Role of 3-Phosphoinositides in the Maturation of Salmonella-containing Vacuoles within Host Cells

Salmonella typhimuriuminvades mammalian cells and replicates within a vacuole that protects it from the host's microbicidal weapons. The Salmonella-containing vacuole (SCV) undergoes a remodelling akin to that of the host cell's endocytic pathway, but SCV progression is arrested prior to fusion with lysosomes. We studied the role of phosphatidylinositol 3-kinase (PI3-K) in SCV maturation within HeLa cells. Phosphatidylinositol 3-phosphate (PI3P), monitored in situ using fluorescent conjugates of FYVE or PX domains, was found to accumulate transiently on the SCV. Wortmannin prevented PI3P accumulation and the recruitment of EEA1 but did not affect the association of Rab5 with the SCV. Importantly, inhibition of PI3-K also impaired fusion of the SCV with vesicles containing LAMP-1. Rab7, which is thought to be required for association of LAMP-1 with the SCV, still associated with SCV in wortmannin-treated cells. We have therefore concluded that a 3-phosphoinositide-dependent step exists following recruitment of Rab7 to the SCV. The data also imply that 3-phosphoinositide-dependent effectors of Rab5 are not an absolute requirement for recruitment of Rab7. Despite failure to acquire LAMP-1, the SCV persists and allows effective replication of Salmonella within wortmannin-treated host cells. These findings imply that PI3-K is involved in the development of the SCV but is not essential for intracellular survival and proliferation of Salmonella. Salmonella typhimuriuminvades mammalian cells and replicates within a vacuole that protects it from the host's microbicidal weapons. The Salmonella-containing vacuole (SCV) undergoes a remodelling akin to that of the host cell's endocytic pathway, but SCV progression is arrested prior to fusion with lysosomes. We studied the role of phosphatidylinositol 3-kinase (PI3-K) in SCV maturation within HeLa cells. Phosphatidylinositol 3-phosphate (PI3P), monitored in situ using fluorescent conjugates of FYVE or PX domains, was found to accumulate transiently on the SCV. Wortmannin prevented PI3P accumulation and the recruitment of EEA1 but did not affect the association of Rab5 with the SCV. Importantly, inhibition of PI3-K also impaired fusion of the SCV with vesicles containing LAMP-1. Rab7, which is thought to be required for association of LAMP-1 with the SCV, still associated with SCV in wortmannin-treated cells. We have therefore concluded that a 3-phosphoinositide-dependent step exists following recruitment of Rab7 to the SCV. The data also imply that 3-phosphoinositide-dependent effectors of Rab5 are not an absolute requirement for recruitment of Rab7. Despite failure to acquire LAMP-1, the SCV persists and allows effective replication of Salmonella within wortmannin-treated host cells. These findings imply that PI3-K is involved in the development of the SCV but is not essential for intracellular survival and proliferation of Salmonella. Salmonella-containing vacuole early endosome autoantigen 1 lysosome-associated membrane protein 1 mannose-6-phosphate receptor phosphate-buffered saline phosphatidylinositol 3-kinase phosphatidylinositol 3-phosphate 40-kDa subunit of the NADPH oxidase streptolysin-O green fluorescent protein lipopolysaccharide fluorescein isothiocyanate Salmonella-induced filament 4-morpholineethanesulfonic acid The enteropathogenic bacterium Salmonella typhimuriumis able to invade host cells, where it resides within a membrane-bound vacuole. By altering the normal traffic of intracellular membranes, the Salmonella vacuole remains isolated from the cellular lysosomes, thereby avoiding contact with their microbicidal contents. Although great progress has been made in elucidating the molecular mechanisms of invasion, the processes whereby the bacteria control the interaction of the vacuole with the endocytic pathway remain incompletely understood and often controversial (1.Ochman H. Soncini F.C. Solomon F. Groisman E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7800-7804Crossref PubMed Scopus (524) Google Scholar, 2.Hayward R.D. Koronakis V. EMBO J. 1999; 18: 4926-4934Crossref PubMed Scopus (245) Google Scholar, 3.Uchiya K. Barbieri M.A. Funato K. Shah A.H. Stahl P.D. Groisman E.A. EMBO J. 1999; 18: 3924-3933Crossref PubMed Scopus (289) Google Scholar, 4.Beuzon C.R. Meresse S. Unsworth K.E. Ruiz-Albert J. Garvis S. Waterman S.R. Ryder T.A. Boucrot E. Holden D.W. EMBO J. 2000; 19: 3235-3249Crossref PubMed Scopus (457) Google Scholar, 5.Hashim S. Mukherjee K. Raje M. Basu S.K. Mukhopadhyay A. J. Biol. Chem. 2000; 275: 16281-16288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).Following attachment to the host cell surface, Salmonellauses a specialized type III secretion system to deliver bacterial effector proteins across the cell plasma membrane into the cytoplasm (6.Kubori T. Matsushima Y. Nakamura D. Uralil J. Lara-Tejero M. Sukhan A. Galan J.E. Aizawa S.I. Science. 1998; 280: 602-605Crossref PubMed Scopus (695) Google Scholar). These virulence factors include phosphatases (7.Norris F.A. Wilson M.P. Wallis T.S. Galyov E.E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14057-14059Crossref PubMed Scopus (351) Google Scholar), Rho-GTPase exchange factors (8.Hardt W.D. Chen L.M. Schuebel K.E. Bustelo X.R. Galan J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar), and other modulators of the host cell cytoskeleton (2.Hayward R.D. Koronakis V. EMBO J. 1999; 18: 4926-4934Crossref PubMed Scopus (245) Google Scholar, 9.Fu Y. Galan J.E. Mol. Microbiol. 1998; 27: 359-368Crossref PubMed Scopus (198) Google Scholar, 10.Zhou D. Mooseker M.S. Galan J.E. Science. 1999; 283: 2092-2095Crossref PubMed Scopus (324) Google Scholar). The net effect of these bacterial proteins is the induction of pronounced ruffling of the host cell membrane, leading to the internalization of the bacteria into a Salmonella-containing vacuole (SCV).1Initially, the composition of the SCV resembles that of the host's early endocytic compartment. Distinctive endosomal markers such as the early endosome autoantigen 1 (EEA1), the transferrin receptor, and Rab5 are detectable on the membrane of the early SCV (5.Hashim S. Mukherjee K. Raje M. Basu S.K. Mukhopadhyay A. J. Biol. Chem. 2000; 275: 16281-16288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11.Steele-Mortimer O. Meresse S. Gorvel J.P. Toh B.H. Finlay B.B. Cell Microbiol. 1999; 1: 33-49Crossref PubMed Scopus (258) Google Scholar). At later stages, the SCV acquires selectively some, but not all, of the proteins known to be present in the host cell late endosomes. Thus, although the lysosome-associated membrane protein 1 (LAMP-1) (12.Garcia-del Portillo F. Zwick M.B. Leung K.Y. Finlay B.B. Infect. Agents Dis. 1993; 2: 227-231PubMed Google Scholar), the vacuolar proton pump (11.Steele-Mortimer O. Meresse S. Gorvel J.P. Toh B.H. Finlay B.B. Cell Microbiol. 1999; 1: 33-49Crossref PubMed Scopus (258) Google Scholar), and Rab7 (13.Meresse S. Steele-Mortimer O. Finlay B.B. Gorvel J.P. EMBO J. 1999; 18: 4394-4403Crossref PubMed Scopus (198) Google Scholar) associate with the SCV, the mannose-6-phosphate receptor (M6PR) (14.Garcia-del Portillo F. Finlay B.B. J. Cell Biol. 1995; 129: 81-97Crossref PubMed Scopus (207) Google Scholar) is largely excluded. More importantly, unlike the late endosome, the SCV fails to merge with lysosomes. This diversion of the endocytic traffic is dictated by an additional set of bacterial proteins encoded by a separate pathogenicity island in the Salmonella genome. These proteins are also delivered via a type III system to the host cell cytosol, protecting the bacteria from exposure to the degradative environment of the lysosome (15.Hensel M. Mol. Microbiol. 2000; 36: 1015-1023Crossref PubMed Scopus (266) Google Scholar).The normal traffic of cellular endomembranes is directed by several types of regulatory proteins, including the Rab family of small GTPases (16.Bock J.B. Matern H.T. Peden A.A. Scheller R.H. Nature. 2001; 409: 839-841Crossref PubMed Scopus (520) Google Scholar). The fusion of early endosomes is directed by Rab5, in conjunction with several effector proteins that include EEA1 (17.Christoforidis S. Miaczynska M. Ashman K. Wilm M. Zhao L. Yip S.C. Waterfield M.D. Backer J.M. Zerial M. Nat. Cell. Biol. 1999; 1: 249-252Crossref PubMed Scopus (498) Google Scholar, 18.Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar). Both Rab5 and EEA1 have been detected on the early SCV (5.Hashim S. Mukherjee K. Raje M. Basu S.K. Mukhopadhyay A. J. Biol. Chem. 2000; 275: 16281-16288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11.Steele-Mortimer O. Meresse S. Gorvel J.P. Toh B.H. Finlay B.B. Cell Microbiol. 1999; 1: 33-49Crossref PubMed Scopus (258) Google Scholar), but their precise roles in vacuolar maturation are not well defined. EEA1 attaches directly to Rab5, but this interaction needs to be stabilized by PI3P, a product of the type III phosphatidylinositol 3-kinase (PI3-K), VPS34. The latter is also thought to be a Rab5 effector (17.Christoforidis S. Miaczynska M. Ashman K. Wilm M. Zhao L. Yip S.C. Waterfield M.D. Backer J.M. Zerial M. Nat. Cell. Biol. 1999; 1: 249-252Crossref PubMed Scopus (498) Google Scholar).Recently, PI3P was reported to be an essential factor in the normal maturation of endosomes (19.Simonsen A. Wurmser A.E. Emr S.D. Stenmark H. Curr. Opin. Cell Biol. 2001; 13: 485-492Crossref PubMed Scopus (405) Google Scholar) and also of phagosomes (20.Vieira O.V. Botelho R.J. Rameh L. Brachmann S.M. Matsuo T. Davidson H.W. Schreiber A. Backer J.M. Cantley L.C. Grinstein S. J. Cell Biol. 2001; 155: 19-26Crossref PubMed Scopus (416) Google Scholar), which more closely resemble the SCV. It therefore appeared likely that this phosphoinositide could also play a role in SCV development and raised the possibility that alterations in the metabolism of PI3P may contribute to the maturation arrest of the vacuole. The purpose of the present experiments was therefore to analyze the metabolism of PI3P during the course of invasion of mammalian cells by Salmonella.It recently became apparent that modular domains within certain proteins have the ability to interact with the headgroups of defined phosphoinositides. Two types of domains have been identified that recognize PI3P with great selectivity and considerable affinity: FYVE domains, such as the one present in EEA1 (21.Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Crossref PubMed Scopus (440) Google Scholar), and PX domains (22.Cheever M.L. Sato T.K. de Beer T. Kutateladze T.G. Emr S.D. Overduin M. Nat. Cell. Biol. 2001; 3: 613-618Crossref PubMed Scopus (311) Google Scholar, 23.Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell. Biol. 2001; 3: 675-678Crossref PubMed Scopus (493) Google Scholar, 24.Xu Y. Hortsman H. Seet L. Wong S.H. Hong W. Nat. Cell. Biol. 2001; 3: 658-666Crossref PubMed Scopus (238) Google Scholar). In this report we have used chimeric constructs of GFP and the FYVE domain of EEA1 and of the PX domain of the 40-kDa subunit of the NADPH oxidase to monitor the distribution and dynamics of PI3P during the formation and progression of the SCV. The enteropathogenic bacterium Salmonella typhimuriumis able to invade host cells, where it resides within a membrane-bound vacuole. By altering the normal traffic of intracellular membranes, the Salmonella vacuole remains isolated from the cellular lysosomes, thereby avoiding contact with their microbicidal contents. Although great progress has been made in elucidating the molecular mechanisms of invasion, the processes whereby the bacteria control the interaction of the vacuole with the endocytic pathway remain incompletely understood and often controversial (1.Ochman H. Soncini F.C. Solomon F. Groisman E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7800-7804Crossref PubMed Scopus (524) Google Scholar, 2.Hayward R.D. Koronakis V. EMBO J. 1999; 18: 4926-4934Crossref PubMed Scopus (245) Google Scholar, 3.Uchiya K. Barbieri M.A. Funato K. Shah A.H. Stahl P.D. Groisman E.A. EMBO J. 1999; 18: 3924-3933Crossref PubMed Scopus (289) Google Scholar, 4.Beuzon C.R. Meresse S. Unsworth K.E. Ruiz-Albert J. Garvis S. Waterman S.R. Ryder T.A. Boucrot E. Holden D.W. EMBO J. 2000; 19: 3235-3249Crossref PubMed Scopus (457) Google Scholar, 5.Hashim S. Mukherjee K. Raje M. Basu S.K. Mukhopadhyay A. J. Biol. Chem. 2000; 275: 16281-16288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Following attachment to the host cell surface, Salmonellauses a specialized type III secretion system to deliver bacterial effector proteins across the cell plasma membrane into the cytoplasm (6.Kubori T. Matsushima Y. Nakamura D. Uralil J. Lara-Tejero M. Sukhan A. Galan J.E. Aizawa S.I. Science. 1998; 280: 602-605Crossref PubMed Scopus (695) Google Scholar). These virulence factors include phosphatases (7.Norris F.A. Wilson M.P. Wallis T.S. Galyov E.E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14057-14059Crossref PubMed Scopus (351) Google Scholar), Rho-GTPase exchange factors (8.Hardt W.D. Chen L.M. Schuebel K.E. Bustelo X.R. Galan J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar), and other modulators of the host cell cytoskeleton (2.Hayward R.D. Koronakis V. EMBO J. 1999; 18: 4926-4934Crossref PubMed Scopus (245) Google Scholar, 9.Fu Y. Galan J.E. Mol. Microbiol. 1998; 27: 359-368Crossref PubMed Scopus (198) Google Scholar, 10.Zhou D. Mooseker M.S. Galan J.E. Science. 1999; 283: 2092-2095Crossref PubMed Scopus (324) Google Scholar). The net effect of these bacterial proteins is the induction of pronounced ruffling of the host cell membrane, leading to the internalization of the bacteria into a Salmonella-containing vacuole (SCV).1 Initially, the composition of the SCV resembles that of the host's early endocytic compartment. Distinctive endosomal markers such as the early endosome autoantigen 1 (EEA1), the transferrin receptor, and Rab5 are detectable on the membrane of the early SCV (5.Hashim S. Mukherjee K. Raje M. Basu S.K. Mukhopadhyay A. J. Biol. Chem. 2000; 275: 16281-16288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11.Steele-Mortimer O. Meresse S. Gorvel J.P. Toh B.H. Finlay B.B. Cell Microbiol. 1999; 1: 33-49Crossref PubMed Scopus (258) Google Scholar). At later stages, the SCV acquires selectively some, but not all, of the proteins known to be present in the host cell late endosomes. Thus, although the lysosome-associated membrane protein 1 (LAMP-1) (12.Garcia-del Portillo F. Zwick M.B. Leung K.Y. Finlay B.B. Infect. Agents Dis. 1993; 2: 227-231PubMed Google Scholar), the vacuolar proton pump (11.Steele-Mortimer O. Meresse S. Gorvel J.P. Toh B.H. Finlay B.B. Cell Microbiol. 1999; 1: 33-49Crossref PubMed Scopus (258) Google Scholar), and Rab7 (13.Meresse S. Steele-Mortimer O. Finlay B.B. Gorvel J.P. EMBO J. 1999; 18: 4394-4403Crossref PubMed Scopus (198) Google Scholar) associate with the SCV, the mannose-6-phosphate receptor (M6PR) (14.Garcia-del Portillo F. Finlay B.B. J. Cell Biol. 1995; 129: 81-97Crossref PubMed Scopus (207) Google Scholar) is largely excluded. More importantly, unlike the late endosome, the SCV fails to merge with lysosomes. This diversion of the endocytic traffic is dictated by an additional set of bacterial proteins encoded by a separate pathogenicity island in the Salmonella genome. These proteins are also delivered via a type III system to the host cell cytosol, protecting the bacteria from exposure to the degradative environment of the lysosome (15.Hensel M. Mol. Microbiol. 2000; 36: 1015-1023Crossref PubMed Scopus (266) Google Scholar). The normal traffic of cellular endomembranes is directed by several types of regulatory proteins, including the Rab family of small GTPases (16.Bock J.B. Matern H.T. Peden A.A. Scheller R.H. Nature. 2001; 409: 839-841Crossref PubMed Scopus (520) Google Scholar). The fusion of early endosomes is directed by Rab5, in conjunction with several effector proteins that include EEA1 (17.Christoforidis S. Miaczynska M. Ashman K. Wilm M. Zhao L. Yip S.C. Waterfield M.D. Backer J.M. Zerial M. Nat. Cell. Biol. 1999; 1: 249-252Crossref PubMed Scopus (498) Google Scholar, 18.Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (905) Google Scholar). Both Rab5 and EEA1 have been detected on the early SCV (5.Hashim S. Mukherjee K. Raje M. Basu S.K. Mukhopadhyay A. J. Biol. Chem. 2000; 275: 16281-16288Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11.Steele-Mortimer O. Meresse S. Gorvel J.P. Toh B.H. Finlay B.B. Cell Microbiol. 1999; 1: 33-49Crossref PubMed Scopus (258) Google Scholar), but their precise roles in vacuolar maturation are not well defined. EEA1 attaches directly to Rab5, but this interaction needs to be stabilized by PI3P, a product of the type III phosphatidylinositol 3-kinase (PI3-K), VPS34. The latter is also thought to be a Rab5 effector (17.Christoforidis S. Miaczynska M. Ashman K. Wilm M. Zhao L. Yip S.C. Waterfield M.D. Backer J.M. Zerial M. Nat. Cell. Biol. 1999; 1: 249-252Crossref PubMed Scopus (498) Google Scholar). Recently, PI3P was reported to be an essential factor in the normal maturation of endosomes (19.Simonsen A. Wurmser A.E. Emr S.D. Stenmark H. Curr. Opin. Cell Biol. 2001; 13: 485-492Crossref PubMed Scopus (405) Google Scholar) and also of phagosomes (20.Vieira O.V. Botelho R.J. Rameh L. Brachmann S.M. Matsuo T. Davidson H.W. Schreiber A. Backer J.M. Cantley L.C. Grinstein S. J. Cell Biol. 2001; 155: 19-26Crossref PubMed Scopus (416) Google Scholar), which more closely resemble the SCV. It therefore appeared likely that this phosphoinositide could also play a role in SCV development and raised the possibility that alterations in the metabolism of PI3P may contribute to the maturation arrest of the vacuole. The purpose of the present experiments was therefore to analyze the metabolism of PI3P during the course of invasion of mammalian cells by Salmonella. It recently became apparent that modular domains within certain proteins have the ability to interact with the headgroups of defined phosphoinositides. Two types of domains have been identified that recognize PI3P with great selectivity and considerable affinity: FYVE domains, such as the one present in EEA1 (21.Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Crossref PubMed Scopus (440) Google Scholar), and PX domains (22.Cheever M.L. Sato T.K. de Beer T. Kutateladze T.G. Emr S.D. Overduin M. Nat. Cell. Biol. 2001; 3: 613-618Crossref PubMed Scopus (311) Google Scholar, 23.Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell. Biol. 2001; 3: 675-678Crossref PubMed Scopus (493) Google Scholar, 24.Xu Y. Hortsman H. Seet L. Wong S.H. Hong W. Nat. Cell. Biol. 2001; 3: 658-666Crossref PubMed Scopus (238) Google Scholar). In this report we have used chimeric constructs of GFP and the FYVE domain of EEA1 and of the PX domain of the 40-kDa subunit of the NADPH oxidase to monitor the distribution and dynamics of PI3P during the formation and progression of the SCV. We thank Dr. M. Yaffe for providing the p40PX-GFP constructs, Drs. B. B. Finlay and J. Brumell (University of British Columbia) for advice and for providing bacterial strains, Jonathan Plumb for preparation of the pool-3 fraction of polyethylenimine used for transfections, and Dr. M. Zerial for providing the Rab7 cDNA.

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