The Mechanism Of Cellular Autophagy Regulated By Coxiella Burnetii Effector Protein CpeB

Coxiella burnetii（C.burnetii）is the causative agent of zoonotic Q fever.It belongs to the genus Coxiella in the family Coxiellaceae of the Legionellales,and is considered as an obligate intracellular parasite until the development of acidified citrate cysteine medium（ACCM）,which supports host cell-fee growth of C.burnetii.C.burnetii is typically transmitted from animals to humans with a very low infection dose via contaminated aerosols or tick bites.People who infected with C.burnetii usually presents with flu-like illness,accompanied by signs such as malaise and high fever.In most cases,it can be resolved spontaneously,but it can also develop into a chronic and persistent infection,with symptoms such as hepatitis,endocarditis and even death.C.burnetii can also infect livestock,not only causing losses in the livestock industry,but also becoming a new source of infection.Therefore,strengthening the research on the pathogenic mechanism of C.burnetii is of great significance for providing new ideas and insights for the prevention and control of Q fever.In vivo,C.burnetii targets alveolar macrophages wherein the pathogen replicates in a giant parasitophorous vacuole（Coxiella-containing vacuole,CCV）.It is found that with the expansion of CCV,the fusion between the autophagosomes and the CCV occurs continuously,thereby enlarging the space for C.burnetii to survive and providing nutrients for its growth.Meanwhile,promoting cellular autophagy can also promote the intracellular replication of C.burnetii,suggesting the important role of autophagy in C.burnetii infection.In infected cells,C.burnetii secretes a variety of effector proteins into the host cytoplasm through its type IVB secretion system（T4BSS）,which is involved in the regulation of various host cell life processes including autophagy,apoptosis,providing a favorable environment for its own replication and survival.Plasmid Qp H1 is an important virulence factor of C.burnetii which encoded multiple effector proteins.CpeB is an effector protein encoded by the gene cbua0013 on plasmid Qp H1,and studies suggest that CpeB may be an important effector protein and play an important role in the intracellular replication of C.burnetii.Therefore,our study focused on exploring the role of effector protein CpeB in the pathogenicity of C.burnetii.Firstly,a eukaryotic expression vector of CpeB was constructed to explore the effect of CpeB on cellular autophagy.Then shuttle vectors were constructed using the plasmid Qp H1 as a template and the mutant strains were generated based on the principle of incompatibility of similar plasmids via transformation of the corresponding shuttle plasmids into wild-type C.burnetii.The biological function of CpeB was detected by analyzing the differences in virulence of mutant strains after infection of cells and SCID mice.Finally,the interaction between CpeB and the host protein Rab11a was confirmed by co-immunoprecipitation and confocal microscopy and the structural domains responsible for their interaction were elucidated,as well as the effect of Rab11a on C.burnetii infection and intracellular replication.Additionally,the effect of ATG4B,an important autophagy regulator,on the intracellular survival of C.burnetii was also investigated.By detecting the expression levels of some important host proteins,we found that the autophagy of cells was continuously activated after C.burnetii infection.With the treatment of bafilomycin-A1（Bafi-A1）or rapamycin（Rapa）,it was found that promoting autophagy using Rapa facilitated the intracellular replication of C.burnetii,while blocking autophagy using Bafi-A1 inhibited its intracellular survival,suggesting that autophagy plays an important role in the intracellular replication of C.burnetii.In He La cells,the effect of CpeB on autophagy was examined by GFP-LC3 single fluorescence indicator system and Western blotting.The results showed that CpeB overexpression led to a significantly aggregated dot-like distribution of GFP-LC3 which was much higher than that of the control group.Meanwhile,the level of LC3-II in CpeB overexpressed cells was significantly increased,indicating the potential role of CpeB in promoting autophagy.With the referring of genetic manipulation methods reported,a C.burnetii plasmid-deficient strain（NMIIpQGK）and a strain generated by introduction of CpeB into NMIIpQGK（NMIIpQGK-cpe B）were obtained.Related infection experiments were carried out after identification of the strains using Q-PCR.It was found that compared with the wild-type C.burnetii,the autophagy level of cells infected with NMⅡpQGK was significantly decreased and the intracellular replication of NMⅡpQGK was also significantly inhibited.However,the autophagy level of host cells and the intracellular replication of NMⅡpQGK-cpe B increased when compared with the NMⅡpQGK strain,indicating that CpeB may benefit intracellular replication of C.burnetii by promoting autophagy.Different from the large CCV formed by the wild-type C.burnetii infection in infected cells,CCVs formed by NMIIpQGK infection failed to fuse and showed the characteristics of multiple and small.Although the introduction of CpeB into NMIIpQGK strain could complement the decreased autophagy and the reduced intracellular growth of the mutant strain to a certain extent,it was unable to compensate for the phenotypic defects of CCV homologous fusion.In SCID mice model,we found that compared with the wild-type C.burnetii,the level of hepatomegaly and splenomegaly in the NMIIpQGK infected mice was significantly decreased,as well as the bacterial load in the liver and spleen,while the virulence of NMIIpQGK-cpe B exhibited an increase,indicating that the expression of CpeB could partially compensate for the decrease in virulence of C.burnetii caused by plasmid deficiency.These results suggest that CpeB is an important virulence factor of C.burnetii in a SCID mouse model.We further conducted an in-depth study of the interaction between CpeB and the host protein.Through an AP-MS experiment,Rab11a,host autophagy-associated protein,was identified as an interactor of CpeB.Using co-immunoprecipitation and confocal microscopy,the interaction of CpeB and Rab11a was confirmed,and the domains responsible for this interaction was explored by construction of truncations.It was found that the interaction domains between Rab11a and CpeB were located between the 113～216 amino acid sites of Rab11a and the 164～204 amino acid sites of CpeB.To reveal the underlying mechanism of the interaction,the influence of CpeB on the expression or function of Rab11a was investigated.It was found that CpeB did not affect the expression level of Rab11a in host cells,and it also exerted no significant effect on the enzyme activity of Rab11a in the form of GTPase activating protein（GAP）or guanine nucleotide exchange factor（GEF）.The effect of Rab11a on cellular autophagy was examined using methods as described above.It was found that the expression level of Rab11a was closely related to the lipidation of LC3 and the intracellular replication of C.burnetii,which suggested that the effect of Rab11a on intracellular growth of C.burnetii is likely to be related to its modulation of autophagy.In order to clarify the effect of the interaction on autophagy,the effect of CpeB on autophagy was detected in the case of interfering with Rab11a expression,and it was found that compared with the control group,knocking down Rab11a expression partially offset the level of autophagy caused by CpeB overexpression,indicating that the promotion of autophagy by CpeB partially depends on Rab11a.Finally,the macrophage Rab11a conditional knockout(Rab11a^-/-CKO)mice were generated,and the C.burnetii infection experiment was carried out via aerosolized intratracheal inoculation route.The splenomegaly and bacterial load in spleens or lungs of Rab11a^-/-CKO mice were much lower than those of wild-type mice.Histopathological analysis also showed that the inflammation and pathological changes in lungs of Rab11a^-/-CKO mice were milder.In summary,these results demonstrated that Rab11a interacts with the effector protein CpeB and plays an important role in the replication of C.burnetii in host cells,and the promotion of autophagy by CpeB is partially dependent on Rab11a.To study the role of ATG4B in the intracellular survival of C.burnetii.ATG4B-targeted si RNAs were designed for the experiment.It was found that knocking down the expression of ATG4B blocked the autophagic flux to a certain extent,and inhibited the intracellular replication of C.burnetii and CCV development,suggesting that ATG4B plays an important role in the intracellular survival of C.burnetii.In conclusion,our study provides a preliminary exploration of the mechanism by which the plasmid effector protein CpeB regulates cellular autophagy and the important role of ATG4B in intracellular survival of C.burnetii.We identified that CpeB could promote cellular autophagy,and was a potential virulence factor in a SCID mouse infection model.The host protein Rab11a is the interacting target of CpeB in host cells,and the interaction domain is located between 113～216 amino acid sites of Rab11a and164～204 amino acid sites of CpeB.Rab11a not only induced autophagy and promoted C.burnetii intracellular replication in cells,but also partly responsible for CpeB-induced autophagy.Additionally,we also demonstrated that the host protein ATG4B plays an important function in the intracellular replication and CCV homologous fusion of C.burnetii.This study revealed the important role of autophagy in the infection and replication of C.burnetii and provided a new insight for the prevention and treatment of Q fever infection.