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The glycerol-3-phosphate dehydrogenases GpsA and GlpD constitute the oxidoreductive metabolic linchpin for Lyme disease spirochete host infectivity and persistence in the tick

['Dan Drecktrah', 'Division Of Biological Sciences', 'University Of Montana', 'Missoula', 'Montana', 'United States Of America', 'Laura S. Hall', 'Bethany Crouse', 'Benjamin Schwarz', 'Laboratory Of Bacteriology']

Date: 2022-05

We have identified GpsA, a predicted glycerol-3-phosphate dehydrogenase, as a virulence factor in the Lyme disease spirochete Borrelia (Borreliella) burgdorferi: GpsA is essential for murine infection and crucial for persistence of the spirochete in the tick. B. burgdorferi has a limited biosynthetic and metabolic capacity; the linchpin connecting central carbohydrate and lipid metabolism is at the interconversion of glycerol-3-phosphate and dihydroxyacetone phosphate, catalyzed by GpsA and another glycerol-3-phosphate dehydrogenase, GlpD. Using a broad metabolomics approach, we found that GpsA serves as a dominant regulator of NADH and glycerol-3-phosphate levels in vitro, metabolic intermediates that reflect the cellular redox potential and serve as a precursor for lipid and lipoprotein biosynthesis, respectively. Additionally, GpsA was required for survival under nutrient stress, regulated overall reductase activity and controlled B. burgdorferi morphology in vitro. Furthermore, during in vitro nutrient stress, both glycerol and N-acetylglucosamine were bactericidal to B. burgdorferi in a GlpD-dependent manner. This study is also the first to identify a suppressor mutation in B. burgdorferi: a glpD deletion restored the wild-type phenotype to the pleiotropic gpsA mutant, including murine infectivity by needle inoculation at high doses, survival under nutrient stress, morphological changes and the metabolic imbalance of NADH and glycerol-3-phosphate. These results illustrate how basic metabolic functions that are dispensable for in vitro growth can be essential for in vivo infectivity of B. burgdorferi and may serve as attractive therapeutic targets.

Lyme disease (borreliosis) is the most common tick-borne disease in the Northern hemisphere and its prevalence is increasing. Borrelia burgdorferi, the etiological agent of Lyme disease, is an enzootic pathogen that alternates between a tick vector and vertebrate host. Humans are considered an incidental host after transmission of B. burgdorferi following the bite of an infected tick. The mechanisms by which B. burgdorferi persists in the Ixodid tick, transmits to a vertebrate host and establishes infection are not well understood. Therefore, identifying virulence factors and uncovering the pathogenic strategies in the spirochete remain important to address the public health concerns of Lyme disease. In this study, we identify an enzyme involved in three-carbon metabolism, GpsA, as a new virulence factor with an effect on persistence in ticks. GpsA and GlpD, another enzyme, constitute a bidirectional metabolic node connecting lipid biosynthesis and glycolysis, which serves as the linchpin for regulating carbon utilization for B. burgdorferi throughout its enzootic cycle. Disruption of this node causes a lethal metabolic imbalance revealing a potential therapeutic target for the treatment of Lyme disease.

Funding: This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R01AI130247 to DSS, DD and MCL) and provided by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases of the National Institutes of Health (CMB and FG). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

In this study we examine the in vivo role of the GpsA/GlpD metabolic node in murine infectivity and tick persistence in an animal model of Lyme disease. Additionally, we molecularly dissect the contributions and interactions in vitro of these two G3PDHs in spirochete survival and morphology as well as broad metabolic regulation using a metabolomics approach.

GlpD shuttles G3P towards glycolysis via DHAP to provide energy while the reverse reaction, reducing DHAP to G3P mediated by another predicted G3PDH, GpsA, connects carbohydrates entering glycolysis to lipid and lipoprotein biosynthesis ( Fig 1 ) [ 5 – 7 ]. In the absence of glycerol, GpsA, using carbohydrates from glycolysis, serves as the only known pathway to provide G3P for lipid and lipoprotein biosynthesis as B. burgdorferi cannot import this phosphorylated sugar alcohol or salvage glycerolipids [ 24 ]. B. burgdorferi must coordinate the activities of GlpD and GpsA to efficiently balance carbon sources and redox cofactors, such as NADH, and respond to the physiological requirements of the spirochete. Thus, the GlpD/GpsA metabolic node regulates carbon flow between lipid biosynthesis and glycolysis in response to the phase of the enzootic cycle as indicated by the available carbon sources.

(A) Schematic overview of the intersection of glycerol metabolism and glycolysis, including the conversion of pyruvate to lactate and the use of glycerol-3-phosphate (G3P) for lipid and lipoprotein biosynthesis. Dihydroxyacetone phosphate (DHAP); glycerol uptake facilitator (GlpF, BB0240); glycerol kinase (GlpK, BB0241); glycerol-3-phosphate dehydrogenase (GlpD, BB0243); glycerol-3-phosphate dehydrogenase (GpsA, BB0368); triose phosphate isomerase (TPI); phosphotransferase systems (PTS); lactate permease (LctP). (B) This redox junction consists of two predicted glycerol-3-phosphate dehydrogenases, GlpD and GpsA. GlpD putatively oxidizes G3P to DHAP, while reducing flavin adenine dinucleotide (FAD) or NAD + , to feed glycerol into glycolysis. GpsA reduces DHAP to G3P, using the reducing power of NAD(P)H, to provide carbohydrates for lipoproteins and glycerophospholipids.

Available carbon sources are a dynamic determinant of B. burgdorferi persistence and transit through the enzootic cycle [ 7 , 8 ]. Glucose is likely the preferred carbon source of B. burgdorferi in the vertebrate host and initially during tick feeding, but other carbohydrates can support growth and have a role during the enzootic cycle [ 9 – 11 ]. In particular, glycerol becomes important for B. burgdorferi persistence in the tick as glucose levels decrease when the blood meal is consumed by the tick and its microbiome. This importance is exemplified by the finding that B. burgdorferi mutants unable to utilize glycerol for glycolysis are significantly compromised for persistence in the tick, yet remain infectious in the vertebrate host [ 12 – 14 ]. Glycerol also supports B. burgdorferi growth in vitro, particularly at 23°C, the temperature often used to mimic tick-like conditions [ 12 , 13 , 15 ]. Glycerol enters the cell through the glycerol uptake facilitator GlpF, is converted to glycerol 3-phosphate (G3P) by the glycerol kinase GlpK and either is shuttled to glycolysis via conversion to dihydroxyacetone phosphate (DHAP) by the glycerol-3-phosphate dehydrogenase (G3PDH) GlpD or serves as the three-carbon backbone for lipid and lipoprotein biosynthesis ( Fig 1 ) [ 5 – 7 ]. The glp operon, consisting of the glpF, glpK and glpD genes, along with bb0242, is controlled by a diverse repertoire of regulatory mechanisms [ 16 ]. Gene expression is induced during nutrient stress by the stringent response mediated by Rel Bbu , (p)ppGpp and the effector protein DksA [ 15 , 17 , 18 ], by the response regulator Rrp1, which produces c-di-GMP [ 13 , 19 ], at 23°C [ 20 ], by glycerol [ 13 , 15 ], and in the tick [ 12 ]. RpoS and BadR both repress levels of glp operon transcripts, although BadR likely exerts its influence through RpoS [ 21 , 22 ]. Additionally, the c-di-GMP effector protein PlzA can either positively or negatively affect glp operon expression depending on its c-di-GMP-binding state [ 23 ]. These regulatory pathways targeting the glp operon represent the best understood strategies that B. burgdorferi uses to persist in the tick and illustrate the central role of glycerol regulation in this phase of the enzootic cycle. Other genes involved in carbohydrate utilization, such as malQ and chbC, are not required for B. burgdorferi in either its vertebrate host or tick vector [ 10 , 11 ].

Lyme disease is the most prevalent arthropod-borne infection in North America with an estimated 476,000 cases annually in the United States [ 1 ]. Borrelia (Borreliella) burgdorferi, the enzootic spirochete that causes Lyme disease [ 2 – 4 ], is maintained in nature by cycling between Ixodes ticks and a vertebrate host reservoir, primarily white-footed mice; the bacterium is neither free-living nor transovarially transmitted by the female ticks to oocytes. The reduced genome of B. burgdorferi reflects the constraints of host dependence where numerous biosynthetic and energy-producing metabolic pathways have been lost, including amino acid synthesis, nucleotide synthesis, fatty acid synthesis, the citric acid cycle, and the electron transport chain [ 5 , 6 ]. Thus, B. burgdorferi has evolved into an unabashed scavenger of amino acids, nucleosides, peptides and various carbon sources including glucose, N-acetylglucosamine (GlcNAc) and glycerol. The metabolic capacity retained by B. burgdorferi to flourish in the disparate environments of the vertebrate host and tick vector is important to understand as these strategies, in the absence of any identified toxins or secreted effectors, determine survival of the spirochete and thus the pathogenesis of Lyme disease.

Results

B. burgdorferi has a reduced genome resulting in limited metabolic capacity where the only identified connection of glycerol metabolism and lipid biosynthesis to glycolytic energy production is the bidirectional oxidoreductase node mediated by the opposing actions of a pair of G3PDHs GlpD and GpsA (Fig 1) [5–7]. Based on sequence homology, GlpD is thought to oxidize G3P to DHAP and concomitantly reduce either NAD+ or FAD. GpsA is predicted to catalyze the reverse reaction to reduce DHAP to G3P using the reducing power of NADH or NADPH. Pappas et al., 2011 [12] and He et al., 2011 [13] have shown that GlpD and the glp operon, respectively, are important for B. burgdorferi growth on glycerol and for persistence in the tick, but dispensable for murine infectivity. The function of GpsA either in vitro or in vivo has not previously been evaluated in B. burgdorferi.

B. burgdorferi gpsA complements the growth defect of an Escherichia coli gpsA mutant To genetically confirm the predicted function of B. burgdorferi GpsA, we heterologously complemented the growth phenotype of an E. coli gpsA mutant. The B. burgdorferi gpsA (bb0368) gene was cloned into the E. coli (Ec) isopropyl β-d-1-thiogalactopyranoside (ITPG)-inducible expression vector pUC18. The E. coli gpsA mutant BB20-14 [25], which is a G3P auxotroph and cannot grow on glucose as the sole carbon source, was made competent and transformed with either the empty vector, pUC18, or the vector carrying the B. burgdorferi gpsA gene, pUC18-gpsA Bb . The strains Ec ΔgpsA (BB20-14), Ec ΔgpsA+pUC18 (empty vector) and Ec ΔgpsA+pUC18-gpsA Bb (expressing B. burgdorferi gpsA) were inoculated at 5 × 107 cells ml-1 and grown in M9 minimal salts media (Fig 2A) or M9 minimal salts media with glucose (Fig 2B). Cultures were grown at 37°C for 9 h with the OD 600 taken every 17 min. In M9 media lacking a carbon source, all three strains grew at approximately the same rate and failed to grow exponentially or reach high cell density (Fig 2A). When glucose was added as the sole carbon source to the M9 media, only the strain expressing B. burgdorferi gpsA (Ec ΔgpsA+pUC18-gpsA Bb ) had sustained exponential growth and grew to high cell density (>5 × 108 cells ml-1) (Fig 2B). These data demonstrate that B. burgdorferi gpsA can heterologously complement the growth phenotype of an E. coli gpsA mutant on glucose as a sole carbon source, providing experimental support for the annotated function of gpsA in B. burgdorferi. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Heterologous complementation of an E. coli gpsA mutant with B. burgdorferi gpsA restores growth in glucose. The E. coli gpsA null mutant, strain BB20-14 (Ec ΔgpsA, white circles), E. coli gpsA null mutant with the inducible pUC18 expression vector (Ec ΔgpsA pUC18, gray circles) or E. coli gpsA null mutant with pUC18 carrying the B. burgdorferi gpsA gene (Ec ΔgpsA pUC18-gpsA Bb ) were grown in M9 minimal media containing 0.1 mM IPTG either without (A) or with 1% glucose (B) at 37°C. Cell density measurements (OD 600 ) were taken every 17 min. Data are the average from two separate cultures for each strain and error bars represent SEM; the experiment shown is representative of three independent biological replicates. The Ec ΔgpsA pUC18-gpsA Bb strain had significantly higher (p < 0.05) OD 600 values compared to the other two strains from 4 h to 8 h of growth, as determined by one-way ANOVA with a Tukey’s post-hoc test. https://doi.org/10.1371/journal.ppat.1010385.g002

ΔgpsA, ΔglpD and ΔgpsA/ΔglpD mutant strains have wild-type growth in vitro To investigate the biological role of the metabolic node that interconverts G3P and DHAP regulated by GpsA and GlpD, the genes gpsA or glpD were mutated alone or together by allelic exchange with antibiotic resistance cassettes to generate ΔgpsA, ΔglpD and the double mutant ΔgpsA/ΔglpD strains. All strains were complemented in cis to yield the full array of complemented strains: gpsA complemented (gpsA+), glpD complemented (glpD+), gpsA complemented in the ΔgpsA/ΔglpD background (gpsA+/ΔglpD), glpD complemented in the ΔgpsA/ΔglpD background (ΔgpsA/glpD+) and both gpsA and glpD complemented in the ΔgpsA/ΔglpD background (gpsA+/glpD+) (S1 Fig). Immunoblot analyses of the mutants using antibodies against GlpD and GpsA were used to confirm the deletion and complementation of the respective genes (S1H and S1I Fig). To genetically evaluate the role of gpsA and glpD during in vitro growth, cultures were grown in Barbour-Stoenner-Kelly II media containing 6% rabbit serum (BSK + RS) for eight days and spirochetes were enumerated using a Petroff-Hauser cell counter. No statistical difference in growth was observed between wild-type, ΔgpsA, ΔglpD or the double mutant ΔgpsA/ΔglpD strains after day one (Fig 3A). These data suggest that neither gpsA nor glpD play a significant role during B. burgdorferi growth in nutrient-rich media in vitro. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Growth of the gpsA and glpD mutants and survival under nutrient stress without or with different carbon sources in vitro. (A) B. burgdorferi strains were inoculated at 1 × 105 cells ml-1 in BSK + RS and grown at 35°C. Cells were enumerated every 24 h and cell density plotted over eight days. Data are the means from three independent biological replicates and error bars represent SEM. No significant difference in cell density was determined except at day 1 between the wild-type (WT) and ΔgpsA/ΔglpD strains: * indicates p = 0.046 between the mean cell density of WT and ΔgpsA/ΔglpD strains at day 1 as determined by one-way ANOVA with a Tukey’s post-hoc test. Strains were grown in BSK + RS at 35°C (to a cell density of 5–9 × 107 cells ml-1) before shifting to RPMI alone (B), or RPMI containing 0.4% glycerol (C) or 0.4% N-acetylglucosamine (GlcNAc) (D) and incubated at 35°C for 24 h. Cultures were plated in semi-solid BSK media and allowed to grow at 35°C in 5% CO 2 before colony enumeration. Data are presented as the percent survival of each strain before (0 h) shifting to the nutrient stress media. Data are the mean of at least three biological replicates and errors bars represent the SEM. Significance determined by one-way ANOVA with a Tukey’s post-hoc test. (* p < 0.0001; ** p < 0.002; # p < 0.01; † p = 0.0075). https://doi.org/10.1371/journal.ppat.1010385.g003

GpsA is required for survival during nutrient stress We hypothesized that GpsA functions in survival during nutrient stress, an in vitro condition used to mimic the tick midgut environment between bloodmeals. To test this, B. burgdorferi strains were grown to 7–9 × 107 cells ml-1 in normal growth medium, collected, resuspended and incubated in RPMI medium (which contains 2 mg ml-1 glucose) for 24 h, as previously described [18]. Cells were plated in semi-solid BSK, and colonies allowed to grow for approximately two weeks before enumeration. B. burgdorferi survival is represented as the percentage survival after 24 h of nutrient stress compared to cells plated before nutrient stress (0 h). ΔgpsA mutants were almost completely compromised for survival during nutrient stress, while the ΔglpD mutant was not significantly affected compared to wild type (Fig 3B). This phenotype is restored in the gpsA+ and, surprisingly, in the ΔgpsA/ΔglpD double mutant (Fig 3B). Complementing the double mutant with glpD, thus essentially constructing an independent gpsA mutant, also resulted in a B. burgdorferi strain unable to survive nutrient stress, similar to the ΔgpsA mutant (ΔgpsA/glpD+ Fig 3B). gpsA complementation of the double mutant and complete complementation of the double mutant significantly increased survival compared to ΔgpsA, but did not fully restore survival to wild-type levels (gpsA+/ΔglpD and gpsA+/glpD+, Fig 3B). These data suggest that gpsA plays a crucial role in survival during nutrient stress in culture while glpD is dispensable. Furthermore, our results suggest we have identified the first suppressor mutation in B. burgdorferi as deleting the glpD gene in a ΔgpsA mutant background restored viability under nutrient stress. Because the link from glycolysis to G3P metabolism is severed in the ΔgpsA mutant, we determined if glycerol could restore survival of the gpsA mutant in nutrient stress medium (RPMI + glycerol). Strains were grown and treated as in Fig 3B except that 0.4% glycerol was added to the RPMI medium. Unexpectedly, glycerol in the nutrient stress medium was cytotoxic to wild-type B. burgdorferi (Fig 3C). In fact, glycerol was toxic to all strains except the ΔglpD mutants (Fig 3C), suggesting that metabolism of G3P by GlpD is necessary for the bactericidal activity of glycerol in this restrictive medium. Next, we examined if GlcNAc, a carbohydrate necessary for in vitro growth [9,10,26], could rescue the gpsA survival defect in RPMI medium. The results with GlcNAc were similar to those with glycerol: survival in GlcNAc was significantly compromised in almost all strains compared to those lacking glpD (Fig 3D). The glpD complement of the double ΔgpsA/ΔglpD mutant (ΔgpsA/glpD+) was significantly compromised for survival compared to the ΔgpsA/ΔglpD mutant (Fig 3D). GlcNAc is not predicted to be metabolized by the action of GlpD, suggesting an unidentified link between GlpD and the metabolism of GlcNAc, possibly involving redox cofactors involved in the GlpD/GpsA oxidoreductase node. Together these results illuminate the importance of the metabolic balance of the intermediates and cofactors involved in the GpsA/GlpD node in B. burgdorferi adaptation to and survival during changing carbohydrate availability.

The gpsA mutant displays increased round body formation and decreased reductase activity To assess the redox potential of the G3PDH mutants, overall reductase activity in individual cells was monitored by microscopy using a fluorescent reporter. Wild-type and mutant strains were grown to ~7–9 × 107 cells ml-1, collected and incubated in RPMI medium (nutrient stress) for 16 h. Cultures were then incubated with BacLight RedoxSensor Green and propidium iodide for 10 min before wet-mounting live cells to be imaged by fluorescence microscopy. RedoxSensor Green stain fluoresces (shown as cyan in Fig 4) when reduced, indicating intracellular reductase activity and cell viability. Almost all (90%) wild-type cells in nutrient stress media for 16 h show reductase activity/viability while only about 30% of ΔgpsA cells fluoresce (cyan staining, Fig 4A, 4B, and 4F). Strikingly, ΔgpsA cells undergo a dramatic morphological change from flat-wave to a condensed spherical form called round bodies (RBs) during nutrient stress that is rarely seen in wild-type B. burgdorferi (Fig 4A, 4B, and 4F). The physiological role of RBs remains unknown but the transition is triggered by environmental stress and may be related to persistence, as this form has been observed in vivo in ticks [18,27–29]. Both the decrease in reductase activity and the increase in RB formation in ΔgpsA mutant cells are restored not only in the gpsA+ strain but also in the ΔgpsA/ΔglpD double mutant (Figs 4C, 4D, and 4F), which is further evidence of glpD functioning as a suppressor mutation of the pleiotropic gpsA phenotypes. Deletion of the glpD gene alone affected neither reductase activity nor morphology during nutrient stress (Fig 4E and 4F). These data further support the findings that GpsA is important for cell survival under nutrient stress, a result likely reflected in reduced reductase activity. Additionally, the dramatic increase in RB formation of the ΔgpsA mutant is evidence that GpsA is a key modulator of morphological changes during nutrient stress. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Decreased reductase activity and increased round body formation in the gpsA mutant. (A) Wild-type (WT), (B) ΔgpsA mutant, (C) gpsA+ complement, (D) ΔgpsA/ΔglpD double mutant and (E) ΔglpD mutant strains were grown in BSK + RS at 35°C (to a cell density of 5–9 × 107 cells ml-1) before shifting to RPMI for 16 h at 35°C. Bacterial reductase activity was detected by staining with RedoxSensor Green and membrane integrity assessed by staining with propidium iodide (PI). Live cells were imaged by fluorescence microscopy for RedoxSensor Green (cyan) and PI (magenta) and overlaid with white light images. (F) The percentage of cells stained with RedoxSensor Green and the percentage of cells in round body (RB) form was quantified. Data are the mean of three biological replicates and error bars represent the SEM. Asterisks signify a significant difference (p ≤ 0.0001) between the mean percent of RedoxSensor-stained ΔgpsA cells compared to all other strains and the mean percent of RBs in the ΔgpsA strain compared to all other strains as determined by one-way ANOVA with a Tukey’s post-hoc test. https://doi.org/10.1371/journal.ppat.1010385.g004 A recent study has implicated GpsA in resistance of Streptococcus pneumoniae to oxidative stress [30]. To examine if gpsA also protects B. burgdorferi from oxidative stress, we measured the viability of wild-type, ΔgpsA and gpsA+ strains following exposure to H 2 O 2 and found no significant differences between the strains, at least under the conditions tested (S2 Fig).

GpsA and GlpD influence the B. burgdorferi metabolome To better understand how GpsA and GlpD affect the global physiology of B. burgdorferi, we performed semi-targeted metabolomics by liquid chromatography-tandem mass spectroscopy on the ΔgpsA and ΔglpD mutants in culture. Strains were grown in BSK + RS at 35°C to a density of ~3 × 107 cells ml-1 before processing and analysis. Comparing the mutant metabolomes to wild type by unbiased principal component analysis, separate axes were readily observed that defined the metabolic effects of ΔgpsA versus the metabolic effects of ΔglpD. Complements of both mutants resulted in a return toward wild type and the ΔgpsA/ΔglpD double mutant comigrated with ΔglpD in agreement with the increased survival phenotype of the ΔgpsA/ΔglpD double mutant (Fig 5A). The first principal component, which defines the separation of ΔgpsA from wild type, is heavily loaded by the opposing behavior of ATP and AMP, positively loaded for NADH, and negatively loaded with G3P and three-carbon glycolytic intermediates (Fig 5B). By contrast, ΔglpD separation along principal component two is positively loaded for G3P and negatively loaded for NADH in support of an opposing reaction directionality between GpsA and GlpD. Interestingly, both primary principal components are positively loaded for AMP and negatively loaded for ATP suggesting that proper functioning of the G3P arm of B. burgdorferi metabolism is essential for energy metabolism (Fig 5B). PPT PowerPoint slide

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TIFF original image Download: Fig 5. GpsA regulates central metabolism and redox balance. (A) Polar metabolomics of bacterial strains subjected to principal component (PC) analysis with pareto-scaling. (B) The corresponding metabolite loading distribution for the analysis displayed in (A) for principal components 1 and 2. (C) Metabolites that significantly vary between ΔgpsA and wild type (WT) with a false discovery rate (FDR) less than 5%. Values in the heatmap at left are displayed as the log 2 (fold change mutant versus WT) and values in the accompanying heatmap at right indicate whether that metabolite passes a 5% FDR filter for the indicated comparison as assessed by a Benjamini-Hochberg correction. (D) Metabolic map of the changes in glycolysis and the glycerol shunt that occur with the loss of GpsA. All measured metabolites in the included pathways are displayed. The log 2 (fold change ΔgpsA versus WT) is displayed as color of the node and the -log(p-value) is displayed as the size of the node. Enzymes in the glycerol arm of metabolism are displayed as diamonds. Data are from four independent biological replicates. https://doi.org/10.1371/journal.ppat.1010385.g005 In further support of the anticipated enzymatic function of GpsA, univariate analysis of the metabolite datasets showed that the largest metabolic changes in the ΔgpsA mutant compared to wild type were localized to the putative redox cofactor (NADH, 23.7-fold increase) and the anticipated product (G3P, 20.5-fold decrease) of GpsA (Fig 5C). Despite its predicted role as a substrate of GpsA, DHAP levels were only 1.7-fold higher in the ΔgpsA mutant. This smaller effect compared to G3P and NADH is likely due to triose phosphate isomerase converting DHAP to glyceraldehyde-3-phosphate (GAP) for glycolysis (Fig 5D). With the exceptions of DHAP, GAP, and fructose-1,6-bisphosphate, glycolytic intermediates, particularly pyruvate (6.6-fold decrease), were decreased in the ΔgpsA mutant. Concomitantly, ATP levels decreased (2.1-fold) and AMP levels increased (2.6-fold). These changes in glycolysis likely drive the decreased energy levels in the cell and may account for the susceptibility of the ΔgpsA mutant strain to nutrient stress (Fig 3). The complete list of metabolite levels in all the mutants is included (S2 Table). The dramatic 23.7-fold increase in NADH levels in the gpsA mutant compared to wild type suggests that GpsA is a dominant regulator of NADH levels in B. burgdorferi. The dysregulation of glycolysis may be a consequence of these elevated levels of NADH, which would inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thus glycolysis in the ΔgpsA mutant [31] (Fig 5D). The changes in metabolite levels in the ΔgpsA mutant were largely restored to wild-type levels in the gpsA+, but energy stress was still apparent in elevated AMP levels (Fig 5C). Metabolite levels in the ΔgpsA/ΔglpD double mutant shifted toward wild-type levels, although not as completely as in the gpsA+. Univariate analysis further supported the action of GlpD as metabolically opposed to GpsA. While the metabolic phenotype in the ΔglpD mutant was of a smaller magnitude compared to the ΔgpsA mutant, DHAP levels in the ΔglpD mutant were 4.9-fold lower and G3P levels 4.2-fold higher than in the wild type (S3A Fig), supporting the predicted GlpD function. NADH levels were lower in the ΔglpD mutant, suggesting that either NADH is the reduced cofactor as G3P is oxidized to DHAP or that the effects of glpD deletion on GAP limit the recovery of NADH by GAPDH (S3B Fig). The effects on glycolysis in the ΔglpD mutant were localized to DHAP, GAP, phosphoenolpyruvate, and pyruvate (S3A Fig). Complementation of the ΔglpD mutant overcorrected the elevated levels of G3P and somewhat restored levels of NADH associated with glpD deletion suggesting the reintroduction of active enzyme (S3A Fig). However, dysregulation of glycolysis and energy metabolism was not restored between the ΔglpD mutant and the glpD+, indicating that there was only partial restoration of the wild-type phenotype even though glpD was complemented in cis with the native glp promoter.

GpsA regulates the NADH/NAD+ ratio To confirm and quantify the GpsA-mediated regulation of nicotinamide cofactor levels observed in the metabolomics analysis, the NADH/NAD+ molar ratios in the ΔgpsA mutant were measured via an in vitro luminescence assay. Strains were grown and samples prepared as described for the metabolomics studies before measuring the NADH and NAD+ levels. The NADH/NAD+ molar ratio was approximately fourfold higher in the ΔgpsA mutant strain than in wild-type cells (Fig 6). This difference was fully restored in the gpsA+, but, curiously, not in the double ΔgpsA/ΔglpD mutant strain (Fig 6). These data support the finding that gpsA highly regulates NADH levels in B. burgdorferi (Fig 5). PPT PowerPoint slide

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TIFF original image Download: Fig 6. GpsA regulates NADH/NAD+ levels. Wild-type (WT, black circles), ΔgpsA mutant (ΔgpsA, white circles), gpsA complemented (gpsA+, dark gray circles) and ΔgpsA/ΔglpD double mutant (light gray circles) strains were grown in BSK + RS at 35°C to ~5 × 107 cells ml-1 and NAD+ and NADH levels were measured with the NAD/NADH-Glo Assay. Each point represents a single biological replicate and bars represent the means. Asterisks represent a significant difference (p < 0.0001) in the means of NADH/NAD+ molar ratios of both the ΔgpsA mutant and the ΔgpsA/ΔglpD double mutant compared to those of both the wild type and the gpsA+ determined by one-way ANOVA with a Tukey’s post-hoc test. https://doi.org/10.1371/journal.ppat.1010385.g006

GpsA and GlpD levels are independent of each other Because GpsA and GlpD catalyze the interconversion of G3P and DHAP, B. burgdorferi may respond to the mutation of one enzyme by altering the levels of the other G3PDH to compensate. To examine this possibility, levels of GpsA protein were examined in the ΔglpD mutant and levels of GlpD protein were examined in the ΔgpsA mutant by immunoblot analyses. Cell lysates analyzed from wild-type, ΔglpD mutant and glpD+ strains grown in normal growth medium (BSK + RS) at 35°C or incubated in nutrient stress medium (RPMI) showed no appreciable difference in GpsA protein levels (Fig 7A). FlaB was used as a loading control (Fig 7A and 7B). Similarly, GlpD levels did not change in the ΔgpsA mutant compared to the wild type or the gpsA+ under similar conditions (Fig 7B). Thus, GpsA and GlpD protein levels are independent of each other, at least during in vitro culture. PPT PowerPoint slide

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TIFF original image Download: Fig 7. GpsA and GlpD levels are independent. (A) Wild-type (WT), ΔglpD mutant (ΔglpD) and glpD complemented (glpD+) strains were grown in BSK + RS at 35°C or shifted to nutrient stress medium (RPMI) for 24 h before total cell lysates were collected. Samples were separated by SDS-PAGE and analyzed by immunoblot with antibodies against GpsA or FlaB (as a control). GpsA is the lower band of the doublet (see S1H Fig). (B) WT, ΔgpsA mutant (ΔgpsA) and gpsA complemented (gpsA+) strains were grown and analyzed as in (A) except antibodies against GlpD were used for the immunoblots in the upper panels. Three independent experiments were done and representative data are shown. https://doi.org/10.1371/journal.ppat.1010385.g007

[END]

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