Teresa Schmidt


B8 Functional analysis of Coxiella burnetii effector proteins

Principal investigator
Anja Lührmann

Roland Lang

Analyzing the expression pattern of the C. burnetii effector CaeA

Coxiella burnetii is a gram negative, obligate intracellular bacterium. The transmission of Coxiella burnetii occurs via aerosol inhalation of contaminated dust particles and animal excretions, which may lead to an acute or chronic Q-fever. Whereas the acute Q-fever progresses asymptomatically or result in a mild, flu-like illness or, the development of chronic disease manifest as endocarditis, hepatitis or pneumonia. Cattle, sheep, goats, cats and ticks are natural hosts for the zoonotic pathogen. After infection by inhalation of bacteria, C. burnetii primarily infects mononuclear phagocytes, in which C. burnetii resides in a parasitophorous vacuole (PV). For intracellular survival of C. burnetii a functional type IV secretion system (T4SS) is essential. The C. burnetii T4SS is required to eject effector proteins through the PV membrane into the host cell cytosol. The effector proteins are believed to manipulate host cell pathways to ensure bacterial replication.  One of the few effector proteins where a function has been assigned is CaeA (C. burnetii anti-apoptotic effector). CaeA is encoded by the open reading frame (ORF) “CBU 1524”. It was shown that the anti-apoptotic activity of CaeA depends on a glutamate-lysine repeat (EK-) motif located in a coiled-coil domain. In the annotation of the Coxiella burnetii Nine Mile I genome, the open reading frame CBU_1524 is combined with the preceding ORF CBU_1523 and is annotated as a pseudogene, as the start of CBU_ 1523 and is not in frame with the stop of CBU_1524. There is growing evidence that this declaration is not true for the Nine Mile II genome. Our group was able to find two transcription starts in transcriptome analysis using RNAseq and RT-PCR, one for CBU_1523 and one for CBU_1524, respectively. We now hypothesize that the ORF CBU_1523 might give raise to a polycistronic mRNA, which is translated into CBU 1523 and CaeA.  In order to clarify this we generated several C. burnetii mutants containing plasmids with different CBU_1523 and CBU_1524 variants and c-terminal HIS tags. Some of these mutants will contain a supposed, upstream located promotor region. Using this strategy we will be able to determine whether CBU_1523 and CBU_1524 encode for two different proteins.

Figure: a) Pseudogene CBU 1523 (1006bp): ATG and TAG are not in frame. b) Hypothesis of transcription of CBU 1523(234bp) and CBU 1524(660bp). c) Expression patterns of the annotated pseudogene CBU_1523 (grey wide arrow) in C. burnetii grown in the cell-free medium ACCM2 (green line), and in Vero cells (red line) for 72 hours. Two apparent transcription start sites (TSSs) that possibly correspond to a small open reading frame (sORF; CBU 1523) and to caeA are indicated by black thin arrows. (Modified according to Rahul Raghavan; "A repeat motif on a Coxiella effector protein facilitates apoptosis inhibition", Virulence, 2016)