Refinement of metabolite detection in cystic fibrosis sputum reveals heme correlates with lung function decline

Abstract

The bacterial growth environment within cystic fibrosis (CF) sputum is complex, dynamic, and shaped by both host and microbial processes. Characterization of the chemical parameters within sputum that stimulate the in vivo growth of airway pathogens (e.g. Pseudomonas aeruginosa) and their associated virulence factors may lead to improved CF treatment strategies. Motivated by conflicting reports of the prevalence and abundance of P. aeruginosa-derived metabolites known as phenazines within CF airway secretions, we sought to quantify these metabolites in sputum using quadrupole time-of-flight mass spectrometry. In contrast to our previous work, all phenazines tested (pyocyanin (PYO), phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide, and 1-hydroxyphenazine) were below detection limits of the instrument (0.1 μM). Instead, we identified a late-eluting compound that shared retention time and absorbance characteristics with PCA, yet generated mass spectra and a fragmentation pattern consistent with ferriprotoporphyrin IX, otherwise known as heme B. These data suggested that UV-vis chromatographic peaks previously attributed to PCA and PYO in sputum were mis-assigned. Indeed, retrospective analysis of raw data from our prior study found that the heme B peak closely matched the peaks assigned to PCA, indicating that the previous study likely uncovered a positive correlation between pulmonary function (percent predicted forced expiratory volume in 1 second, or ppFEV1) and heme B, not PCA or any other phenazine. To independently test this observation, we performed a new tandem mass-spectrometry analysis of 71 additional samples provided by the Mountain West CF Consortium Sputum Biomarker study and revealed a positive correlation (ρ = −0.47, p<0.001) between sputum heme concentrations and ppFEV1. Given that hemoptysis is strongly associated with airway inflammation, pulmonary exacerbations and impaired lung function, these new data suggest that heme B may be a useful biomarker of CF pathophysiology.

Heme protects Pseudomonas aeruginosa and Staphylococcus aureus from calprotectin-induced iron starvation

“…For P. aeruginosa and S. aureus in particular, the current work suggests that heme utilization may prevent iron starvation by CP during infection and may consequently alter their dynamics during coinfection. A recent study indicated that heme levels in the CF lung negatively correlate with lung function ( 60 ). Thus, the possibility that heme enables resistance of S. aureus and P. aeruginosa to iron-sequestering host factors such as lactoferrin and CP in the CF lung, and ultimately contributes to loss of lung function, warrants further investigation.…”

Intraspecies Signaling between Common Variants of Pseudomonas aeruginosa Increases Production of Quorum-Sensing-Controlled Virulence Factors

“…3D ), and we speculate that this is due to increased pyochelin production by ∆ pvdA cells, but future studies will be required to test this model. It was interesting to find that in WT/∆ lasR coculture, heme-related proteins, hasAP , hasS , and hasD , were among the top eight most-upregulated genes by the ∆ lasR strain because the presence of lasR mutants and heme content are both reported biomarkers of disease progression in CF patients ( 13 , 86 ). Coculture-induced lasR mutant phenotypes may link these two correlative observations.…”

Exploiting pyocyanin to treat mitochondrial disease due to respiratory complex III dysfunction

“…injected PYO in BALB/c mice significantly increased glutathione-S-transferase activity only in the heart, but not in other tissues 53 . Although detection of PYO and of its metabolites is not an easy task 54 , our pharmacokinetic profiling was able to reliably detect PYO, and indicated that optimization of PYO administration in mice is required before testing its effect on behavioral parameters in vivo.…”