Simkania negevensis (Sne) is an obligate intracellular, Chlamydia-like emerging pathogen, associated with respiratory diseases. It exhibits a biphasic developmental cycle with extracellular, non-replicative elementary bodies (EBs) and intracellular, replicative reticulate bodies (RBs). After invading the host cell, they multiply within a Sne-containing vacuole [1]. Intracellular bacteria run the risk of being combated by the host immune system. This includes the host induced ubiquitination of bacterial components. Ubiquitination is an essential eukaryotic protein modification that, depending on the ubiquitinylation pattern, regulates many cellular aspects such as proteostasis, DNA repair and NF-κB activation. Increasing evidence suggests that various bacteria introduce effectors into the host cell to hijack the host ubiquitin system and establish an infection [2]. A bioinformatics screen of the Sne genome identified a high number of putative ubiquitin-modifying enzymes including four ubiquitin ligases. In previously published proteomics analysis performed on day 3 post infection (pi), one of these ligases could be identified [3].

In our research, we are especially interested in the activity of this ligase and its role during infection. To analyze this, quantitative real-time PCR, protein purification, autoubiquitinylation- and ubi crest assays as well as mass spectrometry were performed. Additionally, a cell line was generated that inducibly expresses the ligase and various transfection methods were used.

From day 2 to day 4 pi, a downregulation of the expression of the ligase could be measured. Furthermore, the ligase showed no toxic effect on the host cell and the protein seems to localize in the host cytoplasm when it is overexpressed. A specificity of the ligase for K11- and K63- linked ubiquitin chains could be shown.

In the ongoing arm race between host and pathogen, Sne expresses a ubiquitin ligase that possibly interferes with the host ubiquitin system.

References:

M. Vouga et al., Crit. Rev. Microbiol. 43, 62 (2017).Y. Zhou & Y. Zhu, Cell. Microbiol. 17, 26 (2015).J. Herweg et al., Mol. Microbiol. 99, 151 (2016).