Elucidating structure and function of CE-clan protease-related bacterial pathogenicity factors with dual ubiquitin(-like) protease and acetyltransferase activity

Regulation of protein function by ubiquitination and acetylation has been investigated extensively and is known to be exploited by pathogens to hijack their host. Especially gram-negative bacteria (e.g. Yersinia sp., Vibrio sp., Chlamydia sp.) encode for effector proteins that are injected into host cells in order to promote infection and ensure bacterial survival. According to the MEROPS database, many of these bacterial virulence factors belong to the CE-clan, an enzyme family mainly comprised of cysteine proteases with deubiquitinase (DUB) or ubiquitin-like protease (ULP) activity, but also acetyltransferases (AcT). Some effectors even exhibit a dual activity as DUB/ULP and AcT. However, the underlying mechanisms regulating their enzymatic activity on a molecular level are often not completely understood and their exact role within the infection process remains elusive.

By using tools from structural biology, biochemistry and cell biology we elucidate the structure-function relationship of a bacterial CE-clan effector in context of host cell infection. The Chlamydia-like pathogen Simkania negevensis encodes for a CE-clan member called SnCE1. Besides its protease activity as a deubiquitinase and deSUMOylase, we report strong autoacetylation for SnCE1, suggesting a dual specificity similar to Chlamydia effector ChlaDUB1. ChlaDUB1 has been reported to carry out both, AcT and DUB activity, by the same active site. Despite structural homology between these two bacterial effectors, X-ray crystallographic analysis reveals differences in variable regions, therefore hinting at alternative mechanistics in SnCE1. Modelling of SnCE1 in complex with acetyl group donor Coenzyme A (CoA) identifies key residues for CoA-binding, supported by mutational studies and mass-spectrometric data. To further elucidate the physiological role of SnCE1 we set up infection studies in cell culture. Upon infection of HEK293T cells with Simkania, SnCE1 localizes to the ER and also mitochondria. The exact pathways targeted by SnCE1 still have to be identified. If there is a potential cross-talk between acetylation and ubiquitination/SUMOylation, as well as an impact of the acetylation status on infection, remains question of our research.

Our studies contribute to a comprehensive understanding of these type of bacterial virulence factors and their role in infection, as it is crucial for our understanding of host-pathogen interactions.