Identification of hemolysis-related pathogenicity factors of Bartonella bacilliformis

Question

Bartonella bacilliformis is the causative agent of Carrion's disease, a vector-borne illness restricted to the South American Andes. In the acute phase, bacteria infect erythrocytes causing severe hemolytic anemia with case-fatality rates as high as 90% in untreated patients. Erythrocyte invasion is the most important step in the pathogenesis of Carrion's disease and results in its subsequent destruction. Exact knowledge of this process is crucial for drug development.

Methods

To identify genes involved in hemolysis, a Tn5 transposon library was generated and screened for hemolytic activity. Hemolysis mutants were identified and systematically analyzed by loss of function/gain of function experiments. For this, markerless deletion was newly established for B. bacilliformis, and the generated deletion and complementation mutants were tested for hemolytic activity in a novel in vitro based hemolysis assay using human erythrocytes. To study B. bacilliformis hemolysis and to test potential therapeutic drugs in vivo, we currently establish a (humanized) murine infection model.

Results

Two hemolysis-related genes, a porin and a phospholipase, were identified. The loss of one of the two genes led to the complete inhibition of hemolysis whereas the hemolytic activity was restored by plasmid-based complementation. Although B. bacilliformis is believed to be a human-specific pathogen, we were still interested in whether murine erythrocytes can nevertheless be infected by B. bacilliformis in vitro. It turned out that murine erythrocytes undergo hemolysis upon B. bacilliformis infection. Murine infection experiments are ongoing to analyze the course of infection in detail.

Conclusion

Two hemolysis-related genes/proteins of B. bacilliformis have been identified and were functionally characterized. Ongoing work aims to establish a mouse model to study hemolysis in vivo and to evaluate potential anti-virulence strategies.