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Cell adhesion and invasion mechanisms in microbial pathogenesis
Cell adhesion
and invasion mechanisms in microbial pathogenesis B.B. Finlay
Biotechnology Laboratory and Departments of Biochemistry and Microbiology, University of British Columbia, Vancouver, British Columbia, Canada Current Opinion in Cell Biology 1990 2:815420
Introduction
Successful colonization of a host by bacterial or protozoan pathogens is a complex event, usually involving a pathogen-encoded &and and a eukaryotic receptor (Beachey, J Infect Db 1981,143:315-345). A microbe has a choice of three types of host components with which it can interact: secreted cell products, host cell surfaces, or extracellular matrices. Microbial pathogens have developed numerous ways to interact with these components [ 11. By coating themselves in secreted host products, pathogens can avoid the host immune system. They can also use them as a bridge to adhere to cell receptors that bind the secreted products. Binding to extracellular matrices tiords a stable environment for pathogen proliferation. Adherence to spectic cell surface molecules confers upon the pathogen the ability to select preferentially which host component it interacts with. Finally, it is becoming apparent that by interacting with cell receptors that are normally involved in cell-cell contact or cell-matrix contact (especially integrins), pathogens are able to hijack these receptors, enter the cell and exist as intracellular parasites. There are many mechanisms used by parasites to interact with host cells, but this review will concentrate on some examples that involve pathogen interactions with extracellular matrix components or host surface receptors involved in cell-matrix or cell-cell contacts. These interactions have shed new light on host-parasite interactions, and are also providing new techniques to study the mechanisms used for cell-cell and cell-matrix contact.
Binding of pathogens components
to extracellular
matrix
Most eukaryotic matrices such as basement membranes are usually covered by host cells and are not accessible to incoming pathogens. However, if there is any preceding tissue damage (such as a viral infection, bacterial toxins, or mechanical damage) basement membranes
will be exposed to pathogens. Some components of host extracellular matrices, such as fibronectin, are also secreted and found on host cell surfaces. As a result, many pathogens have developed similar mechanisms to adhere to such matrix molecules (Table l), a necessary step in the pathogenesis of these parasites. Two Gram-positive pathogenic bacteria, Strepcoccus @gene-s (group A streptococci) and Stu@yloco~~u.s aurem, bind to the same amino-terminus region of fibronectin (not the Arg-Gly-Asp-containing cell attachment site - see below) but by different mechanisms. Group A streptococci attach by the glycolipid end of lipoteichoic acid [Courtney et al, Rev Infect Dis 1988, lO(supp1 2):S360-S362], w-bile S. aureus encodes a large fibronectir-binding protein (Froman et al, J Biol Chem 1987, 262:6564-6571; Flock et al, EMT30 J 1987, 6:2351-2357). In addition, both these pathogens bind to other components of the basement membrane. S. w genes binds to type IV collagen (Kostrzynska et al, FEMS Microbial Lett 1989, 59:22!&234), libronectin (Vercellotti et al., Am J Path1 1985, 120:13-21) and possesses receptors to laminin (Switalski et aL, J Biol Gem 1984, 259:37343738), and S. aurezts binds fibronectin (Kuusela, Nature 1978, 276:71&720; Herrmann et al, J Infect Db 1988, 158:693-701), laminin (Lopes et al, Science 1985, 229:275-277), and type lV collagen. These two pathogens are not normally intracellular parasites; instead, they adhere to surfaces and secrete many potent cytotoxins. It is possible that the lysis of host cells by these toxins exposes extracellular matrix components to which these organisms can anchor themselves, establishing an infection. It has been shown that Bacteroides gingivalis, a Gram-negative dental pathogen that adheres to enamel and other oral surfaces, also binds fibronectin (Wiier et al., Infect Immun 1987, 55:2721-27261, although the binding components have not been identified. Because fibronectin is present on oral surfaces, fibronectin binding may contribute to B. gingivulis pathogenesis. Fibronectin is a complex molecule consisting of many domains (Yamada, Cur-r @in Cell Biol1989, 1:956-963;
Abbreviations CR-complement
receptor;
FHA-filamentous LFA-lymphocyte
@ Current
Biology
hemagglutinin; function-associated
ICAM-intercellular antigen.
Ltd ISSN 0955+674
adhesion
molecule;
815
816
Cell-to-cell
contact
ad
extracellular
matrix,
. Table
1. Examples
of different
host-parasite
binding
mechanisms.
Mechanism Pathogen non-RCD’ extracellular
binds to sequence matrix
Staphylococcus Streptococcus
of protein
Leishmania Bordetella
Pathogen binds secreted protein which forms bridge beween parasite
letter
amino
Clycoprotein; lipoteichoic /fibronectin
cruzi
80-85
Jreponema pallidurn
Pathogen has RCD sequence which binds to integrin receptor
Pathogen does not have RCD sequence,
aureus pyogenes
Trypanosoma
Pathogen binds to RCD sequence of extracellular matrix protein
‘Single
Molecules involved (Pathogen/host)
Pathogen
Legionella Mycobacterium and
binds
acid
code.
58-68 kD glycoproteins; Pl, P2 and P3 Kibronectin
gp63;
species pertussis
FHA CR3
pneumophila tuberculosis
host Yersinia
but
and
Major
outer
membrane protein; ? /Complement, CR3
pseudotuberculosis
to integrin
Proctor, Rev Infect Dis 1987,9(suppl4):S317-S321). The cell-binding domain of fibronectin contains an Arg-GlyAsp (RGD) sequence that interacts with the fibronectin receptor, a member of the integrin family (see below). Some pathogenic organisms have devised similar mechanisms to bind to fibronectin. For example, the bacterium Treponemu pallidurn, the causative agent in syphilis, binds directly to the cell-binding domain of libronectin by three related adhesins Pl, P2, and P3 (Thomas et al, J Eq Med 1985 161:514-525; Peterson et al., J E3cpMed 1983, 157:1958-1970; Thomas et al, J Exp Med 1985, 162:1715-1719). However, this pathogen does not interact with other extracellular matrix components such as laminin or collagen. It has not yet been established why this pathogen binds to fibronectin. It may use bound fibronectin to attach to host surfaces via the fibronectin receptor (a bridging mechanism), or it may disguise itself (to avoid the host immune system) by coating itself with this host molecule. Protozoan parasites also adhere to extracellular matrix components, especially fibronectin (for a review, see [2]). Trypomastigotes (the infectious stage) of Try pmmsomu cruzi, the causative agent in Chagas’ disease, bind Iibronectin to their surfaces (Ouaissi et a.!, Nature 1984, 308:38&382). The region of iibronectin that interacts with the parasite has been localized to the RGDcontaining cell-binding domain, as in the case of i? pallidum. Synthetic peptides containing the fibronectin ArgGly-Asp&r sequence bind to the surface of T. cruri and are capable of inhibiting cell invasion by this parasite (Ouaissi et al, Science 1986, 234603-607). The fibronectin receptor on this parasite also binds collagen,
acid
/Various
lnvasin PI integrins
although it has not been determined whether this binding occurs via the RGD sequence found in collagen. Tricbomona.s vaginalis and Tritricbomonas fetus are protozoa that colonize human and cattle urogenital tracts, respectively, causing trichomoniasis in these hosts. Both these parasites are able to bind laminin via a surface receptor (Filho et al, Proc Nat1 Acud Sci USA 1988, 85:8042-8046). Iaminin binding significantly enhances parasitic adherence to epithelial ceU surfaces; antibodies directed against laminin decrease this adherence. There is also some evidence that 7: fetus binds libronectin (Benchimol et al., J Submicrasc Cytol Path1 1990, 2:39-45), although the significance of this has not been established.
Role of integrins
in parasite
internalization
IntraceUular parasites encode speciEc products that are necessary for their internalization into host cells (Moulder, Microbial Rev 1985, 49:298-337). These products are usually adhesins that bind to host receptors (or bind a host molecule which then binds to its receptor) that interact with the cytoskeleton. Pathogenic bacteria and parasites are larger in size than most particles (and viruses) usually internalized by host cells. As a result, internalization of these pathogens requires rearrangement of the underlying host cytoskeleton, and usually requires active participation of host microiilaments. The cytoskeleton is linked to the ceU surface by members of the integrin family (Burridge and Fath,
Cell adhesion
Bioessays 1989, 10:104-108). Integrins are a family of integral membrane glycoproteins that mediate cell-cell or cell-extracellular matrix interactions (Ruoslahti and Pierschbacher, Science 1987, 238:491-497), for example, the receptors for Ebronectin, collagen, laminin, vitronectin, and the complement molecule C3bi. There is mounting evidence that integrins are linked to the actin microElament system through a variety of molecules including talin, vinculin, and a-actinin (Burridge and Fath, 1989). It is thought that these linkages form anchors for the eukaryotic ceU to adhere to other cells or the extracellular matrix. In the case of complement-mediated phagocytosis, linkage of the complement receptor to the host microElaments provides the mechanisms necessary for internalization. It is now becoming apparent that intracellular parasites are able to capitalize on these systems, and use these receptors as mechanisms to gain entry into host cells. Lebbmunia are obligate intraceUular parasites that survive within macrophages. It has recently been shown that a surface glycoprotein of Lei&nania promostigotes, gp63, mediates uptake into host macrophages by binding directly to an integrin, complement receptor (CR)3 (Russell and Wright, J Eap Med 1988, 168:279-292). CR3 usually binds a region of complement fragment C3bi that contains the RGD triplet sequence. Interestingly, gp63 encodes an RGD-like sequence, and synthetic peptides of this sequence block the binding of gp63 or promastig otes to macrophages (Russell and Wright, 1988). Antibodies raised to the RGD-like region of gp63 also crossreact with C3bi [3], indicating structural similarities between these two ligands of CR3. A similar story regarding entry into macrophages has begun to emerge with Boraktelh pertuwk, the causative agent of whooping cough. This pathogenic bacterium inhabits the upper respiratory tract, and may persist as an intracellular organism within epithelial cells and monocytes. One of the major surface components of B. pertzasis, Elamentous hemagglutinin (FHA), participates in adherence of bacteria to host surfaces. This large protein contains an RGD sequence that is necessary for FHA-mediated adhesion to ciliated epithelial cells and macrophages [4]. Relman et al. [ 51 have shown for the first time that FHA-mediated adherence occurs via the complement receptor CR3. Peptides encompassing the FHA RGD sequence inhibit Bordetellu adherence to macrophages, as well as CR3-C3bi interactions, indicating the B. pertussis, like Leisbmunia, can mimic the epitope of C3bi which interacts with CR3. Another surface protein of B. pertu.wk, P69, which is believed to be a vimlence factor, also contains two predicted RGD-containing sequences (Charles et al, Proc Nat1 Acud Sci U.. 1989, 86:355&3558). It remains to be determined whether these sequences are also involved in host-parasite interactions. ‘Iwo other intracellular bacterial parasites which use CR3 as a receptor include Legionella pneumophila (Legionnaires’ disease; Payne and Horwitz, J Exp Med 1987, 166:1377-1389) and Myccbacterium tubercuhis (tuberculosis) [6]. However, in contrast to Lehhmunia
and invasion
mechanisms
in microbial
pathogenesis
Finlay
species and B. pertusks, these parasites bind complement tightly to their surface, which then serves as a bridge to facilitate parasitic uptake into macrophages. Interestingly, although both Legionella and Lehbmania use CR3 as a receptor, their intraceUular environments differ. L pneumophih resides inside a vacuole which is studded with mitochondria and ribosomes, and is not acid&d, whereas Letimunia aimovani is contained within an acidified ribosome and a mitochondria-free vacuole. A related attachment mechanism may be used by the rhinovirus, which causes the common cold. Several groups [7-91 have shown that rhinoviruses use the membrane-bound glycoprotein intercellular adhesion molecule (ICAM)- as their receptor, and secreted forms of ICAM-1, or antibodies to it, can inhibit rhinovirus infection [lo]. ICAM- is also a ligand of the integrin lymphocyte function-associated antigen (LFA)-l or alfl2, which shares the same 02 chain as the CR3 receptor. It has not been determined whether the rhinovirus surface glycoprotein which interacts with ICAM- does so in a manner similar to most integrin-receptor interactions. Pkxmodium falciparum, which causes malaria, also uses ICAMas a receptor [ 111, although it can also bind to at least two other receptors: CD36 and thrombospondin. Non-phagocytic cells such as epithelial cells and Ebroblasts do not encode CR3 receptors and do not normally phagocytose particles yet many intracellular pathogens are able to enter into these cells. The mechanisms used by pathogens to enter these cells may be similar to those used during phagocytosis, perhaps involving integrins and the host cytoskeleton. Yersinia and Salmonella species are bacteria which are ingested orally yet penetrate the intestinal epithelium and enter the underlying reticuloendothelial system, causing various gastrointestinal diseases and typhoid fever. Yersinia enter0 coliticu and Yersinia pseudotuberculosis encode a surface protein, invasin, which mediates adherence and uptake of these bacteria into epithelial cells (Isberg and Falkow, Nature 1985, 317:262-264; Isberg et al, Cell 1987, 50:769-778; and reviewed in [12]). It has recently been shown that epithelial cells can bind __..to invasin, and that this interaction involves the integrins a3P1, a4p1, a5p1, and OrbpI, but not a2l31, a$3 or p2 integrins [13]. Although the cell-binding domain of invasin has been localized (Ieong et al., EMBO J 1990,9:1979-1989) it does not encode an RGD-containing sequence. As mentioned above, integrins are linked to the cytoskeleton. Actin filament inhibitors block the uptake into nonphagocytic cells of most pathogenic bacteria, including Ywsinia, Salmonella, and S&gel&a species (Finlay and Falkow, Biocbimie 1988, 70:108%1099). As Salmonella or Shigeh enter epithelial cells, there is a large condensation of actin surrounding the invading organisms (Clerc and Sansonetti, Infect Immun 1987, 55:2681-2688; Finlay et al., J Cell Sci 1989, suppl 11:99-107), similar to that which occurs in phagocytosis. My colleagues and I have recently found that there is also a large condensation of a-actinin, tropomyosin, and talin around invading Salmonella although these bacteria do not use the
817
818
Cell-to&II
contact
and extracellular
matrix’ .
same integrin receptors as the Yersinia invasin (Finlay and Dedhar, submitted). Although it remains to be demonstrated completely for any given system, it is probable that invading organisms attach to integrins or integrin-like molecules on the host surface either directly, or via an extracellular matrix component. This attachment then triggers a signal in the host cell which causes actin filaments to link to the membranebound receptor, possibly by talin and other proteins, which then generates the force necessary for parasite uptake (Fig. 1). Once inside a host cell, some pathogenic bacteria are able to cause significant rearrangement of the cytoskeleton. Both Listmh monocytogenes (a Gram-positive organism which can cause disease in pregnant women and neonates> and Sbigella j7exneri (a Gram-negative organism which causes shigellosis) enter host cells and dissolve the surrounding vacuolar membrane, leaving them free in the host cytoplasm. Both organisms then cause a large rearrangement of intracellular actin which is associated with intercellular bacterial spread [ 14,151. Addition of cytochalasin D inhibits intra- and intercellular spread
of both organisms, indicating that this actin rearrangement is essential for continued infection of other ceils. A large ‘comet’s tail’, made of randomly orientated actin filaments, extends from the end of these bacteria opposite to its direction of movement. When the host cell membrane is encountered, a protrusion appears, with the bacteria at the tip and the actin trail behind. If the protrusion contacts a neighboring cell, it is internalized, and the process can be repeated in the new host cell. Although this actin rearrangement may explain how intercellular bacterial movement occurs, it also raises additional questions. For example, how does the bacteria convince a neighboring cell to internalize the protrusion? The neighboring cell would only see host cell-surface components on the outside of the protrusion, as the bacteria is located inside the cell membrane and is not exposed to the neighboring cell. Another problem is what signals the extent to which the protrusion is internalized into the adjacent cell? Both organisms must be in an acidic environment to dissolve their surrounding vacuole. Thus, the protrusion must be internalized within an acidified membrane-bound inclusion before the organism can repeat the process.
Fig. 1. Generic model of how intracellular pathogens may enter eukaryotic systems, but not proven in any system). The invading parasite encodes integrin, or bind host proteins which interact with integrins. When the (perhaps calcium or phosphor-ylation of proteins) is generated in the host talin) which then form a bridge between the integrin and actin filaments. by a-actinin. The parasite is then internalized into a membrane-bound phagocytosis.
cells. (This model has been compiled from data from various a molecule on its surface which can either bind directly to an pathogen contacts the host cell integrins, an unknown signal cytoplasm. This signal modifies cytoplasmic proteins (possibly The signal also causes large amounts of crosslinking of filaments inclusion by an actin/myosin-dependent mechanism similar to
Cell adhesion
Future
prospects
and invasion
mechanisms
in microbial
pathogenesis
Finlay
A review of RGD-mediated protozoan interactions with host cells.
The ability of a pathogen to interact with host surfaces is essential for its virulence. If one wishes to inhibit a pathogenic organism from colonizing and establishing an infection, blocking initial attachment is a logical place to start. As many pathogens adhere to host matrices and cell surfaces by similar mechanisms, therapeutic approaches may be considered in the future. Such approaches might include the use of synthetic peptides or antibodies directed against binding domains of the parasite ligand, or by interfering with the mechanisms used to internalize intracellular parasites. In the immediate future, the interaction of pathogens with host cells will expand our knowledge of cell-cell and cell-matrix interactions. These parasites provide new tools to study integrin-ligand interactions. It will be interesting to determine how FHA and gp63 interact with CR3, and compare these interactions with its ‘natural’ l&and C3bi. These interactions can also be used to identify the signal which links the integrin with the cytoskeleton. Different parasites probably use different mechanisms. For example, non-viable Yersinia are taken up by epithelial cells (presumably via an invasin-mediated pathway) yet Salmonella species have to be metabolically active for invasion to occur (Finlay el al., Science 1989,243:940-9431, and do not use the same receptor as invasin. Additionally, it seems that intracellular parasites use several pathways for internalization. At least three pathways appear to be used by E pseudotubercuios.& to enter epithelial cells (Isberg, Mol Biol Med 1990, 7:7+82). As demonstrated with Salmonella, intracellular pathogens will also be useful for determining which cytoplasmic molecules in the host ceU form the bridge between membrane-bound receptors and the host cytoskeleton. It will be interesting to compare these molecules and signals with those that are involved in cell-cell and cell-matrix contact formation. The study of host-parasite interactions is entering an exciting phase. Workers in the previously diverse fields of ceU biology and microbial pathogenesis are now finding common interests, and these findings provide important new insights applicable to both fields. Perhaps in the future it will be more acceptable for ceU biologists to ‘contaminate’ their ceU cultures, and for microbiologists to study ceU biology.
RUSSELDG, TAIAMAS-ROHANA P, ZELECHOW~KIJ: Antibodies raised against synthetic peptides from the Arg-GIy-Asp-containing region of the Lefshmanfu surface protein gp63 cross-react with human C3 and Interfere with gp63mediated binding to macrophages. Infecf lmmun 1989, 57:630-632. This paper demonstrates that a parasitic &and which interacts with a host receptor may be similar in structure to the ‘natural’ &and, thereby acquiring its binding ability. 3. l
REMAN DA, DOMENIGHW M, TUOMANENE, RAPPUOUR, FAU(OW S: Fikunentous hemagglutinin of Bordetellu pwturcfs Nucleotide sequence and crucial role in adherence. Prcx Nutl Acud Sci USA 1989, 86:2637-2641. The FHA nucleotide sequence predicts the presence of an RGD sequence; deletion of this sequence eliminated B. perrus& adherence to respitatoly ciliated epithelial cells. 4. l
REMAN DA TUOMANEN E, FALKOW S, G~LENEOCK DT, SAUKKONENK, WRIGHT S: Recognition of a bacterial adhesin by an eukaryotic integrin: CR3 (aMp2, CDllb/CD18) on human macrophages binds filamentous hemagglutinin of Bordetella petiussis. Cell 1990, 61:1375-1382. This paper demonstrates for the first time that bacterial pathogens can produce molecules (in this case FHA) containing RGD sequences that bind directIy to integrins. 5.
l e
SCHLESINGER l.5, BELL~NGER-KAW~ CG, PAYNENR, Ho~wm MA Phagocytosis of Mycobactetium hrbernclosis is mediated by human monocyte complement receptors and complement component C3. J Immunol 1990, 144:2771-2780. Human monocyte CR1 and CR3 complement receptors mediate uptake of M. tuber&& and C3 component is attached to the bacterial surface, mediating bacterial uptake. 6. l
JM, DAVISG, JEXR AM, FORTXCP, Yosr SC, MARLORCW, KAMARCKME, MCCLEUANDk The major human rhinovirus receptor is ICAM-1. Cell 1989, 56:83w7. One of three simultaneous papers that identify ICAM- as the rhinovirus receptor. See [8,9] also. 7.
GREW
l
DE, MERLUZI VJ, ROTHLEINR, MARLINSD, SPRINGER TA: A ceU adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses. Cell 1989, 56:84-53. STAUNI’ON
8. l
see 171.
TOMASSUVI JE, GRAH~~~D. DEWY CM, L~NEBERGER DW, ROOKEY JA, COU%NO RJ: cDNA cloning reveals that the major group rhinovirus receptor on HeLa ceUs is intercellular adhesion molecule 1. Proc Nat1 Acad Sci USA 1989, 86:4907-4911.
9. l
see 171.
10. l
A
MARLIN SD, STALIN~‘ONDE, SPIUNGERTA, S~TOWA C, SOMMERGRUBER W. MEKLUW VJ: A soluble form of intercellular adhesion molecule-l inhibits rhinovirus infection. Nuture 1990, 344:7C-72. secreted form of ICAMl prevents rhinovirus infection.
11.
AR, SIMONS DL TANSF~J, NRWBOIDCl, MARSHK: Intercellular adhesion molecule-l is an endothelial ceU adhesion receptor for Plasmodium falci~atum. Nature 1989, 341:57-59. Transfected cells were used to demonstrate that ICAM-1. in addition to CD36, is a receptor for P. falcrparum. As [CAM-l is widely distributed, it may be one of the receptors used during malarial infections. BERENDT
l
Annotated reading l l e
references
and recommended
Of interest Of outstanding interest
FINIAY BB. FALKOW S: Common themes in microbial l pathogenicity. Micrcbiol Rev 1989, 53:21&230. A review of the principles of bacterial pathogenesis. Recently character. ized factors involved in pathogenesis are compared between pathogens, and bacterial adherence and imasion mechanisms are presented.
ISBERGRR Mammalian ceU adhesion functions and cellular penetration of enteropathogenic YersinIa species. Mok Microbiol 1989, 3:1449-1453. A review of the products and mechanisms used for entry of Yetinia species into cultured cells. A discussion of invasin-integrin interactions is included.
2.
13.
1.
l
OuAtsst 4 CAPRoN L Some aspects of protozoan parasitehost cell interactions with special reference to RGD-mediated recognition process. Microbial Pufbogen 1989, 6:1-5.
12. l
l e
ISBERGRR, LEONGJM: Multiple & chain Integrins are recep tars for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 1990, 60:861-871.
819
820
Cell-to-cell
contact
and extracellular
matrix
This is the first report of direct binding between a pathogen ligand (invasin) and a non-B2 integrin. It clearly demonstrates that imasin binds to several 81 integrins, and that antibodies to these integrins block bacterial adherence and uptake. h-twin does not contain an RGD se. quence.
14.
ML, MOUIUER J, D’HoL~~~.E H. COQLIIS-RONDON M, SANSONEITI PJ: Identification of ica.4, a plasmid locus of Sbigeffa f7exner-i that governs bacterial intra- and intercellular spread through interaction with F-actin. from Nat/ Acad Sci USA 1989, 863867-3871. This paper details the actin rearrangement associated with intercellular. movement of S. JIexneti in cultured epithelial cells using a fluorescent l
BERNARDINI
actin cess.
label, and identif.ies
a bacterial
gene which
is involved
in this pro-
TIINW LG, PORTNOY DA Actin tilaments and the growth, movement and spread of the intracelhtlar bacterial parasite Listeria monocytogenes J Cell Rio1 1989, 109:1597-1608. A transmission electron microscofx study of the morphological events associated with Ltileti invasion and intercellular movement within cultured macrophages. Actin filaments were decorated, resulting in striking micrographs of the ‘comet’ composed of the bacteria and actin ftlamentc. 15. a
and invasion mechanisms in microbial pathogenesis B.B. Finlay
Biotechnology Laboratory and Departments of Biochemistry and Microbiology, University of British Columbia, Vancouver, British Columbia, Canada Current Opinion in Cell Biology 1990 2:815420
Introduction
Successful colonization of a host by bacterial or protozoan pathogens is a complex event, usually involving a pathogen-encoded &and and a eukaryotic receptor (Beachey, J Infect Db 1981,143:315-345). A microbe has a choice of three types of host components with which it can interact: secreted cell products, host cell surfaces, or extracellular matrices. Microbial pathogens have developed numerous ways to interact with these components [ 11. By coating themselves in secreted host products, pathogens can avoid the host immune system. They can also use them as a bridge to adhere to cell receptors that bind the secreted products. Binding to extracellular matrices tiords a stable environment for pathogen proliferation. Adherence to spectic cell surface molecules confers upon the pathogen the ability to select preferentially which host component it interacts with. Finally, it is becoming apparent that by interacting with cell receptors that are normally involved in cell-cell contact or cell-matrix contact (especially integrins), pathogens are able to hijack these receptors, enter the cell and exist as intracellular parasites. There are many mechanisms used by parasites to interact with host cells, but this review will concentrate on some examples that involve pathogen interactions with extracellular matrix components or host surface receptors involved in cell-matrix or cell-cell contacts. These interactions have shed new light on host-parasite interactions, and are also providing new techniques to study the mechanisms used for cell-cell and cell-matrix contact.
Binding of pathogens components
to extracellular
matrix
Most eukaryotic matrices such as basement membranes are usually covered by host cells and are not accessible to incoming pathogens. However, if there is any preceding tissue damage (such as a viral infection, bacterial toxins, or mechanical damage) basement membranes
will be exposed to pathogens. Some components of host extracellular matrices, such as fibronectin, are also secreted and found on host cell surfaces. As a result, many pathogens have developed similar mechanisms to adhere to such matrix molecules (Table l), a necessary step in the pathogenesis of these parasites. Two Gram-positive pathogenic bacteria, Strepcoccus @gene-s (group A streptococci) and Stu@yloco~~u.s aurem, bind to the same amino-terminus region of fibronectin (not the Arg-Gly-Asp-containing cell attachment site - see below) but by different mechanisms. Group A streptococci attach by the glycolipid end of lipoteichoic acid [Courtney et al, Rev Infect Dis 1988, lO(supp1 2):S360-S362], w-bile S. aureus encodes a large fibronectir-binding protein (Froman et al, J Biol Chem 1987, 262:6564-6571; Flock et al, EMT30 J 1987, 6:2351-2357). In addition, both these pathogens bind to other components of the basement membrane. S. w genes binds to type IV collagen (Kostrzynska et al, FEMS Microbial Lett 1989, 59:22!&234), libronectin (Vercellotti et al., Am J Path1 1985, 120:13-21) and possesses receptors to laminin (Switalski et aL, J Biol Gem 1984, 259:37343738), and S. aurezts binds fibronectin (Kuusela, Nature 1978, 276:71&720; Herrmann et al, J Infect Db 1988, 158:693-701), laminin (Lopes et al, Science 1985, 229:275-277), and type lV collagen. These two pathogens are not normally intracellular parasites; instead, they adhere to surfaces and secrete many potent cytotoxins. It is possible that the lysis of host cells by these toxins exposes extracellular matrix components to which these organisms can anchor themselves, establishing an infection. It has been shown that Bacteroides gingivalis, a Gram-negative dental pathogen that adheres to enamel and other oral surfaces, also binds fibronectin (Wiier et al., Infect Immun 1987, 55:2721-27261, although the binding components have not been identified. Because fibronectin is present on oral surfaces, fibronectin binding may contribute to B. gingivulis pathogenesis. Fibronectin is a complex molecule consisting of many domains (Yamada, Cur-r @in Cell Biol1989, 1:956-963;
Abbreviations CR-complement
receptor;
FHA-filamentous LFA-lymphocyte
@ Current
Biology
hemagglutinin; function-associated
ICAM-intercellular antigen.
Ltd ISSN 0955+674
adhesion
molecule;
815
816
Cell-to-cell
contact
ad
extracellular
matrix,
. Table
1. Examples
of different
host-parasite
binding
mechanisms.
Mechanism Pathogen non-RCD’ extracellular
binds to sequence matrix
Staphylococcus Streptococcus
of protein
Leishmania Bordetella
Pathogen binds secreted protein which forms bridge beween parasite
letter
amino
Clycoprotein; lipoteichoic /fibronectin
cruzi
80-85
Jreponema pallidurn
Pathogen has RCD sequence which binds to integrin receptor
Pathogen does not have RCD sequence,
aureus pyogenes
Trypanosoma
Pathogen binds to RCD sequence of extracellular matrix protein
‘Single
Molecules involved (Pathogen/host)
Pathogen
Legionella Mycobacterium and
binds
acid
code.
58-68 kD glycoproteins; Pl, P2 and P3 Kibronectin
gp63;
species pertussis
FHA CR3
pneumophila tuberculosis
host Yersinia
but
and
Major
outer
membrane protein; ? /Complement, CR3
pseudotuberculosis
to integrin
Proctor, Rev Infect Dis 1987,9(suppl4):S317-S321). The cell-binding domain of fibronectin contains an Arg-GlyAsp (RGD) sequence that interacts with the fibronectin receptor, a member of the integrin family (see below). Some pathogenic organisms have devised similar mechanisms to bind to fibronectin. For example, the bacterium Treponemu pallidurn, the causative agent in syphilis, binds directly to the cell-binding domain of libronectin by three related adhesins Pl, P2, and P3 (Thomas et al, J Eq Med 1985 161:514-525; Peterson et al., J E3cpMed 1983, 157:1958-1970; Thomas et al, J Exp Med 1985, 162:1715-1719). However, this pathogen does not interact with other extracellular matrix components such as laminin or collagen. It has not yet been established why this pathogen binds to fibronectin. It may use bound fibronectin to attach to host surfaces via the fibronectin receptor (a bridging mechanism), or it may disguise itself (to avoid the host immune system) by coating itself with this host molecule. Protozoan parasites also adhere to extracellular matrix components, especially fibronectin (for a review, see [2]). Trypomastigotes (the infectious stage) of Try pmmsomu cruzi, the causative agent in Chagas’ disease, bind Iibronectin to their surfaces (Ouaissi et a.!, Nature 1984, 308:38&382). The region of iibronectin that interacts with the parasite has been localized to the RGDcontaining cell-binding domain, as in the case of i? pallidum. Synthetic peptides containing the fibronectin ArgGly-Asp&r sequence bind to the surface of T. cruri and are capable of inhibiting cell invasion by this parasite (Ouaissi et al, Science 1986, 234603-607). The fibronectin receptor on this parasite also binds collagen,
acid
/Various
lnvasin PI integrins
although it has not been determined whether this binding occurs via the RGD sequence found in collagen. Tricbomona.s vaginalis and Tritricbomonas fetus are protozoa that colonize human and cattle urogenital tracts, respectively, causing trichomoniasis in these hosts. Both these parasites are able to bind laminin via a surface receptor (Filho et al, Proc Nat1 Acud Sci USA 1988, 85:8042-8046). Iaminin binding significantly enhances parasitic adherence to epithelial ceU surfaces; antibodies directed against laminin decrease this adherence. There is also some evidence that 7: fetus binds libronectin (Benchimol et al., J Submicrasc Cytol Path1 1990, 2:39-45), although the significance of this has not been established.
Role of integrins
in parasite
internalization
IntraceUular parasites encode speciEc products that are necessary for their internalization into host cells (Moulder, Microbial Rev 1985, 49:298-337). These products are usually adhesins that bind to host receptors (or bind a host molecule which then binds to its receptor) that interact with the cytoskeleton. Pathogenic bacteria and parasites are larger in size than most particles (and viruses) usually internalized by host cells. As a result, internalization of these pathogens requires rearrangement of the underlying host cytoskeleton, and usually requires active participation of host microiilaments. The cytoskeleton is linked to the ceU surface by members of the integrin family (Burridge and Fath,
Cell adhesion
Bioessays 1989, 10:104-108). Integrins are a family of integral membrane glycoproteins that mediate cell-cell or cell-extracellular matrix interactions (Ruoslahti and Pierschbacher, Science 1987, 238:491-497), for example, the receptors for Ebronectin, collagen, laminin, vitronectin, and the complement molecule C3bi. There is mounting evidence that integrins are linked to the actin microElament system through a variety of molecules including talin, vinculin, and a-actinin (Burridge and Fath, 1989). It is thought that these linkages form anchors for the eukaryotic ceU to adhere to other cells or the extracellular matrix. In the case of complement-mediated phagocytosis, linkage of the complement receptor to the host microElaments provides the mechanisms necessary for internalization. It is now becoming apparent that intracellular parasites are able to capitalize on these systems, and use these receptors as mechanisms to gain entry into host cells. Lebbmunia are obligate intraceUular parasites that survive within macrophages. It has recently been shown that a surface glycoprotein of Lei&nania promostigotes, gp63, mediates uptake into host macrophages by binding directly to an integrin, complement receptor (CR)3 (Russell and Wright, J Eap Med 1988, 168:279-292). CR3 usually binds a region of complement fragment C3bi that contains the RGD triplet sequence. Interestingly, gp63 encodes an RGD-like sequence, and synthetic peptides of this sequence block the binding of gp63 or promastig otes to macrophages (Russell and Wright, 1988). Antibodies raised to the RGD-like region of gp63 also crossreact with C3bi [3], indicating structural similarities between these two ligands of CR3. A similar story regarding entry into macrophages has begun to emerge with Boraktelh pertuwk, the causative agent of whooping cough. This pathogenic bacterium inhabits the upper respiratory tract, and may persist as an intracellular organism within epithelial cells and monocytes. One of the major surface components of B. pertzasis, Elamentous hemagglutinin (FHA), participates in adherence of bacteria to host surfaces. This large protein contains an RGD sequence that is necessary for FHA-mediated adhesion to ciliated epithelial cells and macrophages [4]. Relman et al. [ 51 have shown for the first time that FHA-mediated adherence occurs via the complement receptor CR3. Peptides encompassing the FHA RGD sequence inhibit Bordetellu adherence to macrophages, as well as CR3-C3bi interactions, indicating the B. pertussis, like Leisbmunia, can mimic the epitope of C3bi which interacts with CR3. Another surface protein of B. pertu.wk, P69, which is believed to be a vimlence factor, also contains two predicted RGD-containing sequences (Charles et al, Proc Nat1 Acud Sci U.. 1989, 86:355&3558). It remains to be determined whether these sequences are also involved in host-parasite interactions. ‘Iwo other intracellular bacterial parasites which use CR3 as a receptor include Legionella pneumophila (Legionnaires’ disease; Payne and Horwitz, J Exp Med 1987, 166:1377-1389) and Myccbacterium tubercuhis (tuberculosis) [6]. However, in contrast to Lehhmunia
and invasion
mechanisms
in microbial
pathogenesis
Finlay
species and B. pertusks, these parasites bind complement tightly to their surface, which then serves as a bridge to facilitate parasitic uptake into macrophages. Interestingly, although both Legionella and Lehbmania use CR3 as a receptor, their intraceUular environments differ. L pneumophih resides inside a vacuole which is studded with mitochondria and ribosomes, and is not acid&d, whereas Letimunia aimovani is contained within an acidified ribosome and a mitochondria-free vacuole. A related attachment mechanism may be used by the rhinovirus, which causes the common cold. Several groups [7-91 have shown that rhinoviruses use the membrane-bound glycoprotein intercellular adhesion molecule (ICAM)- as their receptor, and secreted forms of ICAM-1, or antibodies to it, can inhibit rhinovirus infection [lo]. ICAM- is also a ligand of the integrin lymphocyte function-associated antigen (LFA)-l or alfl2, which shares the same 02 chain as the CR3 receptor. It has not been determined whether the rhinovirus surface glycoprotein which interacts with ICAM- does so in a manner similar to most integrin-receptor interactions. Pkxmodium falciparum, which causes malaria, also uses ICAMas a receptor [ 111, although it can also bind to at least two other receptors: CD36 and thrombospondin. Non-phagocytic cells such as epithelial cells and Ebroblasts do not encode CR3 receptors and do not normally phagocytose particles yet many intracellular pathogens are able to enter into these cells. The mechanisms used by pathogens to enter these cells may be similar to those used during phagocytosis, perhaps involving integrins and the host cytoskeleton. Yersinia and Salmonella species are bacteria which are ingested orally yet penetrate the intestinal epithelium and enter the underlying reticuloendothelial system, causing various gastrointestinal diseases and typhoid fever. Yersinia enter0 coliticu and Yersinia pseudotuberculosis encode a surface protein, invasin, which mediates adherence and uptake of these bacteria into epithelial cells (Isberg and Falkow, Nature 1985, 317:262-264; Isberg et al, Cell 1987, 50:769-778; and reviewed in [12]). It has recently been shown that epithelial cells can bind __..to invasin, and that this interaction involves the integrins a3P1, a4p1, a5p1, and OrbpI, but not a2l31, a$3 or p2 integrins [13]. Although the cell-binding domain of invasin has been localized (Ieong et al., EMBO J 1990,9:1979-1989) it does not encode an RGD-containing sequence. As mentioned above, integrins are linked to the cytoskeleton. Actin filament inhibitors block the uptake into nonphagocytic cells of most pathogenic bacteria, including Ywsinia, Salmonella, and S&gel&a species (Finlay and Falkow, Biocbimie 1988, 70:108%1099). As Salmonella or Shigeh enter epithelial cells, there is a large condensation of actin surrounding the invading organisms (Clerc and Sansonetti, Infect Immun 1987, 55:2681-2688; Finlay et al., J Cell Sci 1989, suppl 11:99-107), similar to that which occurs in phagocytosis. My colleagues and I have recently found that there is also a large condensation of a-actinin, tropomyosin, and talin around invading Salmonella although these bacteria do not use the
817
818
Cell-to&II
contact
and extracellular
matrix’ .
same integrin receptors as the Yersinia invasin (Finlay and Dedhar, submitted). Although it remains to be demonstrated completely for any given system, it is probable that invading organisms attach to integrins or integrin-like molecules on the host surface either directly, or via an extracellular matrix component. This attachment then triggers a signal in the host cell which causes actin filaments to link to the membranebound receptor, possibly by talin and other proteins, which then generates the force necessary for parasite uptake (Fig. 1). Once inside a host cell, some pathogenic bacteria are able to cause significant rearrangement of the cytoskeleton. Both Listmh monocytogenes (a Gram-positive organism which can cause disease in pregnant women and neonates> and Sbigella j7exneri (a Gram-negative organism which causes shigellosis) enter host cells and dissolve the surrounding vacuolar membrane, leaving them free in the host cytoplasm. Both organisms then cause a large rearrangement of intracellular actin which is associated with intercellular bacterial spread [ 14,151. Addition of cytochalasin D inhibits intra- and intercellular spread
of both organisms, indicating that this actin rearrangement is essential for continued infection of other ceils. A large ‘comet’s tail’, made of randomly orientated actin filaments, extends from the end of these bacteria opposite to its direction of movement. When the host cell membrane is encountered, a protrusion appears, with the bacteria at the tip and the actin trail behind. If the protrusion contacts a neighboring cell, it is internalized, and the process can be repeated in the new host cell. Although this actin rearrangement may explain how intercellular bacterial movement occurs, it also raises additional questions. For example, how does the bacteria convince a neighboring cell to internalize the protrusion? The neighboring cell would only see host cell-surface components on the outside of the protrusion, as the bacteria is located inside the cell membrane and is not exposed to the neighboring cell. Another problem is what signals the extent to which the protrusion is internalized into the adjacent cell? Both organisms must be in an acidic environment to dissolve their surrounding vacuole. Thus, the protrusion must be internalized within an acidified membrane-bound inclusion before the organism can repeat the process.
Fig. 1. Generic model of how intracellular pathogens may enter eukaryotic systems, but not proven in any system). The invading parasite encodes integrin, or bind host proteins which interact with integrins. When the (perhaps calcium or phosphor-ylation of proteins) is generated in the host talin) which then form a bridge between the integrin and actin filaments. by a-actinin. The parasite is then internalized into a membrane-bound phagocytosis.
cells. (This model has been compiled from data from various a molecule on its surface which can either bind directly to an pathogen contacts the host cell integrins, an unknown signal cytoplasm. This signal modifies cytoplasmic proteins (possibly The signal also causes large amounts of crosslinking of filaments inclusion by an actin/myosin-dependent mechanism similar to
Cell adhesion
Future
prospects
and invasion
mechanisms
in microbial
pathogenesis
Finlay
A review of RGD-mediated protozoan interactions with host cells.
The ability of a pathogen to interact with host surfaces is essential for its virulence. If one wishes to inhibit a pathogenic organism from colonizing and establishing an infection, blocking initial attachment is a logical place to start. As many pathogens adhere to host matrices and cell surfaces by similar mechanisms, therapeutic approaches may be considered in the future. Such approaches might include the use of synthetic peptides or antibodies directed against binding domains of the parasite ligand, or by interfering with the mechanisms used to internalize intracellular parasites. In the immediate future, the interaction of pathogens with host cells will expand our knowledge of cell-cell and cell-matrix interactions. These parasites provide new tools to study integrin-ligand interactions. It will be interesting to determine how FHA and gp63 interact with CR3, and compare these interactions with its ‘natural’ l&and C3bi. These interactions can also be used to identify the signal which links the integrin with the cytoskeleton. Different parasites probably use different mechanisms. For example, non-viable Yersinia are taken up by epithelial cells (presumably via an invasin-mediated pathway) yet Salmonella species have to be metabolically active for invasion to occur (Finlay el al., Science 1989,243:940-9431, and do not use the same receptor as invasin. Additionally, it seems that intracellular parasites use several pathways for internalization. At least three pathways appear to be used by E pseudotubercuios.& to enter epithelial cells (Isberg, Mol Biol Med 1990, 7:7+82). As demonstrated with Salmonella, intracellular pathogens will also be useful for determining which cytoplasmic molecules in the host ceU form the bridge between membrane-bound receptors and the host cytoskeleton. It will be interesting to compare these molecules and signals with those that are involved in cell-cell and cell-matrix contact formation. The study of host-parasite interactions is entering an exciting phase. Workers in the previously diverse fields of ceU biology and microbial pathogenesis are now finding common interests, and these findings provide important new insights applicable to both fields. Perhaps in the future it will be more acceptable for ceU biologists to ‘contaminate’ their ceU cultures, and for microbiologists to study ceU biology.
RUSSELDG, TAIAMAS-ROHANA P, ZELECHOW~KIJ: Antibodies raised against synthetic peptides from the Arg-GIy-Asp-containing region of the Lefshmanfu surface protein gp63 cross-react with human C3 and Interfere with gp63mediated binding to macrophages. Infecf lmmun 1989, 57:630-632. This paper demonstrates that a parasitic &and which interacts with a host receptor may be similar in structure to the ‘natural’ &and, thereby acquiring its binding ability. 3. l
REMAN DA, DOMENIGHW M, TUOMANENE, RAPPUOUR, FAU(OW S: Fikunentous hemagglutinin of Bordetellu pwturcfs Nucleotide sequence and crucial role in adherence. Prcx Nutl Acud Sci USA 1989, 86:2637-2641. The FHA nucleotide sequence predicts the presence of an RGD sequence; deletion of this sequence eliminated B. perrus& adherence to respitatoly ciliated epithelial cells. 4. l
REMAN DA TUOMANEN E, FALKOW S, G~LENEOCK DT, SAUKKONENK, WRIGHT S: Recognition of a bacterial adhesin by an eukaryotic integrin: CR3 (aMp2, CDllb/CD18) on human macrophages binds filamentous hemagglutinin of Bordetella petiussis. Cell 1990, 61:1375-1382. This paper demonstrates for the first time that bacterial pathogens can produce molecules (in this case FHA) containing RGD sequences that bind directIy to integrins. 5.
l e
SCHLESINGER l.5, BELL~NGER-KAW~ CG, PAYNENR, Ho~wm MA Phagocytosis of Mycobactetium hrbernclosis is mediated by human monocyte complement receptors and complement component C3. J Immunol 1990, 144:2771-2780. Human monocyte CR1 and CR3 complement receptors mediate uptake of M. tuber&& and C3 component is attached to the bacterial surface, mediating bacterial uptake. 6. l
JM, DAVISG, JEXR AM, FORTXCP, Yosr SC, MARLORCW, KAMARCKME, MCCLEUANDk The major human rhinovirus receptor is ICAM-1. Cell 1989, 56:83w7. One of three simultaneous papers that identify ICAM- as the rhinovirus receptor. See [8,9] also. 7.
GREW
l
DE, MERLUZI VJ, ROTHLEINR, MARLINSD, SPRINGER TA: A ceU adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses. Cell 1989, 56:84-53. STAUNI’ON
8. l
see 171.
TOMASSUVI JE, GRAH~~~D. DEWY CM, L~NEBERGER DW, ROOKEY JA, COU%NO RJ: cDNA cloning reveals that the major group rhinovirus receptor on HeLa ceUs is intercellular adhesion molecule 1. Proc Nat1 Acad Sci USA 1989, 86:4907-4911.
9. l
see 171.
10. l
A
MARLIN SD, STALIN~‘ONDE, SPIUNGERTA, S~TOWA C, SOMMERGRUBER W. MEKLUW VJ: A soluble form of intercellular adhesion molecule-l inhibits rhinovirus infection. Nuture 1990, 344:7C-72. secreted form of ICAMl prevents rhinovirus infection.
11.
AR, SIMONS DL TANSF~J, NRWBOIDCl, MARSHK: Intercellular adhesion molecule-l is an endothelial ceU adhesion receptor for Plasmodium falci~atum. Nature 1989, 341:57-59. Transfected cells were used to demonstrate that ICAM-1. in addition to CD36, is a receptor for P. falcrparum. As [CAM-l is widely distributed, it may be one of the receptors used during malarial infections. BERENDT
l
Annotated reading l l e
references
and recommended
Of interest Of outstanding interest
FINIAY BB. FALKOW S: Common themes in microbial l pathogenicity. Micrcbiol Rev 1989, 53:21&230. A review of the principles of bacterial pathogenesis. Recently character. ized factors involved in pathogenesis are compared between pathogens, and bacterial adherence and imasion mechanisms are presented.
ISBERGRR Mammalian ceU adhesion functions and cellular penetration of enteropathogenic YersinIa species. Mok Microbiol 1989, 3:1449-1453. A review of the products and mechanisms used for entry of Yetinia species into cultured cells. A discussion of invasin-integrin interactions is included.
2.
13.
1.
l
OuAtsst 4 CAPRoN L Some aspects of protozoan parasitehost cell interactions with special reference to RGD-mediated recognition process. Microbial Pufbogen 1989, 6:1-5.
12. l
l e
ISBERGRR, LEONGJM: Multiple & chain Integrins are recep tars for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 1990, 60:861-871.
819
820
Cell-to-cell
contact
and extracellular
matrix
This is the first report of direct binding between a pathogen ligand (invasin) and a non-B2 integrin. It clearly demonstrates that imasin binds to several 81 integrins, and that antibodies to these integrins block bacterial adherence and uptake. h-twin does not contain an RGD se. quence.
14.
ML, MOUIUER J, D’HoL~~~.E H. COQLIIS-RONDON M, SANSONEITI PJ: Identification of ica.4, a plasmid locus of Sbigeffa f7exner-i that governs bacterial intra- and intercellular spread through interaction with F-actin. from Nat/ Acad Sci USA 1989, 863867-3871. This paper details the actin rearrangement associated with intercellular. movement of S. JIexneti in cultured epithelial cells using a fluorescent l
BERNARDINI
actin cess.
label, and identif.ies
a bacterial
gene which
is involved
in this pro-
TIINW LG, PORTNOY DA Actin tilaments and the growth, movement and spread of the intracelhtlar bacterial parasite Listeria monocytogenes J Cell Rio1 1989, 109:1597-1608. A transmission electron microscofx study of the morphological events associated with Ltileti invasion and intercellular movement within cultured macrophages. Actin filaments were decorated, resulting in striking micrographs of the ‘comet’ composed of the bacteria and actin ftlamentc. 15. a