Effect of Shear Stress on Pseudomonas aeruginosa Isolated from the Cystic Fibrosis Lung

Abstract

Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle.

Importance: Biofilm formation by Pseudomonas aeruginosa is one of the hallmarks of chronic cystic fibrosis (CF) lung infections. The biofilm matrix protects this bacterium from antibiotics as well as from the immune system. Hence, the prevention or reversion of biofilm formation is believed to have a great impact on treatment of chronic P. aeruginosa CF lung infections. In the present study, we showed that it is possible to modulate the behavior of a highly adapted transmissible P. aeruginosa CF isolate at both the transcriptomic and phenotypic levels by introducing shear stress in a CF-like environment, driving it from a biofilm to a planktonic lifestyle. Consequently, the results obtained in this study are of great importance with regard to therapeutic applications that introduce shear stress in the lungs of CF patients.

FIG 1

(A) Phenotypes of colonies that were recovered from the RWV bioreactor after 24 h of growth in ASM and subsequently plated on LB medium. Mucoid colonies are indicated by a black arrow, while nonmucoid colonies are indicated by a white arrow. (B) Quantification of bacteria that were recovered from the RWV bioreactor after 24 h of growth in ASM and subsequently plated on LB medium. LS, low fluid shear. HS, high fluid shear. NM, nonmucoid colonies. (M) Mucoid colonies. NS, not statistically significant (P > 0.05).

FIG 2

Scanning electron micrographs of P. aeruginosa CF_PA39 grown under low fluid shear conditions (A to D) or high fluid shear conditions (E to H). Panels B and D represent magnifications of the areas indicated by the white boxes in panels A and C, respectively. Panels F and H represent magnifications of the areas indicated by the white boxes in panels E and G, respectively. The magnification and scale bars are shown below each picture. Images are representative of different biological repeats.

FIG 3

Overview of the genetic regions that contain upregulated (A) or downregulated (B) genes under low fluid shear versus high fluid shear conditions for the key affected functional classes. X and Y represent PA14 genes PA14_33980 and PA14_33970, respectively, which are not present in the PAO1 genome. LS, low fluid shear. HS, high fluid shear. The // symbol indicates that this gene is located at a distant position in the genome. All adjacent genes that are transcribed in the same direction are considered to constitute an operon here.

FIG 4

Hypothetical model of the adaptation of P. aeruginosa CF_PA39 to the low fluid shear conditions at the level of osmoprotection. Under the low fluid shear conditions, P. aeruginosa forms biofilms of closely associated cells that are surrounded by alginate layers. The production of several secreted molecules, as well as extracellular DNA and cell debris from dying cells, creates a local hyperosmotic environment. In order to protect itself against this hyperosmotic condition, P. aeruginosa imports choline via the BetT1 and BetT3 transporters, thereby releasing repression of the betIBA operon (and of choline transporter genes betT1 and betT3) by the BetI repressor, and switches on the genes that are required for glycine betaine biosynthesis, using choline as a substrate. At the same time, the majority of genes involved in the catabolism of glycine betaine to glycine are downregulated, leading to an accumulation of the osmoprotectant glycine betaine. Genes highlighted in green were found to be upregulated whereas genes highlighted in red were found to be downregulated under low fluid shear versus high fluid shear conditions. The genes involved in the biosynthetic process proceeding from choline to glycine betaine are shown at the bottom. Glycine betaine biosynthetic genes betA and betB are shown in green, the betI repressor gene is shown in orange, and the choline transporter genes are shown in blue. The BetI protein is represented by an orange open cylindrical shape. The // symbol indicates that this gene is located at a distant position in the genome. IM, inner membrane.

FIG 5

Production of QS molecules and elastase during growth under different shear stress conditions. (A) 3-Oxo-C₁₂-HSL production. (B) Elastase production. (C) Production of short-chain (C₄-C₈) AHL molecules by P. aeruginosa CF_PA39 grown under low fluid shear (plate shown on the left) or high fluid shear (plate shown on the right) conditions. The picture shown here is representative of the results from all three technical replicates of each biological replicate. LS, low fluid shear. HS, high fluid shear. RFU, relative fluorescence units. *, P < 0.05.