Cellular Microbiology (2006) 8(3), 471–484 doi:10.1111/j.1462-5822.2005.00639.x First published online 28 October 2005 © 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKCMICellular Microbiology 1462-5814© 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd8 3471484Original ArticleM.-A. Bringer et al.AIEC strain LF82 replicates in mature phagolysos- omes Received 31 May, 2005; revised 30 August, 2005; accepted 31 August, 2005. *For correspondence. E-mail a-lise.glasser@ u-clermont1.fr; Tel. (+33) 4 73 17 83 76; Fax (+33) 4 73 17 83 71. The Crohn’s disease-associated adherent-invasive Escherichia coli strain LF82 replicates in mature phagolysosomes within J774 macrophages Marie-Agnès Bringer, 1 Anne-Lise Glasser, 1 * Ching-Hsuan Tung, 2 Stéphane Méresse 3 and Arlette Darfeuille-Michaud 1 1 Pathogénie Bactérienne Intestinale, Laboratoire de Bactériologie, USC INRA 2018, Université d’Auvergne, CBRV, Clermont-Ferrand, France. 2 Center for Molecular Imaging Research, Harvard Medical School, Boston, MA, USA. 3 Centre d’Immunologie de Marseille-Luminy, CNRS- INSERM-University Méditerranée, Parc Scientifique de Luminy, Marseille, France. Summary Adherent-invasive Escherichia coli (AIEC) bacteria isolated from Crohn’s disease patients are able to extensively replicate within macrophages in large vacuoles. The mechanism by which AIEC bacteria sur- vive within phagocytic cells is unknown. This report describes the maturation of AIEC LF82-containing phagosomes within J774 macrophages. LF82- containing phagosomes traffic through the endocytic pathway as shown by the sequential acquisition and loss of EEA1 and Rab7 and by accumulation of Lamp- 1, Lamp-2 and cathepsin D. We demonstrated that AIEC LF82-containing phagosomes mature into active phagolysosomes where bacteria are exposed to low pH and to the degradative activity of cathepsin D. Finally, we showed that an acidic environment is nec- essary for replication of AIEC LF82 bacteria within J774 macrophages. Thus, evidence is provided that AIEC LF82 bacteria do not escape from the endocytic pathway but undergo normal interaction with host endomembrane organelles and replicate within acidic and cathepsin D-positive vacuolar phagolysosomes. Introduction Crohn’s disease (CD) is an inflammatory bowel disease of unknown aetiology in humans (Duchmann and Zeitz, 1999). CD has features that might be the result of a microbial process in the gut (Sartor et al ., 1996; Elson, 2000; Podolsky, 2002; Shanahan, 2002). Some character- istic pathological elements of CD, including aphthous ulcers of the mucosa, mural abscesses, and macrophage and epithelioid cell granulomas also occur in well- recognized infectious disease such as shighellosis, salmonellosis and Yersinia enterocolitis, in which invasive- ness is an essential virulence factor of the bacteria involved (Zumla and James, 1996). However, these patho- genic bacteria have not been found associated with CD. Escherichia coli DNA was detected in 80% of microdis- sected granulomas of CD patients, which suggests a pos- sible role for E. coli in CD lesions (Ryan et al ., 2004). Besides, we recently reported that the ileal mucosa of 36.7% of CD patients is abnormally colonized by patho- genic E. coli strains termed AIEC for a dherent-i nvasive E. coli, which are able to adhere to and to invade intestinal epithelial cells (Darfeuille-Michaud et al., 1998; 2004; Boudeau et al ., 1999). They are also able to replicate extensively in large vacuoles within macrophages without triggering host cell death (Glasser et al ., 2001; Darfeuille- Michaud et al ., 2004). Macrophages serve as the first line of defence by elim- inating indesirable microorganisms. These professional phagocytic cells engulf bacteria within phagosomes that rapidly evolve into bactericidal organelles termed phagoly- sosomes. As they mature into digestive organelles, phagosomes progressively acidify and interact with the endosomal network and/or the biosynthetic pathway (Des- jardins et al ., 1994; Desjardins, 1995; Claus et al ., 1998; Ullrich et al ., 1999; Deretic et al ., 2004; Becker et al ., 2005). Two predominant models exist to explain the bio- genesis of phagolysosomes: the ‘vesicle shuttle’ model, which supports that transport intermediates deriving from endocytic organelles are targeted to phagosomes, and the ‘kiss and run’ model, which proposes that phago- somes undergo transient and partial fusion with endocytic organelles (Desjardins et al ., 1994; Desjardins, 1995; Gu and Gruenberg, 1999; Gruenberg, 2001; Harrison et al ., 2003). This dynamic process is modulated by the sequen- tial appearance and disappearance of proteins on the membrane. First, plasma membrane proteins, including the transferrin receptor (TfR), which initially compose the phagosome, disappear and are replaced sequentially by proteins present in early endosomes (e.g. early endo- somes antigen 1 or EEA1, and Rab5 GTPase). The fol-