A Novel Membrane-asscociated Mechanism Of Eliminating Listeria Monocytogenes In Phagocytes

Intracellular bacterial pathogens entered and invaded host cells to survive and replicate inside the host, which caused the spread of infection via host cells migration. Intracellular bacteria comprise various pathogens such as Mycobacterium tuberculosis, Salmonella enterica, Shigella flexneri and Listeria monocytogenes et al [1, 2]. Intracellular bacterial pathogens cause a spectrum of human disease and harm to human being health. Every year, ten million of people were infected with intracellular bacterial pathogens and that two million people died of diseases associated with intracellular bacterial pathogens.It is well documented that some intracellular bacterial pathogen infection can lead to changes of intracellular Rab protein expression that control intracellular phagosome maturation [3]. For example, intracellular expressions of Rab11, Rab20 and Rab32 increase when Mycobacterium tuberculosis infection [4, 5]. Rab proteins can control the course of intracellular phagosome formation [6]. It has been confirmed that Rab32 play a vital role in Salmonella enterica and Mycobactrium leprase infection [7, 8]. Our preliminary results show that intracellular bacteria propagation increased significantly within DC2.4 cells with the depletion of Rab32 comparing to the control group.Listeria is a typical intracellular bacterial pathogen and can invade phagocytes and non-phagocytes [9, 10]. Host cells can recognize pathogen and engulf bacteria by endocytosis into a phagosome. The fusion of phagosomes and lysosomes releases toxic products that kill most bacteria and degrade them into fragments [11, 12]. Listeriolysin O(LLO) is an essential virulence factor of Listeria monocytogenes. LLO is active at acidic pH and help bacteria escape from the phagosome [13]. Escaped bacteria and vesicles containing bacteria are engulfed by isolated membrane and form autophagosome to degrade bacteria. Published paper show Act A protein secreted by Listeria can help bacteria escape from autophagy [14].To clarify the effect and mechanism of Rab32 in dendritic cells infected with Listeria, we construct the mice with conditional depletion of Rab32 gene in dendritic cells, and transfect DC2.4 cells and RAW264.7 cells with Rab32 gene or LC3 gene by lentivirus vectors. Moreover,we depleted Rab32 gene using siRNA in dendritic cells. Using these cells and mice, we systematically study the effect of Rab32 on Listeria infection within dendritic cells by bacteria load, laser confocal microscopy and high-content imaging system. The main research contents are as follows:1. To investigate the effect of Rab32 on Listeria infection in dendritic cells, we construct conditional knockout mice that depletion of Rab32 gene in dendritic cells. Normal growth and development in the mice are not affected and the number and phenotype of dendritic cells in the spleen and peripheral lymph nodes are not different with wild type mice by flow cytometry. We employ intravenous injection of Listeria, which results in systemic infection and measure bacteria load in the spleen and liver after Listeria infection. The results show that bacterial load increases in the spleen and liver of conditional knockout mice that depletion of Rab32 gene in dendritic cells at three days after Listeria infection. Rab32 in dendritic cells affects Listeria propagation in vivo.2. To explore the effect of Rab32 on Listeria propagation in dendritic cells, we culture bone marrow derived dendritic cells(BMDC) in vitro and measure bacteria load in BMDC after Listeria infection. Listeria proliferation in Rab32 knockout BMDC are more than in wild type BMDC after infection, especially at 2h post infection. To further the interaction between Rab32 and Listeria within dendritic cells, we construct overexpression of EYFP-Rab32 gene in dendritic cells and monitor host cells infected with RFP-Listeria by high-content imaging system. The results show that the dynamic recruitment of Rab32 to enclose Listeria even Listeria escape from phagosome. We further find that Rab32 effect commonly in phagocytes by measuring bacteria load in bone marrow derived macrophages and peritoneal macrophages and by monitoring intracellular Rab32 of EYFP-Rab32 DC2.4 cells after Listeria infection.3. To study the effect of Rab32 vesicle on the life cycle of Listeria, we employ L. monocytegenes mutant defective in ActA production, which is incapable of actin polymerization or cell-cell spreading and another L. monocytegenes mutant defective in LLO production, which can not escape from phagosome. Loss of Rab32 in BMDCs led to increased intracellular proliferation of LLO-deficient Listeria and ActA-deficient Listeria. Rab32 limit intracellular expansion of Listeria during multiple stages of infection. To directly monitor the effect of Rab32 vacuole in the stages of infection, we perform live cell video laser scanning confocal microscope recording. Videos show that Listeria even escaped Listeria are initially tagged by patches of Rab32-containing membrane. And bacteria enclosed in Rab32 vacuole are not dividing. To further examine the functional relationship between Rab32 associated vacuoles and autophagosomes, we use RNA interference and high content imaging analysis to study and the number of Rab32 vacuole are not significantly changed in Atg5-deficiency DC2.4 cells by high content imaging analysis. To study the effect of autophagy on Rab32 vacuoles, intracellular bacterial burden is more elevated in Rab32-deficient dendritic cells when Atg5 is deleted. Then high-content imaging results show that colocalization between Rab32 and LC3 is identified but the number of colocalization is few. Variation trend of Rab32 vacuoles is definitely different with LC3 asscociated autophagosome. Rab32 vacuoles peak at 1h post infection. In contrast, LC3-associated autophagosomes capture Listeria at maximum during the decline of Rab32 association containments. These results suggest that Rab32-mediated enclosure is a pathway that parallels the autophagosome for intracellular bacterial containment.ConclusionsOur studies firstly find that Rab32 is essential for preventing intracellular Listeria propagation. Rab32 is not only in dendritic cells, but is prevalent in phagocytes. Rab32 limit bacteria propagation during the multiple stages of Listeria, even when Listeria escaped from phagosome. Rab32 associated vacuoles play a vital role in preventing bacteria proliferation within phagocytes. In conclusion, Rab32 anti-bacteria mechanism in phagocytes is not totally independent on autophagy and is a novel membrane-associated mechanism.