright © 2012 Yeung et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Robert E. W. Hancock, [email protected]. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that can be found free-living in water and soil and also causes infections in a variety of animals and plants (1). Notably, it is commonly associated with nosocomial infections, particularly lung infections, and is the dominant pathogen in chronic cystic fibrosis (CF) pulmonary infections, persisting in the lungs and inducing serious inflammation that destroys healthy host tissue (2, 3). P. aeruginosa infections are particularly difficult to treat due to the bacterium’s intrinsic resistance to a broad spectrum of antimicrobial agents and its repertoire of virulence factors (4). Motility plays an important role in the pathogenesis of P. aeruginosa (5, 6) and is crucial to the ability of P. aeruginosa to colonize the host and form biofilms (7). P. aeruginosa is known to exhibit three major forms of motility: (i) flagellum-mediated swimming in an aqueous environment and at low agar concentrations ( 0.3% [wt/vol]), (ii) type IV pilus-mediated twitching on solid surfaces (1% [wt/vol] agar) or at the interstitial surface between the agar and plastic or glass (8, 9), and (iii) swarming on semisolid (viscous) surfaces (0.5 to 0.7% [wt/vol] agar), with amino acids serving as the nitrogen source (10). Swarming is a social phenomenon that involves rapid coordinatedmovement of bacteria across a semisolid surface, often typified by dendritic (strain PA14)or solar flare (strain PAO1)-like colonial appearances. Several studies have shown that P. aeruginosa requires its flagella and type IV pili to swarm (10–12), and swarmer cells have two polar flagella and are elongated compared to the normally singly flagellated and shorter swimming cells (10). Biosurfactants produced by the bacteria, such as rhamnolipids and 3-(3hydroxyalkanoyloxy) alkanoic acids (HAAs), are involved in swarming motility, as they aid in overcoming the surface tension between the bacterial cells and their environment (10, 13). The bacterium’s quorum-sensing (QS) systems, las and rhl, also play a role in swarming, possibly by regulating production of rhamnolipids and HAAs (10, 14). In addition to physical changes, swarmer cells overexpress hundreds of genes, including most virulence-related genes, and exhibit elevated adaptive resistance to a variety of antibiotics (11), whilemore than 230 gene products, including 35 regulators, are required for swarming (12), indicating that swarming is a complex adaptation/lifestyle change rather than just a form of motility. RESEARCH ARTICLE May/June 2012 Volume 3 Issue 3 e00073-12 ® mbio.asm.org 1 m b.asm org on M ay 1, 2012 P ubished by m b.asm org D ow nladed rom Besides swimming, twitching, and swarming, in the absence of both flagella and type IV pili, P. aeruginosa has recently been shown to exhibit sliding/spreading motility on semisolid surfaces (15).Murray and Kazmierczak demonstrated that slidingmotility requires rhamnolipid production and responds to many of the same regulatory proteins and environmental cues as swarming motility but is actually inhibited by the presence of pili (15). Due to the difficult nature of studying motility in a living host, previous in vitro motility studies have suggested swarming as a likely mode of motility utilized by P. aeruginosa to colonize the lung based on the conditions that promote swarming motility in vitro (semisolid agar), which mimic the viscous environment of the lung, especially in the case of chronic (mucoid) infections in CF patients, where the lung environment is characterized by the production of copious amounts of mucous. An obvious problem, however, is that under in vivo conditions, agar is absent; instead, the gel-like properties of the mucous layer are contributed mostly by the production of mucin. Mucin is a major component of the respiratory mucus. It is a glycoprotein secreted by themucosal and submucosal glands. The mucin molecule consists of a polypeptide core with branched oligosaccharide side chains, each of which contains 8 to 10 sugars (16). Molecular cross-linking of this structure contributes to the viscoelastic property of mucus (17). In this study, we examined the motility of P. aeruginosa under conditions that mimic in vitro, as closely as possible, the conditions in the CF lung. Motility assay media contained synthetic CF sputummedium (SCFM), developed by Palmer et al. tomimic the nutritional composition of the CF sputum (18), without added NH4Cl but with added mucin and DNA. When mucin was added to SCFM swimming agar, at concentrations as low as 0.05% (wt/ vol), P. aeruginosa was observed to undergo accelerated motility on the surface of the agar. In the presence of mucin, the surface motility colonies of both P. aeruginosa strains, PA14 and PAO1, appeared circular, with bright green centers surrounded by thicker white edges. We found that this form of motility was dependent on the presence of an intact flagellumbut not type IV pili. While quorum sensing (QS) is important, QS-regulated production of rhamnolipids by P. aeruginosa was not required for this form of surface propagation. Microscopic analysis of cells taken from the motility edge revealed that cells were piled up, with the majority of bacterial cells lacking flagella. In contrast, bacterial cells at the center of the motility zone had flagella. Overall, our genetic and phenotypic data led us to suggest that mucinmight be promoting a highly modified form of swarming or a new form of surface motility.