Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae

Abstract

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH(2). The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.

FIG. 1.

Immunoblot analysis of GlpK (A) and GlpD (B) synthesis during growth of M. pneumoniae in the presence of different carbon sources. Antibodies raised against M. pneumoniae GlpK and GlpD were used to determine total amounts of GlpK (A) and GlpD (B) in cells grown in the presence of glucose (lanes 2), glucose and glycerol (lanes 3), or glycerol (lanes 4). The concentrations of the carbon sources were 1% (wt/vol). A total of 50 ng of recombinant Strep-tagged GlpK (A) or His₆-tagged GlpD (B) served as a control (lanes 1).

FIG. 2.

Localization of GlpD. Immunoblot analysis of Triton X-114-treated cell extracts of the wild-type (WT) and glpD mutant (GPM52) with anti-GlpD is shown. A total of 20 μg of protein each from the aqueous phase (C, cytoplasmic fraction) and the detergent phase (M, membrane fraction) was applied to the gel.

FIG. 3.

Localization of GlpD by immunoelectron microscopy. (A) Negative control using gold-labeled anti-rabbit antibody. (B) Positive control using anti-MPN474. (C) Detection of GlpD with anti-GlpD. Some gold particles are indicated by arrows.

FIG. 4.

Isolation of a M. pneumoniae glpD transposon insertion mutant. (A) Schematic representation of the genomic region of the glpD gene in M. pneumoniae and the site of the transposon insertion in the glpD mutant strain GPM52. Probes hybridizing to internal fragments of the glpD and the aac-ahpD genes are depicted as dotted lines. (B) Southern blot analysis to confirm the unique insertion of the minitransposon into the glpD gene of strain GPM52. Chromosomal DNAs of the wild-type (WT) and mutant (GPM52) strains were digested using EcoRV and NdeI. Blots were hybridized with the glpD-specific probe (left) and a probe hybridizing to the aac-ahpD gene of the minitransposon (right). DIG-labeled DNA molecular mass marker III (Roche Applied Science) served as a standard.

FIG. 5.

Growth of M. pneumoniae wild-type (WT) and glpD mutant (GPM52) strains in modified Hayflick medium containing glucose or glycerol as the carbon source. One hundred milliliters of medium was inoculated with 15 mg of cells and incubated for 2, 4, or 6 days at 37°C in 150-cm² cell culture flasks. Glucose and glycerol were added to a final concentration of 1% (wt/vol). Attached cells were collected by scraping, and growth was monitored by determination of the wet weight of the cell pellets. All measurements were done at least twice.

FIG. 6.

Hydrogen peroxide production by wild-type (WT) and glpD mutant (GPM52) strains in the presence of different carbon sources (100 μM) after 20 min. Results are from a representative experiment.

FIG. 7.

Cytotoxicity of M. pneumoniae toward HeLa cell cultures. (A) HeLa cell culture without M. pneumoniae; (B) HeLa cell culture incubated with wild-type M. pneumoniae; (C) HeLa cell culture incubated with the glpD mutant. After 6 days, HeLa cell cultures were stained with crystal violet and photographed.