| Legionella pneumophila is a gram-negative bacterium ubiquitously found in natural environments where it mainly parasitizes amoebae.Upon inhalation of bacteriaridden aerosols,L.pneumophila is able to survive and replicate in lung macrophages,thus causing life-threatening pneumonia,also known as Legionnaires’ disease.After invading the host cell,the bacterium resides in a membrane-enclosed compartment termed the Legionella-containing vacuole(LCV),which avoids fusion with lysosomes and provides nutrients for bacterial proliferation within the cells.The biogenesis and maturation of LCVs are strictly dependent on the Dot/Icm type IV secretion system that injects a large cohort of effector proteins(over 330)into the host cytosol.Once translocated,effector proteins target specific host substrates to alter or hijack diverse host signaling pathways,thereby facilitating the remodeling of LCVs.Therefore,studies on the biochemical functions and biological significance of these effectors are of critical importance to further our understanding on L.pneumophila pathogenesis.Protein ubiquitination is one of the most important posttranslational modifications(PTMs)in eukaryotes and is widely involved in the regulation of various cellular signaling pathways.The intracellular pathogenic bacterium L.pneumophila translocates at least 26 effectors to hijack host ubiquitination network via distinct mechanisms.Among these effectors,SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad,which is critical for the recruitment of ERderived vesicles to the LCVs.However,the ubiquitination targets of SidC/SdcA are largely unknown,which restricts our understanding of the mechanisms used by these effectors to hijack the host vesicle trafficking pathway.In this study,we firstly performed an ubiquitinome analysis of 3×HA-ubiquitin-expressing cells that were infected with wild-type or Δsid C/sdc A mutant L.pneumophila strains.After mass spectrometry analysis,we obtained a list of potential ubiquitination targets of SidC/SdcA.Subsequent biochemical experiments,transfection and infection tests demonstrated that multiple Rab small GTPases and t-SNARE proteins involved in host vesicle trafficking are ubiquitination substrates of SidC/SdcA.SidC/SdcA-mediated ubiquitination of Syntaxin 3 and Syntaxin 4 promotes their unconventional pairing with the v-SNARE protein Sec22 b,thereby contributing to the membrane fusion of ERderived vesicles with the phagosome.In addition,we find that ubiquitination of Rab7 by SidC/SdcA is critical for its anchoring to the phagosomal membrane.Rab7 ubiquitination could impair its binding with the downstream effector RILP,which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7.Our study reveals the biological mechanisms employed by SidC/SdcA contribute to intracellular proliferation by promoting LCV maturation.E3 ligases often target multiple proteins for ubiquitination.OTUB1,a deubiquitinase involved in the regulation of host immunity,is also on our candidate list.In vitro ubiquitination reaction reveals that OTUB1 is indeed ubiquitinated by SidC/SdcA.Strikingly,we found that OTUB1 is subjected to both lysine and serine ubiquitination during L.pneumophila infection.The effectors SidC/SdcA catalyse OTUB1 ubiquitination at multiple lysine residues,resulting in its recruitment to LCVs membrane.Lysine ubiquitination by SidC/SdcA promotes interactions between OTUB1 and Deptor,an endogenous inhibitor of the mTORC1 complex,thus suppressing mTORC1 signalling.The inhibition of mTORC1 leads to suppression of host protein synthesis and promotion of host autophagy during L.pneumophila infection.Member of the Sid E family effectors Sde A catalyse PR-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity.In addition,the levels of the lysine and serine ubiquitination of OTUB1 are further regulated by other effectors that with opposite biochemical functions to antagonize the activities of SidC/SdcA and Sid Es,including Lem27,Dup A/Dup B and Sid J/Sdj A.Taken together,our study reveals an effector-mediated complicated mechanism in regulating the activity of a host DUB.In conclusion,our study confirmed that Rab small GTPases,SNAREs and deubiquitinase OTUB1 are physiological substrates of effector SidC/SdcA through the ubiquitinome analysis of L.pneumophila infected cells combined with biochemical and cell biology experiments.Ubiquitination of different substrates catalyzed by SidC/SdcA contributes to bacteria hijacking host vesicle transport,evading lysosomal fusion and inhibiting the host mTOR pathway,thus promoting intracellular proliferation of L.pneumophila. |
