My current research interests focus on the molecular mechanisms employed by the obligate intracellular bacterium Coxiella burnetii for evasion of host defenses. C. burnetii is the etiological agent of acute and chronic Q (query) fever in humans and resides within the acidified environment of a parasitophorous vacuole (PV) with lysosomal characteristics. In this unique biological niche, bacteria are exposed to a wide variety of damaging agents, such as an acidic pH, hydrolytic enzymes, reactive oxygen species (ROS) and reactive nitrogen species (RNS) released by the host cell as primary defense against invading microorganisms. In order to understand how C. burnetii withstands damage on the cellular level and maintains genomic stability and integrity, our current research projects focus on the functional characterization of ROS detoxifying enzymes and DNA damage repair mechanisms.

1.    Defense mechanisms against oxidative stress.

Bacteria employ a variety of defense mechanisms against oxidative stress, such as superoxide dismutase (SOD) for decomposition of superoxide radicals (O₂^-) and catalase (Kat) or peroxide-scarvenging alkyl hydroperoxide reductase (Ahp) for removal of the resulting hydrogen peroxide (H₂O₂). Genome comparison revealed differences among C. burnetii isolates from distinct genomic groups in regard to defense mechanisms. One of our goals is to understand the impact of a distinct set of defense mechanism on isolate specific virulence.

Compared to other Gram-negative bacteria, C. burnetii exhibits major abbreviations of the SOS response, such as a reduced set of DNA repair genes, their constitutive expression due to the lack of the repressor LexA and a stress-inducible AddAB complex (RecBCD analogue) for recombinational repair. These differences emphasize the high adaptation of C. burnetii to its unique niche. Our research focuses on the functional characterization of genetic events which lead to protection against oxidative stress of C. burnetii within the hostile environment of the PV.

Bacterial defense mechanism against oxidative stress.

C. burnetii encodes for two superoxide dismutase enzymes (SodA, SodC) which mediate the decomposition of superoxide radicals (O₂^-) to hydrogen peroxide (H₂O₂). In the following step H₂O₂ is degraded to H₂O and oxygen by catalase (KatE) or alkyl hydroperoxide reductases (Ahp). Presence of iron (Fe₂⁺) can lead to additional oxidative stress by generation of hydroxyl radicals (OH^-) via the Fenton reaction.

2.    Development of genetic tools for C. burnetii.

Understanding genetic events involved in C. burnetii pathogenesis has been hampered by the lack of standard techniques for genetic manipulations. One of our research goals is to develop new genetic techniques and we recently achieved Himar1-mediated transposition in C. burnetii. This technique is currently employed in our laboratory for the generation of a mutant clone library for identification of potential virulence factors and gene delivery for complementation analysis of natural mutants.

C. burnetii expressing mCherry after Himar1-transposition.

The fluorescence protein mCherry, carried on the Himar1 transposable element, allows easy visualization of successfully transformed bacteria by fluorescence microscopy. Cells were imaged with a Nikon Eclipse TE300 inverted fluorescence microscope; flurescence image for detection of mCherry expression (594nm) and brightfield (40x) were taken separately and merged.

3.    Structure and biosynthesis of the O-specific polysaccharide.

The LPS molecule as a major surface antigen of Gram-negative bacteria remains the only well-characterized virulence factor for C. burnetii. In immune-incompetent hosts, C. burnetii undergoes irreversible phase variation through a variety of chromosomal changes resulting in loss of virulence. Our research interest focuses on the biosynthetic pathway that leads to synthesis and assembly of the O-specific side chain.