Bacterial esterases reverse lipopolysaccharide ubiquitylation to block host immunity.

Aspects of how Burkholderia escape the host's intrinsic immune response to replicate in the cell cytosol remain enigmatic. Here, we show that Burkholderia has evolved two mechanisms to block the activity of Ring finger protein 213 (RNF213)-mediated non-canonical ubiquitylation of bacterial lipopolysaccharide (LPS), thereby preventing the initiation of antibacterial autophagy. First, Burkholderia's polysaccharide capsule blocks RNF213 association with bacteria and second, the Burkholderia deubiquitylase (DUB), TssM, directly reverses the activity of RNF213 through a previously unrecognized esterase activity. Structural analysis provides insight into the molecular basis of TssM esterase activity, allowing it to be uncoupled from its isopeptidase function. Furthermore, a putative TssM homolog also displays esterase activity and removes ubiquitin from LPS, establishing this as a virulence mechanism. Of note, we also find that additional immune-evasion mechanisms exist, revealing that overcoming this arm of the host's immune response is critical to the pathogen.

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.

Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.

Dedicated bacterial esterases reverse lipopolysaccharide ubiquitylation to block immune sensing.

Pathogenic bacteria have evolved diverse mechanisms to counteract cell-autonomous immunity, which otherwise guards both immune and non-immune cells from the onset of an infection1,2. The versatile immunity protein Ring finger protein 213 (RNF213)3-6 mediates the non-canonical ester-linked ubiquitylation of lipopolysaccharide (LPS), marking bacteria that sporadically enter the cytosol for destruction by antibacterial autophagy4. However, whether cytosol-adapted pathogens are ubiquitylated on their LPS and whether they escape RNF213-mediated immunity, remains unknown. Here we show that Burkholderia deubiquitylase (DUB), TssM7-9, is a potent esterase that directly reverses the ubiquitylation of LPS. Without TssM, cytosolic Burkholderia became coated in polyubiquitin and autophagy receptors in an RNF213-dependent fashion. Whereas the expression of TssM was sufficient to enable the replication of the non-cytosol adapted pathogen Salmonella, we demonstrate that Burkholderia has evolved a multi-layered defence system to proliferate in the host cell cytosol, including a block in antibacterial autophagy10-12. Structural analysis provided insight into the molecular basis of TssM esterase activity, allowing it to be uncoupled from isopeptidase function. TssM homologs conserved in another Gram-negative pathogen also reversed non-canonical LPS ubiquitylation, establishing esterase activity as a bacterial virulence mechanism to subvert host cell-autonomous immunity.