# The Ubiquitination of Mycobacterium tuberculosis Rv3717 Promotes Proteasomal Degradation of Interleukin Enhancer-Binding Factor

## TL;DR

This study shows that the tuberculosis bacterium uses a protein called Rv3717 to disrupt host cell defenses by promoting the breakdown of a key immune protein.

## Contribution

This is the first study to show that Rv3717 ubiquitination leads to proteasomal degradation of ILF2, downregulating IL-2 expression.

## Key findings

- Rv3717 interacts with ILF2 and promotes its proteasomal degradation via ubiquitination.
- Ubiquitinated Rv3717 downregulates IL-2 expression in host cells.
- Rv3717-K0 without ubiquitination can reverse these effects.

## Abstract

Mycobacterium tuberculosis Rv3717 facilitates mycobacterial survival in host cells. This study is the first to verify that Rv3717 can be modified by polyubiquitin chains and promote ubiquitin-dependent proteasomal degradation of interleukin enhancer-binding factor 2 (ILF2), thus affecting the expression of IL-2, ILF2, P62, and LC3. More importantly, the changes can be recovered by Rv3717-K0 without ubiquitination. This suggests that ubiquitin-dependent proteasomal degradation induced by Rv3717 is likely to be associated with host cellular defense against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) has developed some strategies to evade host immune responses through ubiquitination, thereby facilitating persistent mycobacterial infection. The Rv3717 protein has been identified as a peptidoglycan (PG) amidase that contributes to mycobacterial survival, but its exact mechanism is still unclear. The findings of this study indicate that Rv3717 inhibits mycobacterial clearance within pulmonary epithelial cells. To elucidate the molecular mechanisms by which Rv3717 facilitates persistent infection, we identified intracellular candidates interacting with Rv3717 using co-immunoprecipitation (Co-IP) combined with liquid chromatography–mass spectrometry (LC-MS/MS). The unique proteins are categorized into three functional networks: mRNA splicing, the immune system process, and the translation process through Protein–Protein Interaction (PPI) analysis. The candidate interacting proteins of Rv3717 are involved in interleukin-2 enhancer-binding factor 2 (ILF2) and TAF15, as well as the polyubiquitin chain (UBC) and E3 ubiquitin ligase TRIM21. Our results suggest that intracellular Rv3717 is likely to influence biological processes through the potential interacting proteins. Our findings confirmed that Rv3717 interacted with interleukin enhancer-binding factor 2 (ILF2) through Co-IP and immunofluorescence assays. Furthermore, Rv3717 was verified to bind with ubiquitin and be degraded through the proteasome system. More importantly, the ubiquitination of Rv3717 accelerated the proteasomal degradation of ILF2 and downregulated the expression of IL-2. This study is the first to propose that the ubiquitination of the mycobacterial membrane vesicle-associated protein Rv3717 facilitates the proteasomal degradation of ILF2, resulting in the downregulation of IL-2 expression. Overall, the role of intracellular Rv3717 in promoting mycobacterial survival is associated with its ubiquitination and the proteasomal degradation of ILF2.