Phenazine Content in the Cystic Fibrosis Respiratory Tract Negatively Correlates with Lung Function and Microbial Complexity

Abstract

Although much is known about how virulence factors affect pathogens and host tissues in vitro, far less is understood about their dynamics in vivo. As a step toward characterizing the chemistry of infected environments, we measured phenazine abundance in the lungs of patients with cystic fibrosis (CF). Phenazines are redox-active small molecules produced by Pseudomonas aeruginosa that damage host epithelia, curb the growth of competing organisms, and play physiologically important roles in the cells that produce them. Here, we quantify phenazines within expectorated sputum, characterize the P. aeruginosa populations responsible for phenazine production, and assess their relationship to CF lung microflora. Chemical analyses of expectorated sputum showed that the concentrations of two phenazines, namely, pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA), were negatively correlated (ρ = -0.68 and -0.57, respectively) with lung function. Furthermore, the highest phenazine concentrations were found in patients whose pulmonary function showed the greatest rates of decline. The constituent P. aeruginosa populations within each patient showed diverse capacities for phenazine production. Early during infection, individual isolates produced more PYO than later during infection. However, total PYO concentrations in sputum at any given stage correlated well with the average production by the total P. aeruginosa population. Finally, bacterial community complexity was negatively correlated with phenazine concentrations and declines in lung function, suggesting a link to the refinement of the overall microbial population. Together, these data demonstrate that phenazines negatively correlate with CF disease states in ways that were previously unknown, and underscore the importance of defining in vivo environmental parameters to better predict clinical outcomes of infections.

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“…Hunter et al (2012) have observed an inverse correlation between phenazine concentrations in sputum of CF patients and lung function during disease progression with similar maximum concentrations for PCA and PYO between 75-100 mM at the highest stage of disease severity. In addition, these authors also observed a strong inverse correlation between phenazine concentration in sputum and microbiome richness.…”

“…This secretion is one of the causes for a chronic polymicrobial infection of the CF airway that, in turn, triggers an immunological response of the host to cause irreversible damage and eventually pulmonary failure (Boucher, 2002;Lyczak et al, 2002;Ratjen and Dö ring, 2003). Throughout the disease progression in conjunction with antibiotic treatments, CF airway microbiomes become less rich in community members and a dominant Pseudomonas aeruginosa population emerges (Hunter et al, 2012). P. aeruginosa plays a central role in CF through many different and complex interactions with the host and the co-habitant microbes in the CF airway microbiome (Sibley et al, 2008a;Hunter et al, 2012).…”

“…Throughout the disease progression in conjunction with antibiotic treatments, CF airway microbiomes become less rich in community members and a dominant Pseudomonas aeruginosa population emerges (Hunter et al, 2012). P. aeruginosa plays a central role in CF through many different and complex interactions with the host and the co-habitant microbes in the CF airway microbiome (Sibley et al, 2008a;Hunter et al, 2012).…”

“…These phenazines consist of four different chemical species with phenazine-1-carboxylic acid (PCA; a precursor for pyocyanin) and pyocyanin (PYO) as important forms with different redox potentials, resulting in different redox-active functions. In general, phenazines have been observed to increase virulence by: (1) distressing airway epithelial cell function via oxidative stress (Wilson et al, 1988;Denning et al, 1998), (2) inhibiting growth of other microbes via oxidative stress (Hassett et al, 1992) and (3) playing physiologically important roles for P. aeruginosa itself (Hunter et al, 2012). For the latter, research has shown specifically for pure cultures of P. aeruginosa that: (i) PCA increases iron availability by reducing Fe(III) to Fe(II) even when bound to human-produced proteins (Wang et al, 2011), (ii) PCA reduces swarming motility and PYO increases biofilm thickness (Ramos et al, 2010), resulting in changes in biofilm formation, (iii) PYO regulates the intracellular redox state under anaerobic conditions by extracellular electron transfer, promoting survival through redox homeostasis and thicker biofilms (Dietrich et al, 2013) and (iv) PYO enables extracellular DNA binding to the surface of the cell to change aggregation characteristics, and thus biofilm formation (Das et al, 2013).…”

“…In addition, these authors also observed a strong inverse correlation between phenazine concentration in sputum and microbiome richness. Finally, they found an important paradoxical relationship because the P. aeruginosa isolates (in laboratory cultures) from the patient group with the most severe stage produced lower rates of PYO compared with the healthy group (Hunter et al, 2012). Could this paradoxical relationship be due to a change in the behavior of P. aeruginosa when grown in a pure culture vs in a mixed culture?…”