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Entry of microbes into the host: using M cells to break the mucosal barrier
Entry of microbes into the host: using M cells to break the mucosal barrier Brad Jones, Lisa Pascopella and Stanley Falkow University of Iowa, Iowa City, Rocky Mountain Laboratories, Hamilton and Stanford University, Stanford, USA Enteric microbial pathogens interact with the gut epithelium to establish infection. Recently, it has become clear that many microorganisms that colonize or traverse the intestinal mucosa do so via the specialized M cells. Recent work has shown that Shigella flexneri and Salmonella typhimurium specifically target M cells to initiate infection of the host. Current Opinion in Immunology 1995, 7:474-478
Introduction A microbial pathogen must enter a host, find a unique niche and circumvent competing microbes and host defense barriers in order to replicate to a sufficient number to allow transmission to a new susceptible host. Pathogens replicate at the expense o f the host's cellular integrity. In most cases the damage is not serious and may go unnoticed, although a proportion of infected individuals can be expected to suffer overt damage or even be killed. The first stage in the interaction of most pathogens with their host is the interaction between the microorganism and the epithelial barrier o f the skin or mucosa. Although many microbes are content to replicate at the mucosal surface, others penetrate the epithelial barrier and replicate within the host. For microbes that must translocate across the epithelial barrier o f the gut, it has become clear that the target for host cell entry is commonly the Peyer's patch and particularly a specialized epithelial cell, the M cell [1-5]. Because M cells have yet to be cultured in vitro, the study of the interactions between M cells and microbes has been restricted to animal models and extrapolated to findings from infection of established tissue culture cell lines. In this review, we will discuss the current understanding o f the interactions between intracellular pathogenic bacteria and M cells of the lymphoid follicles. In the years ahead, we will hope to identify the molecular mechanisms used by microbes to orchestrate their complex interactions with M cells.
Specific M cell surface glycoconjugates The interaction of microbes with M cells requires adhesion as a first step. Cell surface glycoconjugates have been shown to be receptors for some bacterial adhesins
[6]. A glycoconjugate specific for the surface of M cells found within the murine follicle-associated epithelium (FAE) of ileal Peyer's patches has been demonstrated by staining with the lectin Ulex europaeus agglutinin type 1 (UEA1) [7,8°]. Additionally, glycoconjugates that contain Fuc-Ot-(1,2)-Gal; Gal-~-(1,3)-GLc-NAc are more numerous on the surfaces of M cells than non-M cells in murine FAE, as demonstrated by the staining intensity with the lectin from Psophocarpus tetragonolobus WBAII [8°]. Not all M cells are created equal. Colonic and rectal M cells display glycosylation patterns distinct from those of the M cells of Peyer's patches, and are characterized by the presence o f terminal galactose residues [8°]. UEA1 also binds to other cell types besides M cells within caecal patches, and M cells have a different morphology in Peyer's patches compared to caecal patches [9°]. Even within a single Peyer's patch FAE, subpopulations of M cells can be distinguished by distinct probes specific for fucose [8"]. These data are consistent with the finding that Salmonella typhimurium does not interact equally with all M cells in a single lymphoid follicle [10°°].
Role of M cells in the immune response to microbial pathogens One of the roles of M cells is to prime host immunity. Work done with the extracellular pathogen Vibrio cholerae provides information on this function. This pathogen exerts its influence on a host by adhering to the intestinal epithelimn and secreting cholera toxin into the membranes of enterocytes. Experimental infection o f rabbit intestinal loops with V.. cholerae indicates that a small percentage o f the inoculated bacteria are internalized by M cells [11]. These bacteria are transcytosed and introduced as antigen to the lymphoid cells within the M cell pocket. The result is a strong
Abbreviation FAE follicle-associatedepithelium. 474
© Current Biology Ltd ISSN 0952-7915
Using M cells to break the mucosal barrier Jones, Pascopella and Falkow secretory immune response against V. cholerae [12]. Engulfment of large particles appears to be a normal function of M cells as they can pinocytose other bacteria, viruses and even protozoa [13-15].
Different strategies employed by different microbial pathogens Enteric microbial pathogens that interact with M cells utilize different strategies to establish infection. After adhering to M cells, a microbe may be internalized by the M cell mediated antigen samphng mechanism or may actively induce M cell cytoskeletal rearrangements. The rabbit pathogen Escherichia coli RDEC-1 is an example of a microorganism that specifically attaches to, and induces the formation of, actin pedestal structures on the apical surface of M cells [16]. Its preferential adherence to M cells in rabbit Peyer's patches is characterized by its close association with the microvilli that appear to be oriented towards the bacterium. It is believed that specific pih play a role in the pathogen's initial adherence to the epithelium [17]. The attachment of these bacteria to M cells through the actin pedestals may effectively prevent uptake of the bacteria as few, if any, bacteria can be detected within the M cells by electron microscopy. The ability of the human pathogens enteropathogenic E. coli, enteroaggregative E. coli and diffuse-adhering E. coli to adhere to fixed human intestinal tissue has been examined [18]. This study reported increased levels of adherence by enteroaggregative E. coli and diffuse-adhering E. coli to human M cells compared to enterocytes, indicating that many, but not all, pathogenic E. coli strains have developed specific adherence mechanisms to interact with M cells.
enterocolitica, and BCG, use the M cell as a portal of entry into the host. Fujimura [25] demonstrated that BCG specifically adheres to, and is transcytosed through, M cells over ileal Peyer's patches. Y. enterocolitica have also been shown to attach to and pass through M cells [26,27]. The Y. enterocolitica invasin gene (inv) plays an important role in the association of Y enterocolitica with the FAE, as an inv mutant was greatly reduced in its ability to interact with Peyer's patches [28]. Research efforts have defined the events that occur as S. flexneri interacts with intestinal epithelium. Infection of macaque monkeys with wild-type S. flexneri resulted in the destruction of the mucosal surface of lymphoid nodules that was not observed in monkeys infected with an icsA mutant defective in cell-to-cell spreading [29]. A recent paper [30"] has estabhshed that, at the earliest stages of rabbit intestinal loop infection, S. flexneri is found within M cells and lymphoid cells immediately beneath the FAE. Following passage through the epithelium, each of these bacterial pathogens pursues its own unique survival strategy. It seems hkely that organisms such as Campylobacter and Listeria [31] also take advantage of the mucosal samphng system to initiate disease in a host, although it has not been proven,. In marked contrast to the passive uptake of these organisms, Salmonella species are internalized by inducing actin rearrangements in the apical membrane of M cells.
Interactions of invasive Salmonellaspecies with M cells
Many enteric viral pathogens (e.g. reovirus, poliomyelitis virus, coxsackievirus, Astrovirus, and Breda virus) enter host tissue through the Peyer's patches [19-22]. Keovirus is an example of an enteric virus that utilizes the M cell mediated antigen transport system to enter lymphoid follicles [20]. Keovirus attachment to M cells is dependent upon protein conformational changes to the adherence protein hemagglutinin [23"']. These changes are affected by the activity of proteases as experimental infections of ligated loops in the presence of protease inhibitors revealed that the native reovirus particle was incapable of binding to M cells [23"']. It appears likely that other viruses such as hantavirus, arenavirus, hepatitis and HIV would also take advantage of the antigen sampling system of M cells. A recent study [24] has demonstrated that HIV attaches to and enters cells of FAE of lymphoid nodules in a murine explant system. The abihty of HIV to productively interact with these tissues ex vivo suggests that M cells of lymphoid follicles within the human rectal mucosa may be a primary target of the virus.
Salmonella species are among the group of enteric bacterial pathogens that enter and grow within the reticuloendothelial system of a host. In 1974, Carter and Collins [32] traced the oral infection route of Salmonella by examining the interactions of S. enteriditis with murine tissue. Their experiments showed that early in infection, invasive Salmonella associates with the intestinal Peyer's patch tissue rather than with the cells of the intestinal wall. Recently, the interactions between invasive Salmonella species and murine Peyer's patches were examined microscopically. Kohbata et al. [33] demonstrated that invasive S. typhi adheres to and destroys the M cells ofmurine Peyer's patches. Two other studies [10",34°'], published almost simultaneously this past year, examined the interactions between routine intestinal tissue and the mouse-adapted pathogen S. typhimurium [10", 34"]. Confocal microscopy, transmission and scanning electron microscopy were used to demonstrate that invasive S. typhimurium preferentially binds to and enters murine M cells within a 30 minute hgated ileal loop infection (Fig. 1). Entry into the M cells was accompanied by membrane ruffling and actin polymerization similar to that observed when S. typhimurium enters tissue culture cell lines [35-38]. Interactions between bacteria and enterocytes were undetectable within the 30 minute time frame.
A body of data suggests that many invasive enteric bacterial pathogens, including Shigella flexneri, Yersinia
Work by Jones et al. [34"'] examined the interactions of invasive S. typhimurium with murine intestinal Peyer's
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Immunity to infection
Fig. 1. Salmonella typhimurium SL1344 (indicated by the arrow) invading a murine M cell (M) during a 30 minute ligated ileal loop infection. Note cytoskeletal rearrangement and disruption of the M cell. A lymphoid cell (L) can be seen near the luminal surface in the bottom righthand corner of the print.
patches at later time points. At 60 minutes following infection, internalized S. typhimurium had a cytotoxic effect upon the M cells. The destruction o f an M cell formed a hole in the epithelium that allowed bacteria free access to underlying tissue. Penetrating bacteria easily reached the basal lamina of the epithelium and were responsible for the sloughing of large clusters o f enterocytes away from the epithelium o f the Peyer's patch. In addition, bacteria could be found within lymphoid cells of the M cell pocket. Migration of cells containing intracellular bacteria to the lymph nodes, spleen and liver may be the primary method o f systemic dissemination of virulent Salmonella. Tissue culture systems have been used to identify a variety of genes required for Salmonella invasion [37,39-44]. Most o f these genes are located on the
Salmonella chromosome at 59-60 minutes, and include genes that have similarities to genes that encode export proteins for various Shigella invasion determinants [44,45"]. In mice, mutations in some of these genes affect the oral LDs0 dose but not the intraperitoneal LD50 dose of Salmonella, suggesting that they have a specific function for crossing the mucosal barrier. Two such mutants, 0NA and invA, were examined in a murine ligated loop system and were found to have no deleterious effect oi1 nmrine Peyer's patches during ileal loop infections [34"]. After close examination, there was no evidence of M cell entry by these nmtants. This work appears to provide an explanation for the pathology that is observed in many typhoid patients [46"]. Destruction of the FAE by invasive S. typhi often progresses to ulcerations and perforations of the
Using M cells to break the mucosal barrier Jones, Pascopella and Falkow 477 gut. Intestinal perforation often leads to death and is a major cause o f mortality in patients with typhoid fever [46°°].
2.
Egberts HJA, Brinkhoff MGM, Mouwen IMVM: Biology and pathology of the intestinal M-cell. A review. Vet Quart 1985, 7:333-336.
3.
Neutra MR, Kraehenbuhl JP: Transepithelial transport and mucosal defense. I. The role of M cells. Trends Cell Bio11992, 2:134-138.
4.
Owen RL, Jones AL: Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles. Gastroenterology 1974, 66:189-203.
5.
Wolf J, Bye W: The membranous epithelial (M) cell and the mucosal immune system. Annu Rev A4ed 1984, 35:95-112.
6.
Hultgren SJ, Abraham S, Caparon M, Falk P, St Geme JW III, Normark S: Pilus and nonpilus adhesins: assembly and function in cell recognition. Cell 1993, 73:887-901.
7.
Clark MA, Jepson MA, Simmons NL, Booth TA, Hirst BH: Differential expression of lectin-binding sites defines mouse intestinal M cells. J Histochem Cytochem 1993, 41:1679-1687.
Conclusions The intestinal barrier is a relatively impermeable fortress composed of epithelial cells cemented to one another by tight junctions. However, the immune surveillance processes require that macromolecules and particulate antigens in the gut lumen pass through the mucosal barrier to reach the lymphoid system. The specialized FAE serves this purpose. Here, absorptive cells are the predominant cell type and mucus-secreting goblet cells are rare; about 10% o f the cells o f the FAE consist o f membranous epithelial cells called M cells. A vast array of foreign antigens has been shown to attach to and to be taken up by M cells. This uniquely designed antigen delivery system can, however, be exploited by a number of microbial pathogens. It appears that each microbial pathogen that employs M cells as a portal uses a distinctive strategy ranging from the explosive cell ruffling o f Salmonella that leads to M cell death, to the relatively innocuous passive entry o f Y. enterocolitica, which allows the bacteria to reach the underlying central pocket harboring resident lymphocytes and macrophages. We are still in the early stages o f understanding M cells, yet we are beginning to appreciate the genetic and functional basis for entry of pathogenic viruses and bacteria into the host via these cells. At the same time, we are also beginning to appreciate the nature o f the receptors o f specific lectins that bind to M cells within the FAE. It is likely that continuing investigation o f enteric infectious diseases will reveal as much about the path•genesis o f microbial infection as it does about the fundamental aspects of our immune system.
8. •
Giannasca PJ, Giannasca KT, Falk P, Gordon JI, Neutra MR: Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines. Am J Physiol 1994, 267:G1108-G1121. Presents data that shows that different subpopulations of M cells reside within lymphoid follicles of the ileum, colon, and rectum on the basis of binding to specific lectins. Interestingly, differences in binding between M cells and lectins were observed even within the same follicle-ass0ciated epithelium. Clark M A , Jepson MA, Simmons NL, Hirst BH: Differential surface characteristics of M cells from mouse intestinal Peyer's and caecal patches. Histochem J 1994, 26:271-280. Describes how cellular markers of M cells differ in the caecal patches and Peyer's patches, suggesting that local environments may control the development and function of the M cell. 9. •
Clark MA, Jepson MA, Simmons NL, Hirst BH: Preferential interaction of Salmonella typhimurium with mouse Peyer's patch M cells. Res Microbiol 1994, 145:543-552. Demonstrates that invasive Salmonella typhimurium preferentially adheres to and enters the M cells of Peyer's patches. Entry is by a membrane ruffling mechanism. 10. "•
11.
Owen RL, Pierce NF, Apple RT, Cray WJ: M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration. J Infect Dis 1986, 153:1108-1118.
12.
Apter FM, Michet~i P, Winner LS III, Mack JA, Mekalanos JJ, Neutra MR: Analysis of the roles of antilipopolysaccharide and antl-cholera toxin immunoglobulin A (IgA) antibodies in protection against Vibrio cholerae and cholera toxin by use of monoclonal IgA antibodies in vivo. Infect Immun 1993, 61:5279-5285.
13.
Giannasca PJ, Neutra MR: Interactions of microorganisms with intestinal M cells: mucosal invasion and induction of secretory immunity. Infect Agents Dis 1993, 2:242-248.
~14.
Marcial MA, Madara JL: Cryptosporidium: cellular localization, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterology 1986, 90:583-594.
15.
Owen RL, Allen CL, Stevens DP: Phagocytosis of Giardia muris by macrophages in Peyer's patch epithelium in mice. Infect Immun 1981, 33:591~o01.
16.
Inman LR, Cantey JR: Specific adherence of Escherichia coil (strain RDEC-1) to membranous (M) cells of the Peyer's patch in Escherichia coil diarrhea in the rabbit. J Clin Invest 1983, 71:1-8.
17.
Inman LR, Cantey JR: Peyer's patch lymphoid follicle epithelial adherence of a rabbit enteropathogenic Escherlchla coil (strain RDEC-1). Role of plasmid-mediated pill in initial adherence. J Clin Invest 1984, 74:90-95.
18.
Yamamoto T, Koyama Y, Matsumoto M, Sonoda E, Nakayama S, Uchimura M, Paveenkittiporn W, Tamura K, Yokota T, Echeverria P: Localized, aggregative, and diffuse adherence to
Acknowledgements We thank Nafisa Ghori for preparation of tissue samples and printing of Figure 1. B Jones is supported by grant IN-122P from the American Cancer Society and by a grant from the Roy J Carver Charitable Trust. L Pascopella is supported by an IR.TA fellowship through the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. S Falkow is supported by Public Health Service Grant AI26195, the Stanford Digestive Disease Center grant DK38707, and unrestricted gifts from Bristol-Meyers and Praxis Biologicals.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
Bockman DE, Cooper MD: Pinocytosis by epithelium associated with lymphoid follicles in the bursa of Fabricius, appendix, and Peyer's patches. An electron microscopic study. Am JAnat 1973, 136:455-477.
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Immunity to infection HeLa cells, plastic, and human small intestines by Escherichia coil isolated from patients with diarrhea. J Infect Dis 1992, 166:1295-1310. 19.
Heinz BA, Cliver DO: Coxsackievirus-cell interactions thai initiate infection in Porcine ileal explants. Arch Virol 1988, 101:35-47.
epithelial M cells of the Peyer's patches, J Exp Ivied 1994, 180:1 S-23. Describes in detail the interactions between invasive Salmonella typhimurium and murine ileal M cells that lead to the destruction of these cells and bacterial passage through the intestinal epithelium. 35.
Finlay BB, Falkow S: Salmonella interactions with polarized human intestinal Caco-2 epithelial cells. J Infect Dis 1990, 162:1096-1106.
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Wolf JL, Rubin DH, Finberg R, Kauffman RS, Sharpe AH, Trier JS, Fields BN: Intestinal M cells: a pathway for entry of reovirns into the host. Science 1981, 212:471-472.
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Sicinski P, Rowinski J, Warchol JB, larzabek Z, Gut W, Szczygiel B, Bielecki K, Koch G: Poliovirus type 1 enters the human host through intestinal M cells. Gastroenterology 1990, 98:56-58.
Francis CL, Ryan TA, Jones BD, Smith SJ, Falkow S: Ruffles induced by Salmonella and other stimuli direct macropinocylosis of bacteria. Nature 1993, 364:639-642.
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Woode GN, Pohlenz JF, Gourley NE, Fagerland JA: Astrovirns and Breda virus infections of dome cell epithelium of bovine ileum. J Clin MMicrobiol 1984, 19:623-630.
Ginocchio C, Pace J, Galen JE: Identification and molecular characterization of a Salmonella typhlmudum gene involved in triggering the internalization of salmonellae into cultured epithelial cells, Proc Natl Acad SCi USA 1992, 89:5976-5980.
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Jones BD, Paterson HF, Hall A, Falkow S: Salmonella typhlmurlum induces membrane ruffling by a growth factor receptor independent mechanism. Proc Natl Acad SCi USA 1993, 90:10390-10394.
39.
Stone BJ, Garcia CM, Badger JL, Hassett T, Smith RiF, Miller V: Identification of novel loci affecting entry of Salmonella enterlditis into eukaryotic cells. ] Bacteriol 1992, 174:3945-3952.
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Gal,~nJE, Curtiss R II1: Cloning and molecular characterization of genes whose products allow Salmonella typhlmurium to penetrate tissue culture cells. Proc Natl Acad Sci USA 1989, 86:6383-6387.
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Kaniga K, Bossio JC, Galan JE: The Salmonella typhimurlum invasion genes invF and invG encode homologues of the AraC and PulD family of proteins. Mtol MIicrobio11994, 13:555-568.
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Jones BD, Falkow S: Identification and characterization of a Salmonella typhimurium oxygen-regulated gone required for bacterial internalization. Infect Immun 1994, 62:3745-3752.
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Lee CA, Jones BD, Falkow S: identification and characterization of Salmonella typhlmurium invasion locus by selection for hyperinvasive mutants. Proc Natl Acad Sci USA 1992, 89:1847-1851. Groisman EA, Ochman H: Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shlgella flexnerL EMIBO J ]993, 12:3779-3787.
23. ••
Amerongen HM, Wilson GA, Fields BN, Neutra MR: Proteolytic processing of reovirus is required for adherence to intestinal M cells. J Virol 1994, 68:8428-8432. Demonstrated that reovirus must be proteolytically converted to intermediate subviral particles before they are capable of specifically binding to and transcytosing through M cells of murine Peyer's patches. 24.
25.
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Amerongen HM, Weltzin R, Farnet CM, Michetti P, Hasehine WA, Neutra MR: Transepithelial transport of HIV-1 by intestinal M cells: a mechanism for transmission of AIDS. J Acquit Immune Defic Syndr Hum Retrovirol 1991, 4:760-765. Fujimura Y: Functional morphology of microfold cells (M cells) in Peyer's patches - - phagocytosis and transport of BCG by M cells into rabbit Peyer's patches. Gastroenterol 1986, 21:325-335. Hanski C, Kutschka U, Schmoranzer HP, Naumann M, Stallmach A, Hahn H, Menge H, Riecken EO: Immunohistochemical and electron microscopic study of interaction of Yerslnia enterocolltica serotype 0 8 with intestinal mucosa during experimental enteritis. Infect Immun 1989, 57:673-678.
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Fujimora Y, Kihara T, Mine H: Membranous cells as a portal of Yerslnla pseudotuberculosis entry into rabbit ileum. J C/in Electron MMicroscopy 1992, 25:35-45.
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Pepe JC, Miller VL: Yerslnla enterocolltica invasin: a primary role in the initiation of infection. Proc Nat/ Acad SCi USA 1993, 90:6473~477.
44.
29.
Sansonetti PJ, Arondel J, Fontaine A, D'Hauteville H, Bernardini ML: ompB (osmo-regulation) and icsA (cell to cell spread) mutants of Shigella flexneri within HeLa cells: vaccine candidates and probes to study the pathogenesis of shigellosis. Vaccine 1991, 9:41(~422.
45. Galan JF: Interactions of bacteria with non-phagocytic cells. • Curr Opin Immunol 1994, 6:590-595. Discusses the various mechanisms used by pathogenic bacteria to interact with mammalian cells.
30. "•
Perdomo OJJ, Cavaillon JM, Huerre M, Ohayon H, Gounon P, Sansonetti PJ: Acute inflammation causes epithelial invasion and mucosal destruction in experimental shigellosis. J E×p Ivied 1994, 180:1307-1319. Presents work demonstrating that invasive Shigella flexneri breach the intestinal barrier through M cells of the lymphoid follicles. Subsequent destruction of the epithelium appears to be due to infiltration of polymorphonuclear cells. 31.
Marco AJ, Domingo M, Prats M, Briones V, Pumarola M, Dominguez L: Pathogenesis of lymphoid lesions in murine experimental listeriosis. J Comp Pathol 1991, 105:1-15.
32.
Carter PB, Collins FM: The route of enteric infection in normal mice. ] E×p Ivied 1974, 139:1189-1203.
33.
Kohbata S, Yokoyama H, of Salmonella typhi GIFU Peyer's patches in ligated MMicrobiol Immunol 1986,
34. •"
JonesBD, Ghori N, Falkow S: Salmonella typhlmurium initiates murine infection by penetrating and destroying the specialized
Yabuuchi E: Cylopathogenic effect 10007 on M cells of murine ileal ileal loops: an ultrastructural study. 30:1225-1237.
46. Owen RL: M cells - entryways of opportunity for •• enteropathogens. J Exp Ivied 1994, 180:7-9. Reviews the clinical data that indicates that intestinal perforation is a major cause of mortality in typhoid and suggests that Salmonella typhi invasion of Peyer's patch M cells may be responsible.
B Jones, Department of Microbiology, University of Iowa School of Medicine, Iowa City, Iowa 52242-1109, USA. E-mail: [email protected] L Pascopella, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840, USA. E-mail: LisaPasc°pella@rml'niaid'nih'g°v S Falkow, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 54302-5402, USA. E-mail: stanley,falkow@forsythe,stanford.edu
Introduction A microbial pathogen must enter a host, find a unique niche and circumvent competing microbes and host defense barriers in order to replicate to a sufficient number to allow transmission to a new susceptible host. Pathogens replicate at the expense o f the host's cellular integrity. In most cases the damage is not serious and may go unnoticed, although a proportion of infected individuals can be expected to suffer overt damage or even be killed. The first stage in the interaction of most pathogens with their host is the interaction between the microorganism and the epithelial barrier o f the skin or mucosa. Although many microbes are content to replicate at the mucosal surface, others penetrate the epithelial barrier and replicate within the host. For microbes that must translocate across the epithelial barrier o f the gut, it has become clear that the target for host cell entry is commonly the Peyer's patch and particularly a specialized epithelial cell, the M cell [1-5]. Because M cells have yet to be cultured in vitro, the study of the interactions between M cells and microbes has been restricted to animal models and extrapolated to findings from infection of established tissue culture cell lines. In this review, we will discuss the current understanding o f the interactions between intracellular pathogenic bacteria and M cells of the lymphoid follicles. In the years ahead, we will hope to identify the molecular mechanisms used by microbes to orchestrate their complex interactions with M cells.
Specific M cell surface glycoconjugates The interaction of microbes with M cells requires adhesion as a first step. Cell surface glycoconjugates have been shown to be receptors for some bacterial adhesins
[6]. A glycoconjugate specific for the surface of M cells found within the murine follicle-associated epithelium (FAE) of ileal Peyer's patches has been demonstrated by staining with the lectin Ulex europaeus agglutinin type 1 (UEA1) [7,8°]. Additionally, glycoconjugates that contain Fuc-Ot-(1,2)-Gal; Gal-~-(1,3)-GLc-NAc are more numerous on the surfaces of M cells than non-M cells in murine FAE, as demonstrated by the staining intensity with the lectin from Psophocarpus tetragonolobus WBAII [8°]. Not all M cells are created equal. Colonic and rectal M cells display glycosylation patterns distinct from those of the M cells of Peyer's patches, and are characterized by the presence o f terminal galactose residues [8°]. UEA1 also binds to other cell types besides M cells within caecal patches, and M cells have a different morphology in Peyer's patches compared to caecal patches [9°]. Even within a single Peyer's patch FAE, subpopulations of M cells can be distinguished by distinct probes specific for fucose [8"]. These data are consistent with the finding that Salmonella typhimurium does not interact equally with all M cells in a single lymphoid follicle [10°°].
Role of M cells in the immune response to microbial pathogens One of the roles of M cells is to prime host immunity. Work done with the extracellular pathogen Vibrio cholerae provides information on this function. This pathogen exerts its influence on a host by adhering to the intestinal epithelimn and secreting cholera toxin into the membranes of enterocytes. Experimental infection o f rabbit intestinal loops with V.. cholerae indicates that a small percentage o f the inoculated bacteria are internalized by M cells [11]. These bacteria are transcytosed and introduced as antigen to the lymphoid cells within the M cell pocket. The result is a strong
Abbreviation FAE follicle-associatedepithelium. 474
© Current Biology Ltd ISSN 0952-7915
Using M cells to break the mucosal barrier Jones, Pascopella and Falkow secretory immune response against V. cholerae [12]. Engulfment of large particles appears to be a normal function of M cells as they can pinocytose other bacteria, viruses and even protozoa [13-15].
Different strategies employed by different microbial pathogens Enteric microbial pathogens that interact with M cells utilize different strategies to establish infection. After adhering to M cells, a microbe may be internalized by the M cell mediated antigen samphng mechanism or may actively induce M cell cytoskeletal rearrangements. The rabbit pathogen Escherichia coli RDEC-1 is an example of a microorganism that specifically attaches to, and induces the formation of, actin pedestal structures on the apical surface of M cells [16]. Its preferential adherence to M cells in rabbit Peyer's patches is characterized by its close association with the microvilli that appear to be oriented towards the bacterium. It is believed that specific pih play a role in the pathogen's initial adherence to the epithelium [17]. The attachment of these bacteria to M cells through the actin pedestals may effectively prevent uptake of the bacteria as few, if any, bacteria can be detected within the M cells by electron microscopy. The ability of the human pathogens enteropathogenic E. coli, enteroaggregative E. coli and diffuse-adhering E. coli to adhere to fixed human intestinal tissue has been examined [18]. This study reported increased levels of adherence by enteroaggregative E. coli and diffuse-adhering E. coli to human M cells compared to enterocytes, indicating that many, but not all, pathogenic E. coli strains have developed specific adherence mechanisms to interact with M cells.
enterocolitica, and BCG, use the M cell as a portal of entry into the host. Fujimura [25] demonstrated that BCG specifically adheres to, and is transcytosed through, M cells over ileal Peyer's patches. Y. enterocolitica have also been shown to attach to and pass through M cells [26,27]. The Y. enterocolitica invasin gene (inv) plays an important role in the association of Y enterocolitica with the FAE, as an inv mutant was greatly reduced in its ability to interact with Peyer's patches [28]. Research efforts have defined the events that occur as S. flexneri interacts with intestinal epithelium. Infection of macaque monkeys with wild-type S. flexneri resulted in the destruction of the mucosal surface of lymphoid nodules that was not observed in monkeys infected with an icsA mutant defective in cell-to-cell spreading [29]. A recent paper [30"] has estabhshed that, at the earliest stages of rabbit intestinal loop infection, S. flexneri is found within M cells and lymphoid cells immediately beneath the FAE. Following passage through the epithelium, each of these bacterial pathogens pursues its own unique survival strategy. It seems hkely that organisms such as Campylobacter and Listeria [31] also take advantage of the mucosal samphng system to initiate disease in a host, although it has not been proven,. In marked contrast to the passive uptake of these organisms, Salmonella species are internalized by inducing actin rearrangements in the apical membrane of M cells.
Interactions of invasive Salmonellaspecies with M cells
Many enteric viral pathogens (e.g. reovirus, poliomyelitis virus, coxsackievirus, Astrovirus, and Breda virus) enter host tissue through the Peyer's patches [19-22]. Keovirus is an example of an enteric virus that utilizes the M cell mediated antigen transport system to enter lymphoid follicles [20]. Keovirus attachment to M cells is dependent upon protein conformational changes to the adherence protein hemagglutinin [23"']. These changes are affected by the activity of proteases as experimental infections of ligated loops in the presence of protease inhibitors revealed that the native reovirus particle was incapable of binding to M cells [23"']. It appears likely that other viruses such as hantavirus, arenavirus, hepatitis and HIV would also take advantage of the antigen sampling system of M cells. A recent study [24] has demonstrated that HIV attaches to and enters cells of FAE of lymphoid nodules in a murine explant system. The abihty of HIV to productively interact with these tissues ex vivo suggests that M cells of lymphoid follicles within the human rectal mucosa may be a primary target of the virus.
Salmonella species are among the group of enteric bacterial pathogens that enter and grow within the reticuloendothelial system of a host. In 1974, Carter and Collins [32] traced the oral infection route of Salmonella by examining the interactions of S. enteriditis with murine tissue. Their experiments showed that early in infection, invasive Salmonella associates with the intestinal Peyer's patch tissue rather than with the cells of the intestinal wall. Recently, the interactions between invasive Salmonella species and murine Peyer's patches were examined microscopically. Kohbata et al. [33] demonstrated that invasive S. typhi adheres to and destroys the M cells ofmurine Peyer's patches. Two other studies [10",34°'], published almost simultaneously this past year, examined the interactions between routine intestinal tissue and the mouse-adapted pathogen S. typhimurium [10", 34"]. Confocal microscopy, transmission and scanning electron microscopy were used to demonstrate that invasive S. typhimurium preferentially binds to and enters murine M cells within a 30 minute hgated ileal loop infection (Fig. 1). Entry into the M cells was accompanied by membrane ruffling and actin polymerization similar to that observed when S. typhimurium enters tissue culture cell lines [35-38]. Interactions between bacteria and enterocytes were undetectable within the 30 minute time frame.
A body of data suggests that many invasive enteric bacterial pathogens, including Shigella flexneri, Yersinia
Work by Jones et al. [34"'] examined the interactions of invasive S. typhimurium with murine intestinal Peyer's
475
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Immunity to infection
Fig. 1. Salmonella typhimurium SL1344 (indicated by the arrow) invading a murine M cell (M) during a 30 minute ligated ileal loop infection. Note cytoskeletal rearrangement and disruption of the M cell. A lymphoid cell (L) can be seen near the luminal surface in the bottom righthand corner of the print.
patches at later time points. At 60 minutes following infection, internalized S. typhimurium had a cytotoxic effect upon the M cells. The destruction o f an M cell formed a hole in the epithelium that allowed bacteria free access to underlying tissue. Penetrating bacteria easily reached the basal lamina of the epithelium and were responsible for the sloughing of large clusters o f enterocytes away from the epithelium o f the Peyer's patch. In addition, bacteria could be found within lymphoid cells of the M cell pocket. Migration of cells containing intracellular bacteria to the lymph nodes, spleen and liver may be the primary method o f systemic dissemination of virulent Salmonella. Tissue culture systems have been used to identify a variety of genes required for Salmonella invasion [37,39-44]. Most o f these genes are located on the
Salmonella chromosome at 59-60 minutes, and include genes that have similarities to genes that encode export proteins for various Shigella invasion determinants [44,45"]. In mice, mutations in some of these genes affect the oral LDs0 dose but not the intraperitoneal LD50 dose of Salmonella, suggesting that they have a specific function for crossing the mucosal barrier. Two such mutants, 0NA and invA, were examined in a murine ligated loop system and were found to have no deleterious effect oi1 nmrine Peyer's patches during ileal loop infections [34"]. After close examination, there was no evidence of M cell entry by these nmtants. This work appears to provide an explanation for the pathology that is observed in many typhoid patients [46"]. Destruction of the FAE by invasive S. typhi often progresses to ulcerations and perforations of the
Using M cells to break the mucosal barrier Jones, Pascopella and Falkow 477 gut. Intestinal perforation often leads to death and is a major cause o f mortality in patients with typhoid fever [46°°].
2.
Egberts HJA, Brinkhoff MGM, Mouwen IMVM: Biology and pathology of the intestinal M-cell. A review. Vet Quart 1985, 7:333-336.
3.
Neutra MR, Kraehenbuhl JP: Transepithelial transport and mucosal defense. I. The role of M cells. Trends Cell Bio11992, 2:134-138.
4.
Owen RL, Jones AL: Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles. Gastroenterology 1974, 66:189-203.
5.
Wolf J, Bye W: The membranous epithelial (M) cell and the mucosal immune system. Annu Rev A4ed 1984, 35:95-112.
6.
Hultgren SJ, Abraham S, Caparon M, Falk P, St Geme JW III, Normark S: Pilus and nonpilus adhesins: assembly and function in cell recognition. Cell 1993, 73:887-901.
7.
Clark MA, Jepson MA, Simmons NL, Booth TA, Hirst BH: Differential expression of lectin-binding sites defines mouse intestinal M cells. J Histochem Cytochem 1993, 41:1679-1687.
Conclusions The intestinal barrier is a relatively impermeable fortress composed of epithelial cells cemented to one another by tight junctions. However, the immune surveillance processes require that macromolecules and particulate antigens in the gut lumen pass through the mucosal barrier to reach the lymphoid system. The specialized FAE serves this purpose. Here, absorptive cells are the predominant cell type and mucus-secreting goblet cells are rare; about 10% o f the cells o f the FAE consist o f membranous epithelial cells called M cells. A vast array of foreign antigens has been shown to attach to and to be taken up by M cells. This uniquely designed antigen delivery system can, however, be exploited by a number of microbial pathogens. It appears that each microbial pathogen that employs M cells as a portal uses a distinctive strategy ranging from the explosive cell ruffling o f Salmonella that leads to M cell death, to the relatively innocuous passive entry o f Y. enterocolitica, which allows the bacteria to reach the underlying central pocket harboring resident lymphocytes and macrophages. We are still in the early stages o f understanding M cells, yet we are beginning to appreciate the genetic and functional basis for entry of pathogenic viruses and bacteria into the host via these cells. At the same time, we are also beginning to appreciate the nature o f the receptors o f specific lectins that bind to M cells within the FAE. It is likely that continuing investigation o f enteric infectious diseases will reveal as much about the path•genesis o f microbial infection as it does about the fundamental aspects of our immune system.
8. •
Giannasca PJ, Giannasca KT, Falk P, Gordon JI, Neutra MR: Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines. Am J Physiol 1994, 267:G1108-G1121. Presents data that shows that different subpopulations of M cells reside within lymphoid follicles of the ileum, colon, and rectum on the basis of binding to specific lectins. Interestingly, differences in binding between M cells and lectins were observed even within the same follicle-ass0ciated epithelium. Clark M A , Jepson MA, Simmons NL, Hirst BH: Differential surface characteristics of M cells from mouse intestinal Peyer's and caecal patches. Histochem J 1994, 26:271-280. Describes how cellular markers of M cells differ in the caecal patches and Peyer's patches, suggesting that local environments may control the development and function of the M cell. 9. •
Clark MA, Jepson MA, Simmons NL, Hirst BH: Preferential interaction of Salmonella typhimurium with mouse Peyer's patch M cells. Res Microbiol 1994, 145:543-552. Demonstrates that invasive Salmonella typhimurium preferentially adheres to and enters the M cells of Peyer's patches. Entry is by a membrane ruffling mechanism. 10. "•
11.
Owen RL, Pierce NF, Apple RT, Cray WJ: M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration. J Infect Dis 1986, 153:1108-1118.
12.
Apter FM, Michet~i P, Winner LS III, Mack JA, Mekalanos JJ, Neutra MR: Analysis of the roles of antilipopolysaccharide and antl-cholera toxin immunoglobulin A (IgA) antibodies in protection against Vibrio cholerae and cholera toxin by use of monoclonal IgA antibodies in vivo. Infect Immun 1993, 61:5279-5285.
13.
Giannasca PJ, Neutra MR: Interactions of microorganisms with intestinal M cells: mucosal invasion and induction of secretory immunity. Infect Agents Dis 1993, 2:242-248.
~14.
Marcial MA, Madara JL: Cryptosporidium: cellular localization, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterology 1986, 90:583-594.
15.
Owen RL, Allen CL, Stevens DP: Phagocytosis of Giardia muris by macrophages in Peyer's patch epithelium in mice. Infect Immun 1981, 33:591~o01.
16.
Inman LR, Cantey JR: Specific adherence of Escherichia coil (strain RDEC-1) to membranous (M) cells of the Peyer's patch in Escherichia coil diarrhea in the rabbit. J Clin Invest 1983, 71:1-8.
17.
Inman LR, Cantey JR: Peyer's patch lymphoid follicle epithelial adherence of a rabbit enteropathogenic Escherlchla coil (strain RDEC-1). Role of plasmid-mediated pill in initial adherence. J Clin Invest 1984, 74:90-95.
18.
Yamamoto T, Koyama Y, Matsumoto M, Sonoda E, Nakayama S, Uchimura M, Paveenkittiporn W, Tamura K, Yokota T, Echeverria P: Localized, aggregative, and diffuse adherence to
Acknowledgements We thank Nafisa Ghori for preparation of tissue samples and printing of Figure 1. B Jones is supported by grant IN-122P from the American Cancer Society and by a grant from the Roy J Carver Charitable Trust. L Pascopella is supported by an IR.TA fellowship through the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. S Falkow is supported by Public Health Service Grant AI26195, the Stanford Digestive Disease Center grant DK38707, and unrestricted gifts from Bristol-Meyers and Praxis Biologicals.
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46. Owen RL: M cells - entryways of opportunity for •• enteropathogens. J Exp Ivied 1994, 180:7-9. Reviews the clinical data that indicates that intestinal perforation is a major cause of mortality in typhoid and suggests that Salmonella typhi invasion of Peyer's patch M cells may be responsible.
B Jones, Department of Microbiology, University of Iowa School of Medicine, Iowa City, Iowa 52242-1109, USA. E-mail: [email protected] L Pascopella, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840, USA. E-mail: LisaPasc°pella@rml'niaid'nih'g°v S Falkow, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 54302-5402, USA. E-mail: stanley,falkow@forsythe,stanford.edu