Pathogenicity of a minimal organism: Role of protein phosphorylation in Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae is a human pathogen that belongs to the Mollicutes, a group of bacteria with the smallest genomes that are capable of independent life. The reductive evolution of the Mollicutes is reflected by their limited regulatory features for gene expression. Thus, posttranslational regulation might be important for M. pneumoniae to adapt to environmental changes. Among the very few regulatory proteins retained is the HPr kinase (HPrK), which phosphorylates the phosphocarrier protein HPr at the Ser-46 residue. This phosphorylation event is a major signal to trigger carbon catabolite repression in less degenerated bacteria. However, the function of HPr(Ser-P) in M. pneumoniae is unknown. For the protein phosphatase PrpC, an implication in the dephosphorylation of HPr(Ser-P) could be shown. In addition to HPrK, the M. pneumoniae prkC gene encodes another serine/threonine protein kinase C.The determination of the complete phosphoproteome of M. pneumoniae by two-dimensional gel electrophoresis and mass spectrometry allowed the detection of 63 phosphorylated proteins, including many enzymes of central carbon metabolism and proteins related to host cell adhesion. It was also possible to detect 16 phosphorylation sites, among them 8 serine and 8 threonine residues. However, a comparison with the phosphoproteomes of other bacteria revealed that there is only a weak conservation of phosphorylation sites, even if the same proteins are phosphorylated in related organisms. There is only one exception: The phosphorylation of phosphosugar mutases on a conserved serine residue, which could be detected in all studied organisms from archaea and bacteria to man. In the case of the phosphosugar mutase ManB in M. pneumoniae, it could be shown that this protein undergoes autophosphorylation. In conclusion, the results indicate that protein phosphorylation seems to be highly specific for each individual organism.For a more detailed analysis of the phosphorylation network in M. pneumoniae, the phosphoproteomes of the wild type strain and of three isogenic mutants that are affected in the two protein kinases HPrK and PrkC and in the protein phosphatase PrpC were compared. Examination of the phosphorylation profile of the hprK mutant revealed that only HPr is phosphorylated by HPrK, whereas six proteins, including the major adhesin P1 and two cytadherence proteins HMW1 and HMW3, were affected by the loss of PrkC. In contrast, inactivation of PrpC that antagonizes PrkC-dependent phosphorylation resulted in more intensive phosphorylation of the same target proteins. The phenotypic characterization of prkC mutant cells revealed a nonadherent growth type along with a loss of cytotoxicity toward HeLa cells. Thus, posttranslational modification of cytadherence proteins by PrkC is essential for cell adhesion and virulence in M. pneumoniae.The phosphoproteomic analysis demonstrated that several glycolytic enzymes are subject to phosphorylation. M. pneumoniae uses glycolysis as the major pathway for the generation of energy by substrate-level phosphorylation. Using a bacterial two-hybrid approach, the enolase was identified as the central glycolytic enzyme of M. pneumoniae due to its ability to directly interact with all other glycolytic enzymes. Moreover, most of the glycolytic enzymes performed self-interactions. The results support the idea that glycolysis proceeds in a well structured manner even in a minimal organism.In its natural habitat, M. pneumoniae thrives on pulmonary surfaces that are mainly composed of phosphatidylcholine. This phospholipid can be integrated directly into the cell membrane or serve as precursor for cellular processes. M. pneumoniae possesses two potential glycerophosphodiesterases, MPN420 (GlpQ) and MPN566, that are able to cleave deacylated phospholipids to glycerol 3-phosphate and choline. Further glycerol 3-phosphate utilization by enzymes of the glycerol metabolism is crucial for the cytotoxicity of M. pneumoniae due to hydrogen peroxide release. Biochemical studies showed that GlpQ is active as a glycerophosphodiesterase, whereas MPN566 has no enzymatic activity in vitro. Mutants affected in either glycerophosphodiesterase revealed that inactivation of mpn566 did not result in any phenotype. In contrast, the glpQ mutant exhibited a growth defect in glucose-supplemented medium. Moreover, the lack of GlpQ resulted in an absence of hydrogen peroxide formation in the presence of deacylated phospholipids and a loss of cytotoxicity toward HeLa cells. These observations imply that GlpQ is important for the pathogenicity of M. pneumoniae, but also for other functions in the cell. Indeed, proteomic and transcriptomic analyses of the wild type and the glpQ mutant strain suggested a GlpQ-dependent transcription regulation, which led to higher or lower protein amounts of the glycerol facilitator, a subunit of a metal ion ABC transporter, and three lipoproteins. Interestingly, all genes subject to GlpQ-dependent control have a conserved potential cis-acting element upstream of the coding region. Nevertheless, it is open for speculation whether GlpQ or a transcription factor that is controlled by GlpQ is responsible for this regulation.