Robustness and Plasticity of Metabolic Pathway Flux among Uropathogenic Isolates of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a human pathogen that frequently causes urinary tract and catheter-associated urinary tract infections. Here, using 13C-metabolic flux analysis, we conducted quantitative analysis of metabolic fluxes in the model strain P. aeruginosa PAO1 and 17 clinical isolates. All P. aeruginosa strains catabolized glucose through the Entner-Doudoroff pathway with fully respiratory metabolism and no overflow. Together with other NADPH supplying reactions, this high-flux pathway provided by far more NADPH than needed for anabolism: a benefit for the pathogen to counteract oxidative stress imposed by the host. P. aeruginosa recruited the pentose phosphate pathway exclusively for biosynthesis. In contrast to glycolytic metabolism, which was conserved among all isolates, the flux through pyruvate metabolism, the tricarboxylic acid cycle, and the glyoxylate shunt was highly variable, likely caused by adaptive processes in individual strains during infection. This aspect of metabolism was niche-specific with respect to the corresponding flux because strains isolated from the urinary tract clustered separately from those originating from catheter-associated infections. Interestingly, most glucose-grown strains exhibited significant flux through the glyoxylate shunt. Projection into the theoretical flux space, which was computed using elementary flux-mode analysis, indicated that P. aeruginosa metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response.

Large-Scale 13 C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

“…The following labeling strategy allowed elucidation of the type of anaplerotic metabolism in 90% of the marine strains, i.e., in all strains that used the ED pathway. As recently demonstrated, PEP-and pyruvate-based anaplerotic metabolism provides a unique combination of 13 C enrichment in connected pathway intermediates of such bacteria using the ED pathway grown on [1-13 C]glucose (16). Considering the well-defined transition of carbon atoms in the biochemical reactions involved, high 13 C enrichment in pyruvate, together with low 13 C enrichment in oxaloacetate results in PEP carboxylation, whereas the opposite results in pyruvate carboxylation, respectively (Fig.…”

“…The EMP pathway is nearly ubiquitous in the bacterial kingdom (13). It has been argued that this relates to the superior energy efficiency of the EMP pathway (equation 1): it yields twice as much ATP as the ED pathway (equation 2) (14).Glucose ϩ 2 ADP ϩ 2 NAD ϩ → 2 pyruvate ϩ 2 ATP ϩ 2 NADH (1)However, phylogenetically distinct bacteria (Firmicutes, Alphaproteobacteria, and Gammaproteobacteria), including aerobes and anaerobes, autotrophs and heterotrophs, rely on the ED pathway for glucose catabolism (15,16). Quantitative flux through the EMP, ED, and PP pathways is accessible via 13 C fluxomics; in these experiments, quantitative information on fluxes, i.e., the in vivo …”

“…However, phylogenetically distinct bacteria (Firmicutes, Alphaproteobacteria, and Gammaproteobacteria), including aerobes and anaerobes, autotrophs and heterotrophs, rely on the ED pathway for glucose catabolism (15,16). Quantitative flux through the EMP, ED, and PP pathways is accessible via 13 C fluxomics; in these experiments, quantitative information on fluxes, i.e., the in vivo …”

“…Glucose ϩ ADP ϩ NAD ϩ ϩ NADP ϩ → 2 pyruvate ϩ ATP ϩ NADH ϩ NADPH (2) However, phylogenetically distinct bacteria (Firmicutes, Alphaproteobacteria, and Gammaproteobacteria), including aerobes and anaerobes, autotrophs and heterotrophs, rely on the ED pathway for glucose catabolism (15,16). Quantitative flux through the EMP, ED, and PP pathways is accessible via 13 C fluxomics; in these experiments, quantitative information on fluxes, i.e., the in vivo activities of intracellular enzymes and pathways, is obtained from the labeling patterns of metabolites generated during growth on specific 13 C tracer substrates (17,18).…”

“…Such studies have provided valuable insight into the intracellular pathways of microorganisms. For example, industrially relevant microbes have been extensively studied (19), and more recently, fluxomic studies have examined pathogenic (16,20) and ecologically important (21) microorganisms. In contrast, there is still limited knowledge on the intracellular pathways of marine bacteria, and only a few isolates have been investigated so far (22)(23)(24).…”