Fig 1.
Schematic representation of enzymes commonly involved in 2-keto acid metabolism and their corresponding reactions.
The enzymes, shown in green boxes, may require cofactors, shown in gold boxes. Additional substrates and products are shown in blue boxes. CBU0823 and CBU1241 have been included with their respective putative annotations. Adapted from [35].
Fig 2.
Key residues of CBU1241 and CBU0823.
Protein sequence alignment sections containing key residues of (A) CBU1241 compared with MDHs and LDHs and (B) CBU0823 compared with MEs and MLEs. The NAD(P)-binding motifs (blue), substrate binding residues (yellow), catalytic activity residues (green), and metal binding residues (grey) are highlighted, and the boxes indicate unusual residues present within CBU0823 versus other bacterial MEs. Numbering refers to the residue position within the respective protein.
Fig 3.
In vitro GST-CBU1241 activity assays for MDH, LDH or ME function.
(A) MDH activity was exhibited by 2 μg GST-CBU1241 and 0.013 units commercial pig heart mitochondria MDH, whereas 2 μg GST alone exhibited no activity in the MDH standard assay containing 2 mM OAA and 0.5 mM NADH in PBS pH 7.4. (B) LDH activity was not observed for 2 μg GST-CBU1241 or 2 μg GST in the LDH standard assay containing 2 mM pyruvate and 0.5 mM NADH in PBS pH 7.4. Conversely, 0.1 unit of commercial LDH enzyme had measurable activity. (C) Enzyme activity relative to pH 7.4 over a range of pHs, measured using MDH standard assay containing 1 μg GST-CBU1241. Activity was measured using increase or decrease of light absorbance at 340 nm, correlating to NADH oxidation and NAD+ reduction respectively during enzyme activity. Error bars represent standard deviation from the mean from at least three independent experiments.
Fig 4.
Michaelis-Menten plots of enzymatic activity for GST-CBU1241 in relation to substrates OAA (A) and NADH (B).
Enzyme specific activity (μmol/mg per mg protein) measured for varying concentrations of OAA concentrations at 0.5 mM NADH (A), and varying NADH concentrations at 0.6 mM OAA (B). Error bars represent standard deviation from the mean from at least three independent experiments. Linear regression line of best fit for Michaelis-Menten kinetic equation is plotted in grey for NADH (B).
Fig 5.
In vitro 6xHis-CBU0823 activity assays for ME (A), LDH (B) and MDH (C) function, including the influence of varied pH (D), and substituted cofactors (E and F).
(A) ME activity was observed for 20 μg 6xHis-CBU0823 in the ME standard assay containing 3 mM malate, 5 mM NAD+, 1 mM MnCl2. 28.8 μg 6xHis-oMLE and 6xHis-mock purification negative control had no activity. (B) LDH activity was not detected for 20 μg 6xHis-CBU0823 in the LDH standard assay containing 2 mM pyruvate and 0.5 mM NADH, with or without the addition of MnCl2. 28.8 μg 6xHis-oMLE and 6xHis-mock purification also failed to show activity, while 0.1 units commercial LDH enzyme had measurable performance in standard conditions. (C) MDH activity was measured for 20 μg 6xHis-CBU0823 in the MDH standard assay containing 2 mM OAA, 0.5 mM NADH, with improved action on addition of 1 mM MnCl2. 0.013 units commercial pig heart mitochondrial MDH and 1 μg GST-CBU1241 had efficient activity in the assay and 6xHis-mock purification had no detectable activity. (D) Enzyme activity relative to pH 7.4 over a range of pHs, measured using ME standard assay containing 2 μg 6xHis-CBU0823. Activity was measured using increase or decrease of light absorbance at 340 nm, correlating to NADH oxidation and NAD+ reduction respectively during enzyme activity. (E) Removal of MnCl2 and/or substitution with NADP+ cofactor from the standard ME assay reduced 6xHis-CBU0823 activity. Conditions with significantly less activity are indicated. (F) Replacement of the MnCl2 cofactor with 1mM CaCl2, CuCl2, MgCl2, or ZnSO4 achieved varying levels of 6xHis-CBU0823 function. Those with significantly lower cofactor facility compared to 1 mM MnCl2 on ordinary one-way ANOVA are indicated (p < 0.05). Error bars represent standard deviation around the mean from at least three independent experiments.
Fig 6.
Michaelis-Menten plots of enzymatic activity for 6xHis-CBU0823 in relation to malate (A), NAD+ (B), and MnCl2 (C).
Enzyme specific activity (μmol/min per mg protein) measured for (A) varying malate concentrations at 5 mM NAD+ and 1mM MnCl2, (B) varying NAD+ concentrations at 3 mM malate and 1 mM MnCl2, and (C) varying MnCl2 concentrations at 3 mM malate and 5 mM NAD+. Error bars represent standard deviation around the mean from at least three independent experiments.
Fig 7.
6xHis-CBU0823 failed to produce detectable lactate in in vitro assays.
(A) Addition of 6xHis-oMLE produced significantly more pyruvate than 6xHis-CBU0823, and both produced significantly more than negative control and blank reactions. (B) Only 6xHis-oMLE yielded lactate amounts significantly above blank. Malic enzyme activity assays (3 mM malate, 5 mM NAD+ and 1 mM MnCl2) were started with either 54 μg 6xHis-CBU0823, 26 μg 6xHis-oMLE, or equivalent volume of mock purification (negative control) or PBS (blank). Reactions were stopped after 20 minutes. Each reaction was analyzed by GC-MS and mean values compared (* p < 0.05, ** p < 0.01, *** p < 0.001). Error bars represent standard deviation from the mean from six independent replicates.
Fig 8.
Loss of cbu0823 caused alterations in carbon flux through glycolysis and the TCA cycle in C. burnetii.
C. burnetii incubated with [13C]glucose supplemented ACCM-2 for 7 days before GC-MS analysis to determine 13C incorporated into the extracted metabolites. Mean label enrichment of each metabolite detected was compared between wildtype (black), 0823::Tn mutant (blue), and 0823::Tn pFLAG-CBU0823 complemented mutant (red) strains. Error bars represent standard deviation from five biological replicates. Metabolites with a significant difference between wildtype and the 0823::Tn mutant are shaded in yellow. Solid lines represent a single enzyme and dotted lines represent multiple enzymes. The blue line denotes the activity of CBU0823 as a ME. The green line denotes CBU1241 as an MDH. The orange line represents an unestablished conversion of pyruvate to lactate by an LDH-type reaction. PEP = phosphoenolpyruvic acid.
Fig 9.
cbu0823 is not required for efficient intracellular replication of C. burnetii in THP-1 cells.
Intracellular replication within THP-1 macrophage-like cells of C. burnetii wildtype, 0823::Tn mutant and 0823::Tn pFLAG-CBU0823 complemented mutant strains over 7 days depicted in fold change of genome equivalents (GE) measured by qPCR for ompA. (A) Fold change in GE was plotted over 7 days as mean with error bars representing standard deviation from six independent biological replicates. There was no difference in replication between strains (p > 0.05). (B) Average vacuole area, measured in μm2 on representative immunofluorescent images of each strain and plotted as mean with error bars representing standard deviation for four of the six replicates. There was no significant difference between the three strains (p > 0.05). (C) Representative confocal immunofluorescent micrographs of THP-1 cells 3 days post-infection with C. burnetii wildtype, 0823::Tn mutant and 0823::Tn complemented mutant strains. There were no significant differences in CCV size and organism numbers between strains. Anti-Coxiella antibody (red), anti-LAMP-1 antibody (green) and DAPI (blue) were used to stain cells and white asterisks indicate CCVs. The scale bar depicts 10 μm.