Respiratory pathogens adopt a chronic lifestyle in response to bile

Abstract

Chronic respiratory infections are a major cause of morbidity and mortality, most particularly in Cystic Fibrosis (CF) patients. The recent finding that gastro-esophageal reflux (GER) frequently occurs in CF patients led us to investigate the impact of bile on the behaviour of Pseudomonas aeruginosa and other CF-associated respiratory pathogens. Bile increased biofilm formation, Type Six Secretion, and quorum sensing in P. aeruginosa, all of which are associated with the switch from acute to persistent infection. Furthermore, bile negatively influenced Type Three Secretion and swarming motility in P. aeruginosa, phenotypes associated with acute infection. Bile also modulated biofilm formation in a range of other CF-associated respiratory pathogens, including Burkholderia cepacia and Staphylococcus aureus. Therefore, our results suggest that GER-derived bile may be a host determinant contributing to chronic respiratory infection.

Figure 1. Bile exposure causes P. aeruginosa to adopt biofilm mode of growth.

(A) Growth of P. aeruginosa in the presence of increasing concentrations of bile revealed concentrations up to 3% to be subinhibitory to growh of this pathogen. (B) (i) Crystal violet stain of pellicle formation in glass tubes revealed significant increase in the presence of 0.3% (w/v) bile compared to media alone. This response was maintained in two CF clinical isolates, CF242 and CF194. (ii) Microcolony formation in ASM media is significantly increased in the presence of 0.3% bile. (C) Swarming is significantly repressed in the presence of 0.3% bile compared to media alone. Swarm distance was measured and compared to control samples. The control was arbitrarily designated as 1 and the data generated in presence of bile was normalised to this value and presented with the standard error of the mean (SEM; p<0.005). All experiments were performed in triplicate and data presented is representative of at least three independent biological replicates.

Figure 2. QS is triggered in response to bile.

(A)(i) Promoter activity of the pqsA-E biosynthetic operon was significantly increased in the presence of 0.3% bile (p<0.005). (ii) TLC analysis of supernatant extracts from cells grown in the presence and absence of 0.3% bile revealed that the increased expression of the pqsA-E operon in the presence of bile results in increased production of the HHQ and PQS signal molecules. Synthetic HHQ and PQS were loaded as controls to facilitate identification of the signal molecules in the supernatant extract. (Bi & ii) Promoter activity of the rhlI and lasI AHL QS systems was also significantly increased in the presence of 0.3% bile (p<0.005). As with the PQS system, increased promoter activity was evident in early and late exponential phase growth. (Biii) Reversed phase TLC analysis of supernatant extracts taken from bile treated and un-treated PA14 cells grown in LB, and overlaid with soft agar containing the C. violaceum CV026 long chain AHL biosensor strain. Increased production of the long chain AHLs can clearly be seen in the treated sample corresponding to the position of the 3-oxo-C12-HSL standard (25 µg). All experiments were performed in triplicate and data presented is representative of at least three independent biological replicates.

Figure 3. Bile modulates inverse expression of the T6SS and T3SS.

(A) Promoter activity of the T6SS promoter tssA, which has been implicated in chronic infection, was significantly increased in the presence of 0.3% bile (p<0.05). (B) Promoter activity of the T3SS effector exoU, associated with acute infection, was significantly decreased in NTA medium in the presence of 0.3% bile (p<0.005). Previous studies have reported the inverse relationship between expression of these two systems. (C) Real Time RT-PCR expression analysis performed using UPL probes revealed that transcript levels of exoU were reduced >8 fold in NTA medium in response to 0.3% bile (p<0.005). All experiments were performed in triplicate and data presented is representative of at least three independent biological replicates.

Figure 4. Bile modulates biofilm formation in a broad spectrum of CF-associated pathogens.

Addition of subinhibitory concentrations of bile (0.3%) significantly increased biofilm formation in A. baumanii, P. spuronum, B. cenocepacia, and P. aeruginosa. Conversely, biofilm formation was significantly reduced in S. maltophilia and S. aureus at concentrations that are subinhibitory to growth. All experiments were performed in triplicate and data presented is representative of at least three independent biological replicates (**p<0.01, ***p<0.005).

Figure 5. GER-derived bile may influence respiratory pathogen behaviour and biodiversity in the CF pulmonary system.

Bile aspiration linked to GER is highly prevalent in CF patients and has been linked to reduced pulmonary function in these patients. As bile is shown to influence key phenotypes associated with chronic infection in P. aeruginosa, GER-derived bile may constitute a major host factor in chronic respiratory disease. Bile induces biofilm formation, T6SS, and QS in P. aeruginosa, while T3SS and swarming motility are repressed. The transcriptional and phenotypic changes are consistent with a switch towards the chronic lifestyle adopted by P. aeruginosa and other respiratory pathogens in the CF lung. Bile also influences biofilm formation in other respiratory pathogens prevalent in CF patients suggesting that GER-derived bile may influence biodiversity in the CF lung.