Blocking Phosphatidylcholine Utilization in Pseudomonas aeruginosa, via Mutagenesis of Fatty Acid, Glycerol and Choline Degradation Pathways, Confirms the Importance of This Nutrient Source In Vivo

Abstract

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.

Glycerophospholipid synthesis and functions in Pseudomonas

“…Mammalian lungs are naturally coated by indispensable lung surfactant, which is composed of approximately 80% of PC (Bernhard et al, 2001). This lipid serves as an important nutrient for P. aeruginosa during cystic fibrosis lung infection (Sun et al, 2014). P. aeruginosa hemolytic phospholipase C (PlcH) is a secreted hydrolase that degrades hostassociated PC (Jackson et al, 2013;Sage and Vasil, 1997;Wargo et al, 2011).…”

Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators

“…In addition to its role as a nutrient source, choline metabolism can generate glycine betaine (GB), an important osmoprotectant (12) and inducer of virulence factor production (13). The conversion of choline to GB has been shown to be important for Escherichia coli survival in urine (14) and Pseudomonas aeruginosa survival in the mammalian lung (15,16). Many organisms, including Burkholderiales, maintain an intracellular GB pool, potentially as a hedge against future osmotic stress (17).…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…aeruginosa secretes PlcB, a Zn 2ϩ metalloPLC essential for directed twitching motility up a phospholipid gradient of either PE or PC (209). Lung surfactant PC serves as a primary carbon and energy source during P. aeruginosa lung infections (210,211), and therefore, the ability of P. aeruginosa to travel along spatio-chemical gradients by chemotaxis could be critical for fitness and virulence. Hence, PlcB could play a role in pathogenicity during lung infections, although this remains to be assessed.…”

“…Catabolism of the PlcH-released phosphorylcholine provides nutrients and activates biofilm formation and anaerobic metabolism, thus contributing to bacterial fitness and survival in the lungs (210,211,259). Furthermore, SM hydrolysis by PlcH and Cer accumulation in epithelial cells could contribute to pathogenesis of P. aeruginosa lung infections by inhibiting the function of the cystic fibrosis transmembrane conductance regulator Cl Ϫ channel, which leads to thick mucus production that clogs the airways, fostering bacterial growth (49).…”