The Deciphering of Growth-Dependent Strategies for Quorum-Sensing Networks in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is recognized as a significant cause of morbidity and mortality among nosocomial pathogens. In respiratory infections, P. aeruginosa acts not only as a single player but also collaborates with the opportunistic fungal pathogen Aspergillus fumigatus. This study introduced a QS molecule portfolio as a potential new biomarker that affects the secretion of virulence factors and biofilm formation. The quantitative levels of QS molecules, including 3-o-C12-HSL, 3-o-C8-HSL, C4-HSL, C6-HSL, HHQ, PQS, and PYO, measured using mass spectrometry in a monoculture, indicated metabolic changes during the transition from planktonic to sessile cells. In the co-cultures with A. fumigatus, the profile of abundant QS molecules was reduced to 3-o-C12-HSL, C4-HSL, PQS, and PYO. A decrease in C4-HSL by 50% to 170.6 ± 11.8 ng/mL and an increase 3-o-C12-HSL by 30% up to 784.4 ± 0.6 ng/mL were detected at the stage of the coverage of the hyphae with bacteria. Using scanning electron microscopy, we showed the morphological stages of the P. aeruginosa biofilm, such as cell aggregates, maturated biofilm, and cell dispersion. qPCR quantification of the genome equivalents of both microorganisms suggested that they exhibited an interplay strategy rather than antagonism. This is the first study demonstrating the quantitative growth-dependent appearance of QS molecule secretion in a monoculture of P. aeruginosa and a co-culture with A. fumigatus.

Keywords: Aspergillus fumigatus; Pseudomonas aeruginosa; QS system; biofilm; metabolomic analysis; microbial interaction; planktonic cell.

Figure 1

Quantification of extracellular QS molecules secreted during the dynamic growth of a monoculture (A) and bacterial biofilm formation of P. aeruginosa on the hyphae surface of A. fumigatus (B). The bar in the graph represents the averages from two biological replicates. Error bars are presented as the standard deviation. 3-o-C12-HSL, N-(3-oxodecanoyl)-homoserine lactone; 3-o-C8-HSL, N-(3-oxo-octanoyl)-homoserine lactone; C4-HSL, N-butyryl-homoserine lactone; C6-HSL, N-hexanoyl-homoserine lactone; HHQ, 4-hydroxy-2-heptylquinoline; PQS, 2-heptyl-3,4-dihydroxyquinoline.

Figure 2

The secretory levels of pyocyanin in the bacterial monoculture (red line) and its interplay with A. fumigatus (blue line). Pyocyanine graphic: PubChem CID 6817. Atoms are represented by spheres of different colors (black represents carbon, white represents hydrogen, red represents oxygen and blue represents nitrogen).

Figure 3

Quantification via qPCR of microbial biomass in co-cultures. The A. fumigatus culture alone (red line) and in co-culture with P. aeruginosa biofilm (green line), or the P. aeruginosa culture alone (light blue line) and in a co-cultured biofilm with A. fumigatus (dark blue line).

Figure 4

P. aeruginosa growth stages in the monoculture over the course of 48 h. SEM imaging in secondary (a) and backscattered (b–d) electrons. (a) 12 h—motile bacteria with flagella (arrows). (b) 18 h—the bacteria transition to the stationary phase; visible cell wall shrinking. (c) 24 h—aggregates with fibrillar connections; some cells have perforations of the cell wall (arrows). (d) 48 h—the late stationary phase; massive cell aggregates in extracellular polymeric substances with a dense fiber network. Primary magnification of SEM images: 50,000× (a,b,d), 35,000× (c).

Figure 5

SEM imaging in secondary electrons; co-cultures of P. aeruginosa and A. fumigatus. (a) 9 h—bacteria covering fungal hyphae. (b) 12 h—fungal pellets with bacteria covering almost all accessible hyphal surfaces. (c) 12 h—detail of bacteria connected by fibrillar structures (arrows). (d) 18 h—bacteria embedded in extracellular polymeric substances. (e) 18 h—detail of bacteria with pili (arrow) in extracellular polymeric substances. (f) 48 h—detail of fungal pellet without bacteria. Primary magnification of SEM images: 8000× (a), 1200× (b), 50,000× (c), 25,000× (d), 80,000× (e), 3500× (f).