Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung

Abstract

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.Pseudomonas aeruginosa is responsible for chronic infections in the airways of cystic fibrosis (CF) patients (13). During the course of chronic infection, P. aeruginosa forms biofilms, which are thought to promote persistence by protecting the bacterium from antibiotics and host clearance. P. aeruginosa also undergoes phenotypic and genotypic diversification. A manifestation of this diversification is the appearance of colony morphology variants among CF sputum sample isolates. One clear example of this phenomenon, which has been termed "dissociative" behavior (42), is the appearance of mucoid colonies. Mucoidy is characterized by overproduction of the exopolysaccharide (EPS) alginate, a polymer of 1,4-␤-linked mannuronic acid and its epimer, guluronic acid (13). The appearance of mucoid colonies is thought to correlate with a downturn in the p...

Evolution and diversification of Pseudomonas aeruginosa in the paranasal sinuses of cystic fibrosis children have implications for chronic lung infection

“…Several of the mutations and phenotypes that we have observed among sinus isolates, evolved in parallel in the different patients, including those conferring loss of motility and quorum sensing signals (virulence), alginate overproduction, antibiotic resistance and increased biofilm formation (SCVs). (Cabral et al, 1987;Mahenthiralingam et al, 1994;Haussler et al, 1999;Smith et al, 2006;D'Argenio et al, 2007;Starkey et al, 2009;Hoffman et al, 2010). Most of them are generally thought to be beneficial also in the lung environment through the development of resistance to host defenses and antibiotics or to optimized utilization of available nutrients.…”

“…Sinuses are infection foci in CF airways SK Hansen et al studies documented the presence of SCVs in the sinus populations, but CF lung SCV isolates have some additional characteristics besides the small colony form (Haussler et al, 1999;Starkey et al, 2009). Sinus SCV isolates were therefore further analyzed for these specific phenotypes: clumping in liquid culture, decreased motility, enhanced biofilm formation and antibiotic resistance (Figure 3).…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…A mutant screen in Yersinia pestis resulted in the identification of three c-di-GMP-modulating proteins playing a role in attachment and biofilm formation, including the DGC HmsT and the PDE HmsP, which inversely regulate biofilm formation by controlling the synthesis of the polysaccharide poly-␤-1,6-N-acetylglucosamine, both in vitro and in the foregut of its flea vector, as well as y3730, encoding a functional DGC (13,148). While c-di-GMP is an important factor in driving cell-surface and cell-cell interactions, Starkey et al (145) demonstrated that elevated c-di-GMP levels alone are not sufficient to alter the expression of all of the genes affected following wspF inactivation. Using a ⌬wspF single mutant and a nonaggregative ⌬wspF ⌬pel ⌬psl triple mutant, they demonstrated that the regulation of multiple genes is actually dependent on the aggregative phenotype rather than c-di-GMP overproduction.…”

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix

“…We first investigated several PAO1-derived mutant strains, such as Psl-or Pel-negative strains, Psl-inducible strain WFPA801 (PAO1, P BADpsl), mucoid strain PDO300 (alginate overproducer), and a rugose small-colony variant (RSCV) MJK8 that displays increased production of the Pel and Psl [38]. PslG significantly inhibited the biofilm formation of all PAO1-derived strains, which included Psl-negative strains and MJK8-derived ∆psl∆pel strain ( Figure 4A).…”