Desulfurization of mucin by Pseudomonas aeruginosa: influence of sulfate in the lungs of cystic fibrosis patients

Abstract

Pseudomonas aeruginosa is a common cause of chronic respiratory infection in cystic fibrosis (CF) patients. Infection is established within the lung epithelial mucus layer through adhesion to mucins. Terminal residues on mucin oligosaccharide chains are highly sulfated and sialylated, which increases their resistance to degradation by bacterial enzymes. However, a number of microbes, including P. aeruginosa, display mucin sulfatase activity. Using ion chromatography, the levels of sulfation on different respiratory mucins and the availability of inorganic sulfate to pathogens in sputum from CF patients were quantified. The ability of clinical isolates of P. aeruginosa to desulfate mucin was tested by providing mucin as a sole sulfur source for growth. All tested P. aeruginosa strains isolated from the lungs of CF patients were able to use human respiratory mucin as a source of sulfur for growth, whereas other non-clinical species of the genus Pseudomonas were not. However, measured levels of inorganic sulfate in sputum from CF patients suggested that bacteria resident in the lung have sufficient inorganic sulfate for growth and are unlikely to require access to mucin sulfur as a sulfur source during chronic infection. This was confirmed when expression of sulfate-repressed P. aeruginosa genes atsK and msuE was found to be repressed in the sputum of CF patients, which was detected by using quantitative RT-PCR. These results indicate that sulfate starvation is unlikely to occur in pathogens residing in the sputum of CF patients and, therefore, mucin desulfation may have an alternative purpose in the association between P. aeruginosa and the airways of CF patients.

Evidence and Role for Bacterial Mucin Degradation in Cystic Fibrosis Airway Disease

“…Previous studies have shown that mucins can support the carbon demands of P . aeruginosa in vitro [41,42]; however, these studies included autoclaved preparations of commercial porcine gastric mucin (PGM) that contain low molecular mass compounds that are readily utilized. In fact, when PGM preparations were filtered and dialyzed in our study (leaving only large, intact glycoproteins) appreciable growth of P .…”

Evidence and role for bacterial mucin degradation in cystic fibrosis airway disease

“…However, the process of mucin degradation, and its potential contribution to airway disease has not been addressed in detail. Previous studies have shown that mucins can support the carbon demands of P. aeruginosa in vitro [41,42]; however, these studies included autoclaved preparations of commercial porcine gastric mucin (PGM) that contain low molecular mass compounds that are readily utilized. In fact, when PGM preparations were filtered and dialyzed in our study (leaving only large, intact glycoproteins) appreciable growth of P. aeruginosa isolates was not observed.…”

The Flavin Reductase MsuE Is a Novel Nitroreductase that Can Efficiently Activate Two Promising Next-Generation Prodrugs for Gene-Directed Enzyme Prodrug Therapy

“…, homodimeric, NAD(P)H-consuming, flavin-cofactor-dependent, cytoplasmic oxidoreductases [14,23]). One such candidate was MsuE from P. aeruginosa , previously characterised as an NADH-dependent flavin mononucleotide (FMN) reductase that provides reduced FMN to power desulfonation of alkanesulfonates [24,25]. In an initial rapid qualitative test for nitroreductase activity, employing the nitro-quenched fluorophore FSL 61 as a generic nitroreductase substrate (as per [14]), a nitroreductase gene knockout strain of E. coli over-expressing MsuE from plasmid pUCX exhibited comparable levels of fluorescence to isogenic strains over-expressing E. coli NfsA (NfsA_Ec) or NfsB_Ec (Figure 1).…”