Cyclic Di-GMP Signaling Contributes to Pseudomonas aeruginosa-Mediated Catheter-Associated Urinary Tract Infection

Abstract

Bis-(3'-5') cyclic dimeric GMP (c-di-GMP) controls the lifestyle transition between the sessile and motile states in many Gram-negative bacteria, including the opportunistic human pathogen Pseudomonas aeruginosa. Under laboratory conditions, high concentrations of c-di-GMP decrease motility and promote biofilm formation, while low concentrations of c-di-GMP promote motility and decease biofilm formation. Here we sought to determine the contribution of c-di-GMP signaling to biofilm formation during P. aeruginosa-mediated catheter-associated urinary tract infection (CAUTI). Using a murine CAUTI model, a decrease in CFU was detected in the bladders and kidneys of mice infected with strains overexpressing the phosphodiesterases (PDEs) encoded by PA3947 and PA2133 compared to those infected with wild-type P. aeruginosa. Conversely, overexpression of the diguanylate cyclases (DGCs) encoded by PA3702 and PA1107 increased the number of bacteria in bladder and significantly increased dissemination of bacteria to the kidneys compared to wild-type infection. To determine which of the DGCs and PDEs contribute to c-di-GMP signaling during infection, a panel of PA14 in-frame deletion mutants lacking DGCs and PDEs were tested in the CAUTI model. Results from these infections revealed five mutants, three containing GGDEF domains (ΔPA14_26970, ΔPA14_72420, and ΔsiaD) and two containing dual GGDEF-EAL domains (ΔmorA and ΔPA14_07500), with decreased colonization of the bladder and dissemination to the kidneys. These results indicate that c-di-GMP signaling influences P. aeruginosa-mediated biofilms during CAUTI.

Importance: Biofilm-based infections are an important cause of nosocomial infections, since they resist the immune response and traditional antibiotic treatment. Cyclic di-GMP (c-di-GMP) is a second messenger that promotes biofilm formation in many Gram-negative pathogens, including Pseudomonas aeruginosa. Here we determined the contribution of c-di-GMP signaling to catheter-associated urinary tract infection (CAUTI), an animal model of biofilm-based infection. P. aeruginosa with elevated levels of c-di-GMP during the initial infection produces an increased bacterial burden in the bladder and kidneys. Conversely, low concentrations of c-di-GMP decreased the bacterial burden in the bladder and kidneys. We screened a library of mutants with mutations in genes regulating c-di-GMP signaling and found several mutants that altered colonization of the urinary tract. This study implicates c-di-GMP signaling during CAUTI.

FIG 1

Overexpression of DGCs and PDEs during initial infection affects colonization of the bladder and kidneys. (A) In vitro biofilms for DGCs and PDEs expressed on pMMB plasmids in the presence or absence of 1 mM IPTG. (B and C) Bladders (B) and kidneys (C) from mice infected with PA14 (circles), PA14 pMMB PA3702 (squares), PA14 pMMB PA1107 (diamonds), PA14 pMMB PA3947 (triangles), or PA14 pMMB PA2133 (stars). Each symbol represents an individual organ. Bars represent geometric means. Asterisks indicate P values of ≤0.05. Dashed lines represent the limit of detection.

FIG 2

Deletion of specific DGCs and PDEs affects the colonization of the bladder and dissemination to the kidneys during CAUTI. Results for bladders (A) and kidneys (B) from mice infected with PA14 with in-frame deletions in genes containing the GGDEF domain only (circles), GGDEF and EAL (squares), EAL only (triangles), or HD GYP only (diamonds) are shown. Each symbol represents an individual organ. Bars represent geometric means. Dashed lines represent the limit of detection. Deletion mutants highlighted in green are those mutants with at least 1-log increase in CFU detected in either the bladder or the kidneys compared to that for wild-type PA14. Deletion mutants highlighted in blue are those mutants with at least a 1-log decrease in CFU detected in the bladder compared to that for wild-type PA14.

FIG 3

Type IV pili contribute to dissemination to the kidneys. Results for bladders (A) and kidneys (B) from mice infected with wild-type PA14, PA14 ΔfliC, or PA14 ΔpilA are shown. Each symbol represents an individual organ. Bars represent geometric means. Dashed lines represent the limit of detection.