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Journal of Investigative Medicine:
doi: 10.2310/6650.2005.53609
EB 2005 Symposium

Bacterial Penetration of the Mucosal Barrier by Targeting Lipid Rafts

Abraham, Soman N.; Duncan, Matthew J.; Li, Guojie; Zaas, David

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From the Departments of Pathology (S.N.A., G.L.), Molecular Genetics and Microbiology (S.N.A. and M.J.D.), and Medicine (D.Z.), Duke University Medical Center, Durham, NC.

Presented in part at the American Federation for Medical Research‐sponsored symposium during Experimental Biology 2005, San Diego, CA, April 2‐6, 2005.

Address correspondence to: Dr. Soman N. Abraham, Department of Pathology, Duke University Medical Center, Box 3020, Durham NC 27710; e‐mail: Soman.Abraham@duke.edu.

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Abstract

ABSTRACT: Several traditionally extracellular pathogens not known to possess invasive capacity have been shown to invade various mucosal epithelial cells. The mucosal epithelium performs an important barrier function and is not typically amenable to bacterial invasion. Valuable clues to the underlying basis for bacterial invasion have emerged from recent studies examining the invasion of bladder epithelial cells by uropathogenic Escherichia coli and alveolar epithelial cells by Pseudomonas aeruginosa. In both cases, bacterial invasion is achieved through targeting of molecules specifically found within distinct glycosphingolipid‐ and cholesterol‐enriched microdomains called lipid rafts. The importance of lipid rafts in promoting bacterial invasion was shown as disruptors of lipid rafts blocked cellular invasion by both E. coli and P. aeruginosa. In addition, molecular components of lipid rafts were found to be highly enriched in membranes encasing these intracellular bacteria. Furthermore, caveolin proteins, which serve to stabilize and organize lipid raft components, are necessary for bacterial entry. Taken together, targeting of lipid rafts appears to be an effective but poorly recognized mechanism used by pathogenic bacteria to circumvent the mucosal barriers of the host.

Mucosal surfaces of the respiratory, gastrointestinal, and genitourinary tracts represent the major sites of initiating microbial infections. Each of these mucosal surfaces possesses effective barriers against infection, including the powerful flushing action from the flow of mucosal secretions and luminal contents. To avoid early removal by these mechanical forces, pathogens tend to seek refuge within mucosal cells. Many invasive bacteria, such as Listeria monocytogenes, Salmonella typhimurium, and Shigella flexneri, possess distinct and well‐characterized mechanisms for invading the mucosal epithelial cells.1‐3Listeria bind E‐cadherin of intestinal cells, which triggers rapid uptake by the host cells followed by escape of the pathogen from the endosome into the cytoplasm of the host cell.2Salmonella and Shigella use their type III secretion systems to inject effector proteins, triggering membrane ruffling of the M cells and gut epithelial cells, respectively, enabling the pathogens to slip inside intracellular compartments that favor bacterial growth.1,3 However, many mucosal bacteria are not traditionally regarded as intracellular pathogens and do not appear to possess any specialized machinery for invading and multiplying within host cells. Yet their capacity to persist and infect mucosal surfaces would suggest that these pathogens possess at least a limited capacity to invade mucosal epithelial cells. If so, how is this achieved? Recent studies examining the interactions of Escherichia coli with the bladder epithelium and Pseudomonas aeruginosa with alveolar epithelial cells have revealed that these pathogens possess the capacity to invade host epithelial cells, and this capacity furthermore appears to involve distinct cellular entities of the plasma membrane called lipid rafts.4,5

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LIPID RAFTS

The plasma membrane on eukaryotic cells, including epithelial cells, is organized into dynamic detergent‐resistant lipid‐enriched microdomains called lipid rafts. These entities contain cholesterol, glycosphingolipids, and resident integral membrane proteins, such as caveolin.6‐8 The tightly packed lipid environment of lipid rafts favors their association with saturated acyl chains of glycosylphosphatidylinositol (GPI)‐anchored proteins such as CD59 and CD55, acylated cytosolic proteins such as Src kinases, and small and heterotrimeric G proteins.6‐8 In certain endothelial and epithelial cells, these lipid rafts can assume a distinct cave‐like structure ranging in diameter from 50 to 100 nm in the plasma membrane called caveolae.6,8 The primary physiologic function ascribed to lipid rafts is to serve as signaling platforms enriched in preassembled signaling complexes. This trait enables these platforms to rapidly and efficiently transduce signals, the trigger being extracellular ligation of lipid raft components.9 Because the invaginated cave‐like structure of caveolae is suggestive of an endocytic activity, some have examined uptake of extracellular macromolecules by cells via caveolae. Certain hormones have been shown to be internalized via caveolae and then translocated to the nucleus.8,10 Albumin was shown to be internalized by caveolae and subsequently translocated across the cell to be released outside at the basolateral surface.11 Thus, there is now a growing body of data showing that caveolae and lipid rafts possess endocytic activity and that they are capable of endocytosing not only host components but also microbal products, such as cholera toxin, as well as microbes, such as simian virus 40.12

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INVASION OF BLADDER EPITHELIAL CELLS BY E. COLI

The urinary tract is typically a sterile environment, largely because of the powerful hydrokinetic forces associated with urine flow. The bladder epithelium is also highly impermeable to invasion because of the role of the bladder as a storage organ for urine.13,14 The presence of “plaques” comprising uroplakin proteins localized on the luminal surface of superficial bladder cells and their association with clusters of cholesterol and sphingolipids is believed to contribute to a more ordered lipid structure in the plasma membrane, lowering membrane fluidity and permeability.15‐17E. coli is by far the most common causative agent of urinary tract infections. The virulence of E. coli in the bladder has been linked to the expression of FimH, a mannose‐binding lectin on the tips of fimbrial appendages of the bacteria.18 FimH binds to uroplakin 1a on the plasma membranes of bladder cells, promoting not only bacterial attachment but also bacterial invasion of these relatively impermeable cells.18,19 The involvement of lipid rafts in the invasion of the bladder epithelium by E. coli was deduced from the observation that the FimH receptor uroplakin 1a was specifically localized to lipid raft fractions of bladder cell extract.5 More direct indications of lipid raft involvement came from the confocal microscopy observations of bladder cells that revealed integral components of lipid rafts: cholesterol, ganglioslides (GM‐1), and caveolin 1 were found to be highly enriched in the membrane encasing the recently endocytozed bacteria.5 Functional evidence specifically implicating lipid rafts in E. coli entry into bladder cells comes from the observation that sterol binding or chelating drugs block entry of E. coli but not L. monocytogenes into bladder cells.5 In vivo evidence implicating lipid rafts in E. coli infection of the bladder epithelium comes from the finding that instillation of methyl‐β‐cyclodextrin, a disruptor of lipid raft structure, into mouse bladders via a catheter dramatically reduced the capacity of uropathogenic E. coli but not L. monocytogenes to cause infection.5 This capacity of FimH‐expressing E. coli to invade the relatively impermeable bladder epithelium is consistent with earlier reports implicating lipid rafts or caveolae in E. coli invasion of macrophages and mast cells.20,21 These studies have revealed that lipid raft‐ mediated entry of E. coli was not harmful to the pathogen because, unlike the clathrin‐mediated uptake of antibody‐coated bacteria, where endocytosed bacteria are trafficked into lysosomes and degraded, bacteria ingested via lipid rafts retained their viability because they avoided fusion with lysosomes.21 Thus, lipid raft‐mediated entry of E. coli into host cells may be relevant not only at the early stages of the infectious process, when the pathogen is attempting to establish colonization, but also at subsequent stages, when the immune system has been activated.

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INVASION OF ALVEOLAR EPITHELIAL CELLS BY P. AERUGINOSA

P. aeruginosa is a major cause of pneumonia in patients with cystic fibrosis and other immunocompromising conditions.22 The mortality associated with Pseudomonas pneumonia is often due to the spread of the bacteria to the alveolar epithelium of the distal lungs, followed by widespread dissemination via the bloodstream.23 The virulence of Pseudomonas is multifactorial and includes their ability to express pili and flagella.24 Although widely considered an extracellular pathogen, the capacity of P. aeruginosa to invade host cells has been reported.25,26Pseudomonas has been shown to invade nasal and upper airway epithelial cells,25,26 but it is not known if it has the capacity to invade the unicellular layer of cells that comprise the alveolar epithelium, possibly followed by its dissemination into the bloodstream.

The alveolar epithelium is the largest host epithelial surface exposed to the external environment. Approximately 95% of this surface area is lined by specialized type 1 pneumocytes.27 The cell membrane of type 1 pneumocytes has a high concentration of specialized lipid rafts or caveolae that occupy nearly 70% of the plasma membrane.28,29 Recently, we showed that direct instillation of Pseudomonas into the lower lobes of the rat lung resulted in significant association of Pseudomonas with alveolar epithelial cells.4 Confocal microcopy of lung sections revealed airway epithelial cells apparently harboring intracellular Pseudomonas.4 In vitro studies employing cultured type 1‐like pneumocytes confirmed the invasive capacity of Pseudomonas and revealed that, following entry, the Pseudomonas was capable of multiplying within these cells. The localization of lipid raft components GM‐1, caveolin 1, and caveolin 2 in membranes encasing intracellular bacteria was highly suggestive of lipid raft involvement in Pseudomonas entry.4 It is noteworthy that some have suggested that GM‐1 is the putative receptor for adherent Pseudomonas on host cells,30 indicating that the bacterial receptor, as in the case of uropathogenic E. coli, is localized in the lipid raft structure. Disruption of lipid rafts in these type 1‐like pneumocyte cells with low doses of nystatin, filipin, and methyl‐β‐cyclodextrin resulted in significant inhibition of the entry of Pseudomonas but not L. monocytogenes, which invades host cells via a lipid raft‐independent mechanism.4 Thus, entry of Pseudomonas into alveolar epithelial cells, like the entry of E. coli into bladder epithelial cells, was lipid raft dependent.

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MOLECULAR MECHANISMS OF BACTERIAL INVASION

Although lipid raft involvement in E. coli and Pseudomonas invasion of epithelial cells has been clearly demonstrated, less is known regarding the mechanism underlying bacterial entry via lipid raft‐mediated endocytosis. The signaling function of lipid rafts has previously been ascribed to their content of clustered preassembled macromolecular signaling complexes.9 In a similar fashion, it is conceivable that the preexisting macromolecular complexes found in lipid rafts interact with cytoskeletal proteins such as actin, which are essential downstream components of the endocytic process. Crosslinking of extracellular lipid raft components by pathogens may thus trigger controlled signaling in these macromolecular aggregates within this raft structure, culminating in endocytosis of the microbe. Partial support for this notion indeed comes from the observation that proteome analyses of lipid raft fractions from different cell types have consistently revealed the presence of actin subunits.31 To explain the involvement of lipid rafts in the uptake of E. coli and Pseudomonas, which use distinct receptors in plasma membrane, we hypothesize that the endocytic machinery within rafts is activated by bacteria as long as the bacterial receptor is located within the raft structure or moves into lipid rafts following ligation by the pathogen. A diagrammatic view of the role of these preassembled lipid raft macromolecules in bacterial uptake is presented in Figure 1.

Figure 1
Figure 1
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Caveolin proteins, which are regarded as key structural proteins involved in maintaining the stability and organization of lipid rafts, have been implicated in the entry of both E. coli and Pseudomonas.4,5 Two isoforms of caveolin, caveolin 1 and caveolin 2, are coexpressed in most cell types.32,33 A third isoform, caveolin 3, is expressed primarily on muscle cells.34 Caveolin 1 has been implicated in the entry of E. coli because the protein is recruited to membranes encasing internalized bacteria in bladder cells and because E. coli exhibit limited entry into bladder cells where expression of caveolin 1 has been knocked down using ribonucleic acid (RNA) interference.5 Caveolin 2 has been implicated in the entry of Pseudomonas into type 1‐like pneumocyte cells, also by using RNA interference.4 Furthermore, tyrosine phosphorylation of caveolin 2 was an important requirement for Pseudomonas invasion, suggesting that caveolin 2 plays a critical role in the complex signaling events that regulate lipid raft‐mediated endocytosis of Pseudomonas. Because of their capacity to serve as scaffolding proteins, the role of caveolins in the endocytosis of bacteria may lie in organizing and concentrating molecules critical to the endocytic process.

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CONCLUSIONS

In spite of their powerful barrier function, epithelial cells of the bladder and alveoli appear susceptible to penetration by bacteria that target specific lipid raft components. Attachment and subsequent crosslinking of lipid raft molecules of epithelial cells by bacteria are sufficient to activate the intrinsic endocytic properties of these cellular entities. Very little is currently known regarding the signaling events leading to lipid raft‐mediated endocytic activity or of the physiologic relevance of this endocytic activity. Nevertheless, it is clear that pathogenic microbes have evolved mechanisms to coopt this property. The recognition that sterol binding and modulating drugs can impede lipid raft‐mediated uptake may therefore support novel strategies to prevent and treat microbial infections of mucosal surfaces.

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References

1 Stebbins CE, Galan JE. Structural mimicry in bacterial virulence. Nature 2001;412:701-5.

2 Cossart P. Molecular and cellular basis of the infection by Listeria monocytogenes: an overview. Int J Med Microbiol 2002;291:401-9.

3 Cossart P, Sansonetti PJ. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 2004;304:242-8.

4 Zaas DW, Duncan MJ, Li G, et al. Pseudomonas invasion of type I pneumocytes is dependent on the expression and phosphorylation of caveolin-2. J Biol Chem 2005;280:4864-72.

5 Duncan MJ, Li G, Shin JS, et al. Bacterial penetration of bladder epithelium through lipid rafts. J Biol Chem 2004;279:18944-51.

6 Couet J, Belanger MM, Roussel E, Drolet MC. Cell biology of caveolae and caveolin. Adv Drug Deliv Rev 2001;49:223-35.

7 Brown DA, London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 2000;275:17221-4.

8 Anderson RG. The caveolae membrane system. Annu Rev Biochem 1998;67:199-225.

9 Bini L, Pacini S, Liberatori S, et al. Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering. Biochem J 2003;369:301-9.

10 Harada S, Smith RM, Jarett L. Mechanisms of nuclear translocation of insulin. Cell Biochem Biophys 1999;31:307-19.

11 Schnitzer JE, Oh P, Pinney E, Allard J. Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol 1994;127:1217-32.

12 Duncan MJ, Shin JS, Abraham SN. Microbial entry through caveolae: variations on a theme. Cell Microbiol 2002;04:783-91.

13 Lewis SA. Everything you wanted to know about the bladder epithelium but were afraid to ask. Am J Physiol Renal Physiol 2000;278:F867-74.

14 Lewis SA, Diamond JM. Na+ transport by rabbit urinary bladder, a tight epithelium. J Membr Biol 1976;28:1-40.

15 Lande MB, Donovan JM, Zeidel ML. The relationship between membrane fluidity and permeabilities to water, solutes, ammonia, and protons. J Gen Physiol 1995;106:67-84.

16 Ketterer B, Hicks RM, Christodoulides L, Beale D. Studies of the chemistry of the luminal plasma membrane of rat bladder epithelial cells. Biochim Biophys Acta 1973;311:180-90.

17 Vergara J, Zambrano F, Robertson JD, Elrod H. Isolation and characterization of luminal membranes from urinary bladder. J Cell Biol 1974;61:83-94.

18 Martinez JJ, Mulvey MA, Schilling JD, et al. Type 1 pilus-mediated bacterial invasion of bladder epithelial cells. EMBO J 2000;19:2803-12.

19 Wu XR, Sun TT, Medina JJ. In vitro binding of type 1-fimbriated Escherichia coli to uroplakins Ia and Ib: relation to urinary tract infections. Proc Natl Acad Sci U S A 1996;93:9630-5.

20 Shin JS, Gao Z, Abraham SN. Involvement of cellular caveolae in bacterial entry into mast cells. Science 2000;289:785-8.

21 Baorto DM, Gao Z, Malaviya R, et al. Survival of FimH-expressing enterobacteria in macrophages relies on glycolipid traffic. Nature 1997;389:636-9.

22 Chastre J, Fagon JY. Ventilator-associated pneumonia. Am J Respir Crit Care Med 2002;165:867-903.

23 Kurahashi K, Kajikawa O, Sawa T, et al. Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia. J Clin Invest 1999;104:743-50.

24 Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2000;02:1051-60.

25 Grassme H, Jendrossek V, Riehle A, et al. Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts. Nat Med 2003;09:322-30.

26 Kowalski MP, Pier GB. Localization of cystic fibrosis transmembrane conductance regulator to lipid rafts of epithelial cells is required for Pseudomonas aeruginosa-induced cellular activation. J Immunol 2004;172:418-25.

27 Schneeberger EE. Alveolar type 1 cells. In: Crystal RG, West JB, editors. The lung: scientific foundations. Philadelphia: Lippincott-Raven; 1997. p. 535-42.

28 Campbell L, Hollins AJ, Al-Eid A, et al. Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures. Biochem Biophys Res Commun 1999;262:744-51.

29 Newman GR, Campbell L, von Ruhland C, et al. Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function. Cell Tissue Res 1999;295:111-20.

30 Comolli JC, Waite LL, Mostov KE, Engel JN. Pili binding to asialo-GM1 on epithelial cells can mediate cytotoxicity or bacterial internalization by Pseudomonas aeruginosa. Infect Immun 1999;67:3207-14.

31 Nebl T, Pestonjamasp KN, Leszyk JD, et al. Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes. J Biol Chem 2002;277:43399-409.

32 Scherer PE, Lewis RY, Volonte D, et al. Cell-type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J Biol Chem 1997;272:29337-46.

33 Razani B, Engelman JA, Wang XB, et al. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J Biol Chem 2001;276:38121-38.

34 Scherer PE, Lisanti MP. Association of phosphofructokinase-M with caveolin-3 in differentiated skeletal myotubes. Dynamic regulation by extracellular glucose and intracellular metabolites. J Biol Chem 1997;272:20698-705.

Key Words:: caveolae; lipid rafts; bacteria; phagocytosis

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