Novel virulence determinants in Mycoplasma pneumoniae: Contribution of transport systems and H2S production to viability and hemolysis
by Stephanie Großhennig
Date of Examination:2015-01-20
Date of issue:2015-06-02
Advisor:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Jörg Stülke
Referee:PD Dr. Michael Hoppert
Persistent Address:
http://dx.doi.org/10.53846/goediss-5106
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Abstract
English
The human pathogen Mycoplasma pneumoniae is a remarkable minimal organism which primarily colonizes the human lung tissue. To reach distant infection sites, it probably also enters the blood stream. The Mycoplasma genome has constantly undergone reductive changes due to strong adaptation to the convenient conditions and high nutrient availability in its habitat. In turn, this adaptation renders the bacterium dependent on the exploitation of the host tissue. This dependence is reflected by a high number of genes coding for transport proteins with most of them being essential. In the initial part of this work, the importance of several M. pneumoniae transporters for survival and virulence was addressed. However, except for two putative amino acid transporters and one potential Mg2+ transporter, none of the isolated transporter mutants showed a significant effect indicating that the influence of the analyzed transporters in growth and cytotoxicity is negligible. Following the analysis of a putative hemolysin transporter, general hemolytic activities in M. pneumoniae were investigated in the second part of this work. It appeared that the organism causes β-hemolysis on blood agar plates but α-hemolysis (hemoxidation) in liquid culture. Surprisingly, the long assumed hemolysin H2O2 caused only hemoxidation and no real hemolysis. Addition of L-cysteine to liquid blood culture unexpectedly resulted in strong hemoxidation followed by complete hemolysis. In several oral pathogens, such as Treponema denticola, similar effects are caused by production of H2S by so called L-cysteine desulfhydrases. H2S is a toxic gas which leads to modification of hemoglobin and subsequent hemolysis. For M. pneumoniae, none of these processes in response to cysteine have been described before. In this work, the bacteria were proven to be capable of producing H2S. The search for a potential H2S producing enzyme in Mycoplasma retrieved MPN487, an essential putative L-cysteine desulfurase which is actually supposed to be involved in FeS-cluster formation. In enzyme assays, it turned out that MPN487 indeed produces H2S in a Michaelis-Menten-like behavior and additionally forms alanine and pyruvate. Thus, MPN487 was renamed in HapE (H2S, alanine and pyruvate producing enzyme). Since alanine formation is a feature of L-cysteine desulfurases, whereas pyruvate- and H2S production are performed by L-cysteine desulfhydrases, HapE might be a novel enzyme which combines both functions or has changed its activity in a process of reductive evolution. In hemolysis assays, HapE showed strong hemoxidation and hemolysis of erythrocytes when incubated with cysteine. This in vitro hemolysis rate even exceeded that of the major virulence factor H2O2, produced by the enzyme GlpD. Even though the in vivo effect in the human host can only be hypothesized by now, the formation of H2S and pyruvate by HapE might represent an important new way to ensure virulence and energy generation of this minimal pathogen.
Keywords: Mycoplasma pneumoniae; pathogen; bacteria; hemolysis; microbiology; hydrogen sulfide; pneumonia; virulence; transport system; Mollicutes; lung tissue; erythrocytes; cysteine; HapE (MPN487); L-cysteine desulfhydrase; hemoxidation; enzyme; blood