Subversion Of Host Intracellular Vesicle Trafficking By Burkholderia Pseudomallei

Melioidosis is an emerging zoonotic infectious disease caused by the bacterium Burkholderia pseudomallei.Melioidosis can be transmitted to a susceptible host through digestive tract,respiratory tract or damaged epidermis,causing severe diseases such as pneumonia,multiple lung abscesses,spleen abscesses,and acute sepsis.Given the melioidosis can present as a spectrum of clinical diseases with high mortality rates and there is presently no effective vaccine,B.pseudomallei have been classified as category B bioterrorism agents by Centers for Disease Control and Prevention(CDC)in the United States.Melioidosis is highly endemic in tropical and subtropical regions of the world,including southeast Asia and northern Australia.Since melioidosis poses a serious threat to global public health and there are no effective therapeutic strategies for the disease,the development of the underlying pathogenic mechanisms of B.pseudomallei is a priority.The innate immune system is the first line of defense against invading pathogens.Professional phagocytic cells(e.g.,macrophages,neutrophils,and dendritic cells)are critical components of innate immunity system.Following phagocytosis by macrophages,microbial pathogens is contained in the phagosome,which subsequently fuses with lysosomes for the clearance and degradation of pathogens.As a facultative intracellular pathogen,B.pseudomallei efficiently evades host immune defenses and clearance,which has become a key step for its survival and dissemination during infection.Therefore,understanding the intracellular infection mechanism of B.pseudomallei is essential in developing future therapeutic strategies against B.pseudomallei infection.Following uptake,the intracellular bacterial pathogen B.pseudomallei evades phagosome/lysosome maturation pathways in host cells by camouflaging in a specialized organelle,the B.pseudomallei-containing vacuole(BCV),which is essential for intravacuolar proliferation colonization within host cells.Rab proteins are key regulators of intracellular vesicular transport pathways and regulate phagosome trafficking and maturation in macrophages.However,the exact mechanistic details of how B.pseudomallei adapts to the intraphagosomal environment and manipulates the phagocytic process remain unknown.Therefore,in order to identify host cell molecules and pathways utilized by B.pseudomallei for intracellular survival,we initially investigated the localization and expression of 19 Rab GTPases,which are critical regulators of vesicular transport pathways.In this study,we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30 c in B.pseudomallei infected macrophages.Subsequently,we showed that B.pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features.And Rab32 enhances phagosome acidification and promotes the fusion of B.pseudomallei-containing phagosomes with lysosomes to activate cathepsin D,resulting in restricted intracellular growth of B.pseudomallei.We also demonstrated that miR-30b/30 c is associated with the modulation of phagosome maturation and affects the intracellular survival of B.pseudomallei by targeting Rab32 in host innate immune cells.A newly prominent area of research is the identification of pathogenic effector proteins that regulate the host-pathogen interactions.Like many other Gram-negative bacterial pathogens,B.pseudomallei uses a type Ⅲ secretion system(T3SS)to deliver effector proteins into the host cells to manipulate host cell functions,and ultimately establish infection.Especially in the third set of type Ⅲ secretion system(T3SS-3)effector proteins facilitate B.pseudomallei escape from the phagosomes to avoid fusion with lysosomes,although the mechanism has not been fully elucidated.To further identify the effector proteins that play critical roles in regulating phagosome trafficking and maturation,we used homologous recombination to knock out genes that encode T3SS-3 effector proteins(BopA、BopC、BopE、BapA,and BapC),respectively.And the ability of the mutant strains to escape the phagosome during infection was assayed.Notebly,the BapC deletion mutant strain avoided trafficking to lysosomes and phagosome escape was diminished.Moreover,we performed immunoprecipitation-mass spectrometry analyses(IP-MS)and identified the microtubule protein tubulin as a potential host cell interacting protein for BapC.However,it is surprising that BapC does not co-localize with tubulin,but instead localizes in the nucleus.Furthermore,we found that genetic deletion of BapC led to significantly decreased proliferation in macrophages and the murine infection model.In addition,we found that BapC deletion mutant strain elicited a severe host inflammatory response and organ damage in mice.Taken together,the above results suggest that the BapC may play an important role in the process of B.pseudomallei infection.In summary,we aim to better understand B.pseudomallei interferes with intracellular trafficking of host phagosomes and evades immune system-mediated clearance,and as a starting point to analyze the regulation of BCV transport and screening for key effector proteins of T3SS-3 regulating phagosome transport in host cells.We found that the infection of macrophages with B.pseudomallei resulted in the upregulation of Rab32 expression through the inhibition of mi R-30b/30 c expression.Subsequently,Rab32 is recruited to the B.pseudomallei-containing phagosomes and promotes the fusion of the phagosomes with lysosomes,which results in the increased exposure of B.pseudomallei to lysosomal acid hydrolases CTSD,thus limiting the intracellular growth of B.pseudomallei in an early phase of infection in macrophages.And in the meantime,T3SS-3 effector protein,BapC facilitates B.pseudomallei escape from phagosomes.And BapC not only relies on tubulin for nuclear import but also affects the host immune response during B.pseudomallei infection.These studies provide an important theoretical basis for understanding the interactions between B.pseudomallei and the host and for the prevention and treatment of B.pseudomallei infection.