| Legionella is a commonly occurring facultative intracellular zoonotic pathogen in the environment that can cause Legionnaires’ disease,with atypical pneumonia as its main symptom,posing a threat to public health.The disease progresses rapidly and has a high mortality rate.Legionella can survive and replicate inside various host cells,such as amoeba and macrophages.Once engulfed by a host cell,Legionella can secrete large amounts of effector proteins into the host cell through its Dot/Icm type IV secretion system(T4SS),hijacking normal host signaling pathways to help form the Legionellacontaining vacuole(LCV)needed for intracellular growth and replication.Currently,our understanding of the interaction between Legionella and its host is mainly based on studies of Legionella pneumophila.Legionella longbeachae is one of the most common pathogens causing Legionnaires’ disease,with higher virulence than L.pneumophila in some countries and regions.However,there is less research on the pathogenic mechanism of L.longbeachae,and the differences between the pathogenic mechanisms of L.longbeachae and L.pneumophila remain unclear.Starting from the perspective of pathogenic bacteria interfering with the host ubiquitination signal pathway,this study used immunofluorescence detection to find that interference with the ubiquitin-proteasome system significantly reduces the proportion of replicative LCV inside cells infected with L.longbeachae.It was also found that the LCV membrane surface can recruit polyubiquitinated proteins in a T4 SSdependent manner,indicating the importance of the host cell’s ubiquitin system in its intracellular replication and suggesting that L.longbeachae may hijack host ubiquitin signaling to promote LCV maturation through its encoded effector proteins.Based on this,two methods were further used in this study to systematically identify effector proteins with deubiquitylating enzyme(DUB)activity in L.longbeachae.Firstly,potential DUBs in L.longbeachae lysate were captured and identified by mass spectrometry using a ubiquitin-vinyl methyl ester(Ub-VME)probe.Secondly,bioinformatics analysis of 992 putative proteins in the L.longbeachae genome resulted in the identification of 20 potential DUBs.Potential DUBs of L.longbeachae were obtained and purified using a prokaryotic expression system,and their biochemical functions were confirmed by in vivo and in vitro deubiquitylation assays.Using the properties of covalent binding of the active cysteine of DUBs with Ub-VME and Ub-PA probes,it was found that Sid Es LLO(LLO＿0424,LLO＿0425,LLO＿0426,LLO＿3092 and LLO＿3095),LLO＿3391,LLO＿1014,LLO＿2238 and LLO＿2491 were reactive to both probes,indicating that these proteins are DUBs encoded by L.longbeachae.Further investigation using diubiquitin revealed that Sid Es LLO can hydrolyze ubiquitin chains linked via K6,K11,K33,K48,and K63;LLO＿33911009-1200 can cleave all types of di-ubiquitin except for K29 linkage;LLO＿1014 specifically cleaves K63-linked di-ubiquitin;and LLO＿2238exhibits hydrolysis activity towards any type of di-ubiquitin.In vivo experiments,transfection with LLO＿1014,LLO＿2238,LLO＿2491 and LLO＿3391 significantly decreased the level of intracellular polyubiquitinated proteins.Although Rav ZLLO did not exhibit DUB activity in vitro,it significantly reduced the formation of polyubiquitinated proteins in cells.To further explore the biological role of L.longbeachae DUBs in its cell infection,LLO＿1014,LLO＿2238 and Rav ZLLO were first confirmed to be recognized and transported into host cells by T4 SS during infection using a β-lactamase reporter system.Subsequently,immunofluorescence detection revealed that LLO＿1014,LLO＿2238 and Rav ZLLO are all located on the LCV membrane,and their DUB activity negatively regulates the recruitment of polyubiquitinated proteins to the LCV.In addition,expressing homologous LLO＿1014 and LLO＿2238 in L.pneumophila Δceg23 andΔlem27,respectively,can restore the increase in polyubiquitinated protein recruitment to LCV caused by the deletion of ceg23 and lem27,indicating the conservation of biological function between homologous DUB proteins in L.pneumophila and L.longbeachae.Rav ZLLO and its homolog Rav ZLP in L.pneumophila share a high sequence similarity and both possess a conserved His-Asp-Cys catalytic triad,but they exhibit significant differences in regulating the ubiquitination of the LCV.Rav ZLP can inhibit host cell autophagy through its cysteine protease activity during L.pneumophila infection.Therefore,this study further investigated the effect of Rav ZLLO on host cell autophagy.The results showed that Rav ZLLO can inhibit cell autophagy in an activitycysteine-dependent manner under both transfection and L.longbeachae infection conditions.Moreover,overexpression of Rav ZLLO in L.pneumophila Δrav ZLP can restore the inhibitory effect of Rav ZLP on autophagy.These findings demonstrate the functional conservation of Rav ZLLO and Rav ZLP in inhibiting host cell autophagy.In summary,L.longbeachae uses multiple DUBs to establish and promote the growth and replication of LCV.This study provides a theoretical basis for a systematic understanding of the pathogenic mechanism of L.longbeachae. |
