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. 2018 Sep 11;9(5):e00961-18.
doi: 10.1128/mBio.00961-18.

The Pseudomonas Aeruginosa Complement of Lactate Dehydrogenases Enables Use of D- And l-Lactate and Metabolic Cross-Feeding

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Free PMC article

The Pseudomonas Aeruginosa Complement of Lactate Dehydrogenases Enables Use of D- And l-Lactate and Metabolic Cross-Feeding

Yu-Cheng Lin et al. mBio. .
Free PMC article

Abstract

Pseudomonas aeruginosa is the most common cause of chronic, biofilm-based lung infections in patients with cystic fibrosis (CF). Sputum from patients with CF has been shown to contain oxic and hypoxic subzones as well as millimolar concentrations of lactate. Here, we describe the physiological roles and expression patterns of P. aeruginosa lactate dehydrogenases in the contexts of different growth regimes. P. aeruginosa produces four enzymes annotated as lactate dehydrogenases, three of which are known to contribute to anaerobic or aerobic metabolism in liquid cultures. These three are LdhA, which reduces pyruvate to d-lactate during anaerobic survival, and LldE and LldD, which oxidize d-lactate and l-lactate, respectively, during aerobic growth. We demonstrate that the fourth enzyme, LldA, performs redundant l-lactate oxidation during growth in aerobic cultures in both a defined MOPS (morpholinepropanesulfonic acid)-based medium and synthetic CF sputum media. However, LldA differs from LldD in that its expression is induced specifically by the l-enantiomer of lactate. We also show that the P. aeruginosa lactate dehydrogenases perform functions in colony biofilms that are similar to their functions in liquid cultures. Finally, we provide evidence that the enzymes LdhA and LldE have the potential to support metabolic cross-feeding in biofilms, where LdhA can catalyze the production of d-lactate in the anaerobic zone, which is then used as a substrate in the aerobic zone. Together, these observations further our understanding of the metabolic pathways that can contribute to P. aeruginosa growth and survival during CF lung infection.IMPORTANCE Lactate is thought to serve as a carbon and energy source during chronic infections. Sites of bacterial colonization can contain two enantiomers of lactate: the l-form, generally produced by the host, and the d-form, which is usually produced by bacteria, including the pulmonary pathogen Pseudomonas aeruginosa Here, we characterize P. aeruginosa's set of four enzymes that it can use to interconvert pyruvate and lactate, the functions of which depend on the availability of oxygen and specific enantiomers of lactate. We also show that anaerobic pyruvate fermentation triggers production of the aerobic d-lactate dehydrogenase in both liquid cultures and biofilms, thereby enabling metabolic cross-feeding of lactate over time and space between subpopulations of cells. These metabolic pathways might contribute to P. aeruginosa growth and survival in the lung.

Keywords: biofilms; lactate isomers; pyruvate; pyruvate fermentation.

Figures

FIG 1
FIG 1
The P. aeruginosa genome encodes several enzymes that interconvert pyruvate and lactate. (Left) Reactions catalyzed by P. aeruginosa’s lactate dehydrogenases; (right) chromosomal loci encoding each of the corresponding enzymes. LdhA catalyzes the reduction of pyruvate during anaerobic survival. LldE catalyzes the oxidation of d-lactate during aerobic growth. Unlike E. coli, which contains only one gene encoding an l-lactate dehydrogenase, P. aeruginosa contains two orthologues for this enzyme. LldD catalyzes the oxidation of l-lactate during aerobic growth. This study describes a role for LldA in catalyzing the oxidation of l-lactate during aerobic growth.
FIG 2
FIG 2
Expression of loci associated with pyruvate and lactate metabolism during aerobic, liquid-culture growth. Strains engineered to express GFP under the control of promoters upstream of lldP (which is cotranscribed with lldD and lldE) or lldA were grown in MOPS medium, with the indicated compounds provided as sole carbon sources. Background fluorescence from a strain with a promoterless reporter was subtracted before normalization to the OD at 500 nm. Error bars, which are often obscured by the point markers, represent the standard deviations from biological triplicates. AU, arbitrary units.
FIG 3
FIG 3
Physiological roles of enzymes that interconvert pyruvate and lactate during growth in shaken liquid cultures and biofilms. (A) Aerobic growth of the indicated strains in MOPS medium with d-glucose, l-lactate, or d-lactate provided as the sole carbon source. Error bars, which are obscured by the point marker in most cases, represent the standard deviations from biological triplicates. (B) Growth and morphological development of the indicated strains under an oxic atmosphere on MOPS medium containing the dyes Congo red and Coomassie blue and amended with d-glucose, l-lactate, or d-lactate. Images were taken after 4 days of incubation. WT, wild type.
FIG 4
FIG 4
PA14 utilizes the l-lactate in synthetic cystic fibrosis sputum medium (SCFM) for growth. (A) Growth of the indicated strains in SCFM. Error bars represent the standard deviations from at least four biological replicates and are omitted in cases where they would be obscured by point markers. P values of the ΔlldDE ΔlldA double mutant versus the wild type and ΔlldDE ΔlldA::lldDE strain are <0.0001 based on an unpaired, two-tailed t test. (B) Expression of the indicated reporter constructs in SCFM. Background fluorescence from a strain with a promoterless reporter was subtracted before normalization to the OD at 500 nm.
FIG 5
FIG 5
Growth of PA14 on self-produced d-lactate. (A) Design schematic for a pyruvate/d-lactate cross-feeding experiment in liquid cultures. P. aeruginosa PA14 was incubated in anoxic liquid medium (MOPS plus 0.1% tryptone plus 40 mM sodium pyruvate) to promote the fermentation and production of d-lactate. The supernatants collected from these cultures were used as the growth medium for oxic cultures, in which the d-lactate then served as a carbon source for PA14 growth. (B) Growth of the indicated strains on supernatants obtained from anaerobic cultures that had fermented pyruvate. Error bars represent the standard deviations from three biological replicates. P values for the ΔlldDE mutant versus the wild type, ΔlldA mutant, and ΔldhA mutant are 4.6 × 10−4, 2.2 × 10−4, and 1.1 × 10−3, respectively, and are based on unpaired, two-tailed t test results with equal variances. (C) Expression of the indicated reporter constructs during growth on the supernatant described in panel A. Background fluorescence from a strain with a promoterless reporter was subtracted before normalization to the OD at 500 nm.
FIG 6
FIG 6
Self-produced d-lactate induces lldPDE in biofilms. (A) Design schematic for a pyruvate/d-lactate cross-feeding experiment in liquid cultures in colony biofilms. Colonies were initially grown atop filter membranes on plates containing 1% tryptone, 1% agar plus 40 mM sodium pyruvate for 2 days under an oxic atmosphere to establish biomass and then transferred to fresh plates of medium containing 0.1% tryptone, 1% agar plus 40 mM sodium pyruvate in an anoxic chamber to stimulate pyruvate fermentation and d-lactate production for 2 days. Finally, the plates were moved from the chamber and back into an oxic atmosphere for 1 day. The procedure was carried out at room temperature. (B) Fluorescence quantification (top) and images (bottom) of colonies grown using the procedure shown in panel A. Background fluorescence was normalized to the “no reporter” control. Error bars represent the standard deviations from three biological replicates. P values for lldPDE reporter versus no reporter and lldA reporter are 1.0 × 10−4 and 2.3 × 10−4, respectively, and are based on unpaired, two-tailed t test results with equal variance.
FIG 7
FIG 7
Expression of enzymes for interconversion of pyruvate and lactate over biofilm depth. Expression of ldhA (which catalyzes pyruvate reduction under anaerobic conditions) and lldE (which catalyzes d-lactate oxidation under aerobic conditions) as reported by promoter-gfp fusions in thin sections from wild-type PA14 biofilms. Biofilms were grown for 3 days on 1% tryptone medium with or without 10 mM pyruvate. Quantification of fluorescence over depth is shown at the right. This experiment was repeated in biological triplicate, and representative results are shown. Cyt cred, with reduced cytochrome c; Cyt cox, with oxidized cytochrome c.

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