Nature © Macmillan Publishers Ltd 1997 Survival of FimH-expressing enterobacteria in macrophages relies on glycolipid trafﬁc David M. Baorto*, Zhimin Gao*, Ravi Malaviya*, Michael L. Dustin*, Anton van der Merwe†, Douglas M. Lublin* & Soman N. Abraham*‡§ Departments of * Pathology and ‡ Molecular Microbiology, Washington University School of Medicine, St Louis, Missouri 63110, USA † MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK ......................................................................................................................... Strains of Escherichia coli persist within the human gut as normal commensals, but are frequent pathogens and can cause recurrent infection 1–3 . Here we show that, in contrast to E. coli subjected to opsonic interactions stimulated by the host’s immune response, E. coli that bind to the macrophage surface exclusively through the bacterial lectin FimH can survive inside the cell following phagocytosis. This viability is largely due to the attenuation of intracellular free-radical release and of phagosome acidiﬁcation during FimH-mediated internalization, both of which are trig- gered by antibody-mediated internalization. This different pro- cessing of non-opsonized bacteria is supported by morphological evidence of tight-ﬁtting phagosomes compared with looser, anti- body-mediated phagosomes. We propose that non-opsonized FimH-expressing E. coli co-opt internalization of lipid-rich microdomains following binding to the FimH receptor, the glycosylphosphatidylinositol-linked protein CD48, because (1) the sterol-binding agents ﬁlipin, nystatin and methyl -cyclodex- trin speciﬁcally block FimH-mediated internalization; (2) CD48 and the protein caveolin both accumulate on macrophage mem- branes surrounding bacteria; and (3) antibodies against CD48 inhibit FimH-mediated internalization. Our ﬁndings bring the traditionally extracellular E. coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies. Escherichia coli are normal residents of the alimentary canal but they have also been implicated in opportunistic and often recurrent infections in man and animals 1–3 . E. coli, as well as many other enteric bacteria, express on their surface type-1 ﬁmbriae, 1–2-m- long ﬁlaments composed of several subunits including FimH, a mannose-binding lectin that is responsible for promoting bacterial adherence and colonization of mucosal surfaces 4,5 . Paradoxically, however, FimH also promotes adhesion to phagocytic cells, an event that should presumably result in early bacterial clearance. With two exceptions 6,7 , most in vitro studies have reported that type-1 ﬁmbrial-mediated association with phagocytic cells results in bac- terial killing 8–10 . As the effect of opsonic serum components or the contribution of other bacterial surface antigens that promote phagocytosis was not excluded, these ﬁndings may not be deﬁnite. Nevertheless, expression of FimH is viewed by some as being detrimental to bacterial virulence in vivo 10,11 , an assumption that could not account for the invariable expression of type 1 ﬁmbriae by E. coli isolated from clinical specimens 12,13 . To determine whether FimH-mediated binding to phagocytic cells is a fatal error for the bacterium, we developed a system directly to compare the fate of intracellular E. coli in mouse bone-marrow- derived macrophages following internalization by a FimH- mediated, non-opsonic mechanism, to the fate of the same organ- ism internalized by an antibody-mediated opsonic mechanism. After phagocytosis, extracellular bacteria were pulsed with enough gentamicin to eradicate them, allowing exclusive observation of intracellular E. coli. In contrast to antibody-internalized E. coli, which are effectively killed in the ﬁrst hour, E. coli internalized by a FimH-mediated mechanism showed almost no sign of killing after letters to nature 636 NATURE | VOL 389 | 9 OCTOBER 1997 Figure 1 Intracellular survival of FimH-internalized E. coli in macrophages compared to opsonized E. coli. Intracellular E. coli can survive in cultured macrophages when they are internalized via FimH, in contrast to opsonized E. coli internalized via antibody, which are effectively killed (a, model strain; b, uropathogenic-clinical isolate). Bacterial viability stains also demonstrate increased intracellular survival of the FimH-internalized bacteria at 1 hour (c, antibody-internalized; d, FimH-internalized). Viable bacteria stain green and dead bacteria stain red. § Present address: Department of Pathology, Box 3712, Duke University Medical Center, Durham, North Carolina 27710, USA.