Identification of essential and virulence genes in Mycoplasma pneumoniae

by Cedric Blötz
Doctoral thesis
Date of Examination:2019-04-02
Date of issue:2019-04-16
Advisor:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Carsten Lüder
Referee:Dr. Michael Pd Hoppert
Referee:Prof. Dr. Fabian Commichau
Referee:Prof. Dr. Stefanie Pöggeler
Referee:Prof. Dr. Henning Urlaub
Persistent Address: http://dx.doi.org/10.53846/goediss-7398

 

 

Files in this item

Name:Dissertation_Cedric Blötz 2019.pdf
Size:3.66Mb
Format:PDF
Description:Identification of essential and virulence genes in Mycoplasma pneumoniae

The following license files are associated with this item:

Abstract

English

The bacterial group of Firmicutes includes many pathogenic bacteria, as Mycoplasma pneumoniae. The Gram-positive, but cel wall-less bacterium is characterized by a minimal genome with only 694 genes. Elucidating the function of all genes encoded in M. pneumoniae would allow the understanding of a cell in its entire complexity. Unfortunately, these bacteria grow very slowly and are hard to manipulate genetically. Only a few techniques and tools are available for the manipulation of M. pneumoniae. In this work, we extended the toolbox for M. pneumoniae with the first self-replicating plasmid pGP2756. It enabled for the first-time plasmid-based protein overexpression, expression of fusion proteins and complementation assays in Mycoplasma, to analyze unknown virulence factors contributing to mycoplasmal pathogenicity. Even if Mycoplasmas contain only down to 482 genes, one-third thereof is of unknown function and has no homology to any available nucleotide sequence so far. For instance, they lack common detoxification enzymes, such as catalase or superoxide dismutase, but many Mycoplasmas use hydrogen peroxide as a virulence factor. Accordingly, we investigated how they can tolerate high peroxide concentrations. Recently, peroxiredoxins were identified in the genomes of Mycoplasmas, which act as antioxidant enzymes. We identified and characterized two similar genes mpn625 and mpn668 to have specific detoxification activities for hydrogen peroxide and organic peroxide, respectively. Analyzing their specific regulation in M. pneumoniae, which has in general only a few transcription factors, revealed that the trigger enzyme GlpQ and the protein kinase C are involved in peroxide stress response. Strikingly, GlpQ and the protein kinase C have a strong influence on each other’s expression. In addition, we identified, mpn329 encoding a zinc responsive regulator (Zur) rather a ferrous iron regulator (Fur), which could alter gene expression in response to the intracellular ion homeostasis and peroxide concentration. For effective pathogenicity, M. pneumoniae needs to escape the host immune system using immunoglobulin binding proteins. In this work, the novel immunoglobulin binding protein of M. pneumoniae IbpM was identified. Our experiments show that IbpM is a multi-binding protein with high affinity for human immunoglobulins, plasminogen and fibronectin. IbpM represents a new surface protein of M. pneumoniae likely responsible for the efficient immune evasion properties of this bacterium often leading to chronic infections. Pathogenicity was shown to be strongly connected to c-di-AMP metabolism in many Firmicutes. Our results show that this essential messenger is present even in the near-minimal bacterium M. pneumoniae, controlling potassium homeostasis by binding to the potassium uptake protein KtrC and might contribute to mycoplasmal pathogenicity. With this work we have shown that even if M. pneumoniae contains only 694 genes, there is more controlled gene expression, second messenger-regulated protein functions and undiscovered virulence mechanisms as ever thought of.
Keywords: Mycoplasma; Mycoplasma pneumoniae; virulence; essential genes; pathogenicity; peroxide; immunoglobulin binding; mpn400; replicative plasmid; mpn329; mpn625; mpn668; pGP2756; c-di-AMP
 
Statistik