Replication of Brucella abortus and Brucella melitensis in fibroblasts does not require Atg5-dependent macroautophagy

BMC Microbiology

Michel Jadot²  Jean-Jacques Letesson³  Xavier De Bolle³  Emeline Goffin¹  Isabelle Hamer² 

[1]Present address: Faculty of Veterinary Medicine-Department of infectious and parasitic diseases, Laboratory of Immunology and Vaccinology, University of Liège, Liège, Belgium

[2]Research Unit in Molecular Physiology (URPhyM), NAmur Research Institute for LIfe Sciences (NARILIS), University of Namur, Namur, Belgium

[3]Research Unit in Biology of Microorganisms (URBM), NAmur Research Institute for LIfe Sciences (NARILIS), University of Namur, Namur, Belgium

关键词: Atg5;    Macroautophagy;    Replication;    Intracellular trafficking;    Brucella melitensis;    Brucella abortus;   

Others  :  1140422
DOI  :  10.1186/s12866-014-0223-5

 received in 2014-04-17, accepted in 2014-08-13,  发布年份 2014

【 摘 要 】

Background

Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate inside the cells. Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a worldwide extended chronic zoonosis. Following invasion, Brucella abortus is found in a vacuole that interacts first with various endosomal compartments and then with endoplasmic reticulum sub-compartments. Brucella establishes its replication niche in ER-derived vesicles. In the past, it has been proposed that B. abortus passed through the macroautophagy pathway before reaching its niche of replication. However, recent experiments provided evidence that the classical macroautophagy pathway was not involved in the intracellular trafficking and the replication of B. abortus in bone marrow-derived macrophages and in HeLa cells. In contrast, another study showed that macroautophagy favoured the survival and the replication of Brucella melitensis in infected RAW264.7 macrophages. This raises the possibility that B. abortus and B. melitensis followed different intracellular pathways before replicating. In the present work, we have addressed this issue by comparing the replication rate of B. abortus and B. melitensis in embryonic fibroblasts derived from wild-type and Atg5^?/? mice, Atg5 being a core component of the canonical macroautophagic pathway.

Results

Our results indicate that both B. abortus S2308 and B. melitensis 16M strains are able to invade and replicate in Atg5-deficient fibroblasts, suggesting that the canonical Atg5-dependent macroautophagic pathway is dispensable for Brucella replication. The number of viable bacteria was even slightly higher in Atg5^?/? fibroblasts than in wild-type fibroblasts. This increase could be due to a more efficient uptake or to a better survival rate of bacteria before the beginning of the replication in Atg5-deficient cells as compared to wild-type cells. Moreover, our data show that the infection with B. abortus or with B. melitensis does not stimulate neither the conversion of LC3-I to LC3-II nor the membrane recruitment of LC3 onto the BCV.

Conclusion

Our study suggests that like Brucella abortus, Brucella melitensis does not subvert the canonical macroautophagy to reach its replicative niche or to stimulate its replication.

【 授权许可】

   
2014 Hamer et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Cemma M, Brumell JH: Interactions of pathogenic bacteria with autophagy systems. Curr Biol 2012, 22(13):R540-R545.
• [2]Vergne I, Fratti RA, Hill PJ, Chua J, Belisle J, Deretic V: Mycobacterium tuberculosis phagosome maturation arrest: mycobacterial phosphatidylinositol analog phosphatidylinositol mannoside stimulates early endosomal fusion. Mol Biol Cell 2004, 15(2):751-760.
• [3]Amer AO, Swanson MS: Autophagy is an immediate macrophage response to Legionella pneumophila. Cell Microbiol 2005, 7(6):765-778.
• [4]Romano PS, Gutierrez MG, Beron W, Rabinovitch M, Colombo MI: The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell. Cell Microbiol 2007, 9(4):891-909.
• [5]Schnaith A, Kashkar H, Leggio SA, Addicks K, Kronke M, Krut O: Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death. J Biol Chem 2007, 282(4):2695-2706.
• [6]Starr T, Ng TW, Wehrly TD, Knodler LA, Celli J: Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment. Traffic 2008, 9(5):678-694.
• [7]Arellano-Reynoso B, Lapaque N, Salcedo S, Briones G, Ciocchini AE, Ugalde R, Moreno E, Moriyon I, Gorvel JP: Cyclic beta-1,2-glucan is a Brucella virulence factor required for intracellular survival. Nat Immunol 2005, 6(6):618-625.
• [8]Celli J, de Chastellier C, Franchini DM, Pizarro-Cerda J, Moreno E, Gorvel JP: Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J Exp Med 2003, 198(4):545-556.
• [9]Pizarro-Cerda J, Moreno E, Gorvel JP: Invasion and intracellular trafficking of Brucella abortus in nonphagocytic cells. Microbes Infect 2000, 2(7):829-835.
• [10]Celli J: Surviving inside a macrophage: the many ways of Brucella. Res Microbiol 2006, 157(2):93-98.
• [11]Pizarro-Cerda J, Meresse S, Parton RG, van der Goot G, Sola-Landa A, Lopez-Goni I, Moreno E, Gorvel JP: Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect Immun 1998, 66(12):5711-5724.
• [12]Starr T, Child R, Wehrly TD, Hansen B, Hwang S, Lopez-Otin C, Virgin HW, Celli J: Selective subversion of autophagy complexes facilitates completion of the Brucella intracellular cycle. Cell Host Microbe 2012, 11(1):33-45.
• [13]Pizarro-Cerda J, Moreno E, Sanguedolce V, Mege JL, Gorvel JP: Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect Immun 1998, 66(5):2387-2392.
• [14]Lamb CA, Yoshimori T, Tooze SA: The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol 2013, 14(12):759-774.
• [15]Mizushima N, Yoshimori T, Ohsumi Y: The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011, 27:107-132.
• [16]Hanada T, Noda NN, Satomi Y, Ichimura Y, Fujioka Y, Takao T, Inagaki F, Ohsumi Y: The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem 2007, 282(52):37298-37302.
• [17]Mizushima N, Kuma A, Kobayashi Y, Yamamoto A, Matsubae M, Takao T, Natsume T, Ohsumi Y, Yoshimori T: Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate. J Cell Sci 2003, 116(Pt 9):1679-1688.
• [18]Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T: Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 2001, 152(4):657-668.
• [19]Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000, 19(21):5720-5728.
• [20]Tanida I, Sou YS, Ezaki J, Minematsu-Ikeguchi N, Ueno T, Kominami E: HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates. J Biol Chem 2004, 279(35):36268-36276.
• [21]Tanida I, Ueno T, Kominami E: Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes. J Biol Chem 2004, 279(46):47704-47710.
• [22]Guo F, Zhang H, Chen C, Hu S, Wang Y, Qiao J, Ren Y, Zhang K, Wang Y, Du G: Autophagy favors Brucella melitensis survival in infected macrophages. Cell Mol Biol Lett 2012, 17(2):249-257.
• [23]Seglen PO, Gordon PB: 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A 1982, 79(6):1889-1892.
• [24]Wu YT, Tan HL, Shui G, Bauvy C, Huang Q, Wenk MR, Ong CN, Codogno P, Shen HM: Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem 2010, 285(14):10850-10861.
• [25]Caro LH, Plomp PJ, Wolvetang EJ, Kerkhof C, Meijer AJ: 3-Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism. Eur J Biochem 1988, 175(2):325-329.
• [26]Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, Kanaseki T, Komatsu M, Otsu K, Tsujimoto Y, Shimizu S: Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 2009, 461(7264):654-658.
• [27]Salcedo SP, Chevrier N, Santos Lacerda TL, Ben Amara A, Gerart S, Gorvel VA, de Chastellier C, Blasco JM, Mege JL, Gorvel JP: Pathogenic Brucellae replicate in human trophoblasts. J Infect Dis 2013, 207(7):1075-1083.
• [28]de Barsy M, Jamet A, Filopon D, Nicolas C, Laloux G, Rual JF, Muller A, Twizere JC, Nkengfac B, Vandenhaute J, Hill DE, Salcedo SP, Gorvel JP, Letesson JJ, De Bolle X: Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2. Cell Microbiol 2011, 13(7):1044-1058.
• [29]Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, Ohsumi Y, Tokuhisa T, Mizushima N: The role of autophagy during the early neonatal starvation period. Nature 2004, 432(7020):1032-1036.
• [30]Cloeckaert A, Zygmunt MS, Dubray G, Limet JN: Characterization of O-polysaccharide specific monoclonal antibodies derived from mice infected with the rough Brucella melitensis strain B115. J Gen Microbiol 1993, 139(7):1551-1556.