Membrane-Bound Nitrate Reductase Is Required for Anaerobic Growth in Cystic Fibrosis Sputum

Abstract

The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen Pseudomonas aeruginosa. To form a chronic CF lung infection, P. aeruginosa must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances P. aeruginosa biofilm formation and antibiotic resistance, it is important to examine P. aeruginosa physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant P. aeruginosa growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for P. aeruginosa anaerobic growth with nitrate in the CF lung.Cystic fibrosis (CF) is a heritable disease characterized by accumulation of large volumes of mucus (sputum) within the lungs and persistent colonization with opportunistic pathogens. While numerous bacterial species transiently colonize CF sputum or persist at low cell densities, the opportunistic pathogen Pseudomonas aeruginosa establishes a chronic infection and can attain densities of Ͼ10 9 cells/ml of sputum (19). Chronic P. aeruginosa infection is the leading cause of morbidity and mortality in CF patients (20). Once established, P. aeruginosa CF lung infections are highly refractory to antibiotic treatments, and even aggressive therapies fail to fully eradicate the bacterium from the lung (20). Several aspects of P. aeruginosa physiology are believed to contribute to its high antibiotic resistance in vivo, including biofilm formation and overproduction of the viscous exopolysaccharide alginate (23).P. aeruginosa CF lung infections are frequently modeled under aerobic laboratory conditions; however, direct oxygen measurements within CF lung sputum in situ indicate that sputum contains hypoxic, and potentially anaerobic, regions (38). In addition, strict anaerobic bacteria have been detected in sputum samples from multiple CF patients (21, 29), suggesting that environments capable of sustaining these species are present in vivo. There are several factors that could potentially reduce oxygen levels within CF sputum: cultured CF lung epithelial cells consume oxygen at a higher rate than non-CF lung epithelial cells, oxygen diffusion through sputum is restricted, and oxygen is consumed by resident sputum microorganisms, including P. aeruginosa (38). Collectively these factors may effectively reduce oxygen to levels insufficient for aerobic respiration. Interestingly, ...

Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis

“…Supporting this hypothesis, 21 of 24 Vibrio genes that have been predicted from bioinformatics to be regulated by the nitrite-responsive elements NarPQ during anaerobic respiration (26) have an increased expression in the symbionts during the morning. A number of pathogens, including V. cholerae, also apparently respire nitrate/nitrite when hostassociated, and this metabolism plays a critical, but as yet uncharacterized, role in their virulence (27)(28)(29). The source of nitrate/nitrite in these associations is not known; however, in the squid host it may come from a spontaneous oxidation of NO produced by the light-organ epithelium (30).…”

Discovery of a biofilm electrocline using real-time 3D metabolite analysis

“…NapA is the periplasmic NO 3 − reductase important for reduction of NO 3 − under aerobic conditions (35); thus, the P. aeruginosa napA mutant is unable to divert electrons to NO 3 − under aerobic conditions. As expected, NO 3 − had no impact on the PYO electrocline in the napA mutant (Fig.…”

Low oxygen induces the type III secretion system in Pseudomonas aeruginosa via modulation of the small RNAs rsmZ and rsmY

“…Anr is a homologue of the Escherichia coli anaerobic response master regulator FNR, and it functions by regulating a large number of genes required for anaerobic respiration, including dnr and the narXL two-component system (Sawers, 1991;Trunk et al, 2010;Zimmermann et al, 1991). During lowoxygen stress, the NarL response regulator is involved in coordinating energy metabolism through induction of nitrate metabolism (Palmer et al, 2007;Schreiber et al, 2007;Sharma et al, 2006) and repression of less efficient modes of energy generation such as pyruvate and arginine fermentation (Benkert et al, 2008).…”