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.Mycoplasma pneumoniae causes infections of the upper and lower respiratory tracts. These bacteria are responsible for a large fraction of community-acquired pneumonias. Although usually harmless for adult patients, M. pneumoniae may cause severe disease in children or elderly people. In addition, M. pneumoniae is involved in extrapulmonary complications such as pediatric encephalitis and erythema multiforme (for reviews, see references 15, 21, and 34).M. pneumoniae and its relatives, the Mollicutes, are all characterized by the lack of a cell wall and a very close adaptation to a life within a eukaryotic host. This close adaptation is reflected by degenerative genome evolution that resulted in an extreme genome reduction. As a result, the Mollicutes are the organisms that are capable of independent life with the smallest known genome. M. pneumoniae has a genome of 816 kb and encodes only 688 proteins (18). This genome reduction is taken even further in the close relative Mycoplasma genitalium, which has only 482 protein-coding genes (18). Thus, the analysis of the Mollicutes allows us to study a minimal form of natural life. This question has recently attracted much interest and resulted in the determination of the essential gene sets of M. pneumoniae, M. genitalium, and, more recently, Mycoplasma pulmonis (6,20). In M. genitalium, with the most reduced genomes, only 100 out of the 482 protein-coding genes are dispensable, ...

Functional Characterization of Osmotically Inducible Protein C (MG_427) from Mycoplasma genitalium

“…Previously, glycerol oxidation by mycoplasmas was shown to generate H 2 O 2 , a major product for cytotoxicity (37,38). Figure 4 shows that although both strains grew well with glucose, they exhibited slow growth with glycerol, as was previously reported for M. pneumoniae (37), with the ⌬MG_427 mutant exhibiting significantly slower growth. These data suggest a protective role of MG_427 against H 2 O 2 .…”

“…Further, we demonstrated that the ⌬MG_427 strain of M. genitalium exhibited hypersensitivity to t-BHP and increased sensitivity to H 2 O 2 and this sensitivity could be reversed in the mutant strain complemented with an intact MG_427 gene, similar to osmC mutants of E. coli (24) and M. smegmatis (25). We also observed a slower growth of the ⌬MG_427 mutant than the wild type in SP-4 medium containing glycerol as the carbon source, a growth condition known to generate H 2 O 2 by mycoplasmas (37,38). Altogether, these data suggest that MG_427 encodes a hydroperoxide reductase which likely plays an important role in protecting M. genitalium against oxidative stress.…”

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

“…The formation of a unique tip-like structure comprising a complex of adhesins and adhesion-related proteins was shown to be the basis for effective adherence of bacteria to the respiratory mucosa (Krause & Balish, 2004). The further colonization process is accompanied by the release of hydrogen peroxide (Hames et al, 2009;Schmidl et al, 2011) and of the pertussis toxin-like CARDS toxin (Kannan & Baseman, 2006), resulting in vacuolization and destruction of epithelial cells.…”

Comparative proteomics of twoMycoplasma hyopneumoniaestrains andMycoplasma flocculareidentified potential porcine enzootic pneumonia determinants

“…mycoides [28,29]. In line with that, it was recently demonstrated that pathogenic strains of M. hyopneumoniae were able to produce hydrogen peroxide from glycerol metabolism, but that the non-pathogenic strain J and M. flocculare were not [11].…”