SFB796 - Sub project B8

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Functional analysis of Coxiella burnetii type IV effector-induced inhibition of host cell apoptosis.

 

Project summary

Coxiella burnetii is a Gram-negative, obligate intracellular pathogen that causes Q-fever, a worldwide zoonotic disease. Q-fever is a mild flu-like illness, but can be associated with chronic or even fatal outcomes. Intracellular replication and inhibition of host cell apoptosis depends on a functional type IV secretion system (T4SS), used to translocate bacterial proteins into the host cell in order to manipulate host cell pathways. Inhibition of host cell apoptosis may play a role in establishing chronic disease by ensuring a productive infection of C. burnetii. In order to understand C. burnetii pathogenesis it is crucial to investigate the function of effector proteins in particular those with anti-apoptotic activities.

We demonstrated that the T4SS effector AnkG inhibits host cell apoptosis possibly by binding to the pro-apoptotic host cell protein p32 (gC1qR). However, the precise mechanism is still unclear. Our recent data suggests that AnkG contains at least two anti-apoptotic domains, as deletion of amino acid 1-5 (AnkGΔ1-5) results in an intermediary anti-apoptotic phenotype. These first five amino acid residues are predicted to contain an IAP-binding domain (IBM), suggesting that AnkG interferes with apoptosis by modulating inhibitor of apoptosis protein (IAP) function. Additionally, we identified two effector proteins interfering with the intrinsic apoptotic pathway. For one of the anti-apoptotic effector proteins (CaeB - C. burnetii anti-apoptotic effector protein B) we narrowed down the point of action, which is located downstream of mitochondria activation and upstream of caspase 3 activation.

In the second phase of the CRC796 we aim to (1) elucidate how CaeB inhibits apoptosis and will therefore identify the precise point of action. This will be done by transfection of CaeB expressing cell lines with plasmids encoding inducible pro-apoptotic proteins. Together with the identification of targeted host cell protein using yeast two-hybrid or/and GST-pull down followed by mass spectrometry, it will allow us to unravel the molecular mechanism applied by CaeB to inhibit apoptosis. Furthermore, we will (2) analyze the molecular activity of the AnkG IBM domain (AnkG1-5), and use the very recently generated AnkG mutant (AnkGR22/23S) which is unable to bind p32 to investigate whether and how the AnkG-p32 interaction results in inhibition of apoptosis. This will be done using RT-PCR analysis, western blot analysis, co-immunoprecipitation assays, apoptosis assays, immunofluorescence analysis, GST-pull down or co-immunoprecipitation coupled with mass spectrometry. In order to verify the obtained results from aim 1 and 2 it will be necessary to (3) generate C. burnetii mutants lacking either the T4SS or the anti-apoptotic effector proteins AnkG and CaeB. This will be accomplished by newly developed transposon mutagenesis approach since a method to generate site-directed knockdown is not available for this obligate intracellular bacteria. Completion of the proposed aims will help to understand how C. burnetii can establish persistent infection and thereby chronic disease.

 

Project relevant publications

Bisle S, Klingenbeck L, Borges V, Sobotta K, Schulze-Luehrmann J, Menge C, Heydel C, Gomes JP, Lührmann A.   (2016).   The inhibition of the apoptosis pathway by the Coxiella burnetii effector protein CaeA requires the EK repetition motif, but is independent of survivin.   Virulence 13: 1-13.

Berens C, Bisle S, Klingenbeck L, Lührmann A.   (2015).   Applying an inducible expression system to study interference of bacterial virulence.   J Vis Exp 100: e52903.

Eckart RA, Schulze-Luehrmann J, Bisle S, Wittmann I, Jantsch J, Schmid B, Berens C, Lührmann A.   (2014).   The anti-apoptotic activity of the Coxiella burnetii effector protein AnkG is controlled by p32-dependent trafficking.   Infect Immun 82: 2763-2771.

Klingenbeck L, Eckart RA, Berens C, Lührmann A.   (2013).   The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level.   Cell Microbiol 15: 675-687.

Carey KL, Newton HJ, Lührmann A, Roy CR.   (2011).   The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication.   PLoS Pathogens 7: e1002056.

Lührmann A, Nogueira C, Carey KL, Roy CR.   (2010).   Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein.   Proc Natl Acad Sci USA 107: 18997-19001.

Pan X, Lührmann A, Satoh A, Laskowski-Arce MA, Roy CR.   (2008).   Ankyrin repeat proteins comprise a diverse family of bacteria type IV effectors.   Science 320: 1651-1654.

Lührmann A and Roy CR.   (2007).   Coxiella burnetii inhibits activation of host cell apoptosis through a mechanism that involves preventing cytochrome c release from mitochondria.   Infect Immun. 75: 5282-5289.