Glycerol metabolism and pathogenicity of <i>Mycoplasma pneumoniae</i>

Mycoplasma pneumoniae, der Erreger der atypischen Pneumonie, kann neben Glukose und Fruktose auch Glycerol als Kohlenstoffquelle nutzen. Das Glycerol wird über erleichterte Diffusion in die Zelle geschleust und anschließend durch die Glycerol-Kinase phosphoryliert. Das Glycerol-3-phosphat wird dann zu Dihydroxyacetonphosphat umgewandelt, welches in der Glykolyse zur Gewinnung von Energie in Form von ATP weiter umgesetzt wird.In dieser Arbeit sollte das Enzym, das für die Oxidation von Glycerol-3-phosphat zuständig ist (annotiert als GlpD), in M. pneumoniae näher untersucht werden. Es stellte sich heraus, dass dieses Protein keine Dehydrogenase-, sondern eine Oxidase-Aktivität besitzt, d.h. Sauerstoff als Elektronenakzeptor verwendet. Hierbei wird dann Wasserstoffperoxid freigesetzt, welches als Virulenzfaktor die Wirtszellen nachweislich schädigen kann. Eine glpD-Mutante ist weniger zytotoxisch gegenüber HeLa-Zellen als der Wildtyp. Die Glycerol-3-phosphat-Oxidase ist demnach der Produzent eines primären Virulenzfaktors in M. pneumoniae.Neben der glpD-Mutante wurden noch weitere Mutanten auf ihre Zytotoxizität untersucht. Es stellte sich heraus, dass die HPr-Kinase und eine Ser/Thr-Kinase (PrkC) wie GlpD einen Einfluss auf die Etablierung der Pathogenität von M. pneumoniae haben. Die Mutanten dieser Gene waren alle weniger zytotoxisch als der Wildtyp. Dies zeigt, dass Phosphorylierungen offensichtlich eine wichtige regulatorische Rolle für die Pathogenität in M. pneumoniae spielen.Eine nähere Untersuchung der Kinase PrkC hat gezeigt, dass sie Ähnlichkeiten zu einer Ser/Thr-Kinase, die in vielen Gram-positiven Organismen zu finden ist, aufweist. Diese Kinase übernimmt wichtige regulatorische Funktionen in den Zellen. In dieser Arbeit konnte gezeigt werden, dass PrkC eine wichtige Eigenschaft, nämlich die Autophosphorylierung, mit den bereits beschriebenen PrkCs gemeinsam hat. Es war jedoch nicht möglich, Substrate der Kinase zu identifizieren. Aber die Isolierung einer prkC-Mutante war erfolgreich, die nicht mehr in der Lage ist, adhäsiv an Oberflächen zu wachsen und sich deshalb nicht mehr an die Wirtszellen anhaften kann. Dies gibt einen Hinweis darauf, dass PrkC eine wichtige Rolle bei dem Aufbau der so genannten Tipstruktur, dem Anhaftungsorganell von M. pneumoniae, spielen könnte

Regulation of carbon metabolism in the mollicutes and its relation to virulence

The mollicutes are cell wall-less bacteria that live in close association with their eukaryotic hosts. Their genomes are strongly reduced and so are their metabolic capabilities. A survey of the available genome sequences reveals that the mollicutes are capable of utilizing sugars as source of carbon and energy via glycolysis. The pentose phosphate pathway is incomplete in these bacteria, and genes encoding enzymes of the tricarboxylic acid cycle are absent from the genomes. Sugars are transported by the phosphotransferase system. As in related bacteria, the phosphotransferase system does also seem to play a regulatory role in the mollicutes as can be concluded from the functionality of the regulatory HPr kinase/phosphorylase. In Mycoplasma pneumoniae, the activity of HPr kinase is triggered in the presence of glycerol. This carbon source may be important for the mollicutes since it is available in epithelial tissues and its metabolism results in the formation of hydrogen peroxide, the major virulence factor of several mollicutes. In plant-pathogenic mollicutes such as Spiroplasma citri, the regulation of carbon metabolism is crucial in the adaptation to life in plant tissues or the insect vectors. Thus, carbon metabolism seems to be intimately linked to pathogenicity in the mollicutes. Copyright (c) 2007 S. Karger AG, Basel

In Vivo Activity of Enzymatic and Regulatory Components of the Phosphoenolpyruvate:Sugar Phosphotransferase System in Mycoplasma pneumoniae

Mycoplasma pneumoniae is a pathogenic bacterium that is highly adapted to life on mucosal surfaces. This adaptation is reflected by the very compact genome and the small number of regulatory proteins. However, M. pneumoniae possesses the HPr kinase/phosphorylase (HPrK/P), the key regulator of carbon metabolism in the Firmicutes. In contrast to the enzymes of other bacteria, the HPrK/P of M. pneumoniae is already active at very low ATP concentrations, suggesting a different mode of regulation. In this work, we studied the ability of M. pneumoniae to utilize different carbohydrates and their effects on the activity of the different phosphotransferase system (PTS) components. Glucose served as the best carbon source, with a generation time of about 30 h. Fructose and glycerol were also used but at lower rates and with lower yields. In contrast, M. pneumoniae is unable to use mannitol even though the bacterium is apparently equipped with all the genes required for mannitol catabolism. This observation is probably a reflection of the continuing and ongoing reduction of the M. pneumoniae genome. The general enzymatic and regulatory components of the PTS, i.e., enzyme I, HPr, and HPrK/P, were present under all growth conditions tested in this study. However, HPrK/P activity is strongly increased if the medium contains glycerol. Thus, the control of HPrK/P in vivo differs strongly between M. pneumoniae and the other Firmicutes. This difference may relate to the specific conditions on lipid-rich cell surfaces

Multiple-Mutation Reaction: a Method for Simultaneous Introduction of Multiple Mutations into the glpK Gene of Mycoplasma pneumoniae

In Mycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a translation stop site. This often makes it difficult to express Mycoplasma proteins in E. coli isolates. In this work, we developed a strategy for the one-step introduction of several mutations. This method, the multiple-mutation reaction, is used to simultaneously replace nine opal codons in the M. pneumoniae glpK gene

The Stability of Cytadherence Proteins in Mycoplasma pneumoniae Requires Activity of the Protein Kinase PrkC▿ †

Mycoplasma pneumoniae belongs to the mollicutes, a group of bacteria that have strongly reduced genomes but that are nevertheless capable of independent life. With only three transcription factors, the regulatory features of these bacteria are very limited. Thus, posttranslational regulation might be important for M. pneumoniae. In addition to the highly specific HPr kinase, the M. pneumoniae prkC gene encodes the serine/threonine protein kinase C. In order to study the function(s) of this kinase, we isolated an M. pneumoniae mutant affected in PrkC. This mutation resulted in nonadherent growth and loss of cytotoxicity. Examination of the phosphorylation profile of the prkC mutant suggested that phosphorylation of cytadherence proteins was affected by the loss of this kinase. In contrast, inactivation of the prpC gene affecting the protein phosphatase that antagonizes PrkC-dependent phosphorylation resulted in more intensive phosphorylation of the cytadherence proteins HMW1 and HMW3 of the major adhesin P1 and of the surface protein MPN474. Moreover, loss of PrkC affects not only the phosphorylation state of the cytadherence proteins but also their intracellular accumulation. However, the expression of the corresponding genes was not affected by PrkC, suggesting that PrkC-dependent phosphorylation results in stabilization of the cytadherence proteins. The HMW proteins and P1 are part of the so-called terminal organelle of M. pneumoniae that is involved in gliding motility, cell division, and adhesion to host epithelial tissues. Our observations suggest that the posttranslational modification of cytadherence proteins by PrkC is essential for the development and function of the M. pneumoniae terminal organelle