Role Of Fibronectin-binding Mscramms In Bacterial

Matrix Biology 18 Ž1999. 211᎐223

Mini-review

Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells Danny Joha,1, Elisabeth R. Wanna , Bernd Kreikemeyer a,2 , Pietro Spezialeb, Magnus Hook ¨¨ a,U a

Center for Extracellular Matrix Biology, Albert B. Alkek Institute of Biosciences and Technology, Texas A & M Uni¨ ersity System Health Science Center, 2121 West Holcombe Boule¨ ard, Houston, TX 77030, USA b Department of Biochemistry, Uni¨ ersity of Pa¨ ia, 27100 Pa¨ ia, Italy Received 11 February 1999; accepted 16 February 1999

Abstract Most bacterial infections are initiated by the adherence of microorganisms to host tissues. This process involves the interaction of specific bacterial surface structures, called adhesins, with host components. In this review, we discuss a group of microbial adhesins known as Microbial Surface Components Recognizing Adhesive Matrix Molecules ŽMSCRAMMs. which recognize and bind FN. The interaction of bacteria with FN is believed to contribute significantly to the virulence of a number of microorganisms, including staphylococci and streptococci. Several FN-binding MSCRAMMs of staphylococci and streptococci exhibit a similar structural organization and mechanism of ligand recognition. The ligand-binding domain consists of tandem repeats of a ; 45 amino acid long unit which bind to the 29-kDa N-terminal region of FN. The binding mechanism is unusual in that the repeat units are unstructured and appear to undergo a conformational change upon ligand binding. Apart from supporting bacterial adherence, FN is also involved in bacterial entry into non-phagocytic mammalian cells. A sandwich model has been proposed in which FN forms a molecular bridge between MSCRAMMs on the bacterial surface and integrins on the host cell. However, the precise mechanism of bacterial invasion and the roles of FN and integrins in this process have yet to be fully elucidated. 䊚 1999 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Extracellular matrix; FN; MSCRAMM; Bacterial adherence; Bacterial invasion

Abbre¨ iations: ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; BHK, baby hamster kidney; FI, type I module in FN; FN-N29, the N-terminal 29-kDa fragment of FN; GST, glutathione-S-transferase; MSCRAMM, microbial surface component recognizing adhesive matrix molecules U Corresponding author. Tel.: q1-713-677-7551; fax: q1-713-677-7576. E-mail address: [email protected] ŽM. Hook ¨¨ . 1 Current address: Xenogen Corporation, 860 Atlantic Avenue, Alameda, CA 94501, USA 2 Current address: Department of Medical Microbiology and Immunology, University of Ulm, Robert-Koch-Strabe 8, D-89081, Ulm, Germany 0945-053Xr99r$ - see front matter 䊚 1999 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 9 9 . 0 0 0 2 5 - 6

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1. Introduction

The process of microbial infection involves a complex series of events that can result in host tissue malfunction or destruction. The adherence of the microorganisms to host tissues represents a critical first step in this process. Non-adherent bacteria can be readily eliminated from the body by such cleansing mechanisms as peristalsis and excretion. Bacteria that adhere to host tissues may remain extracellular or may be internalized into an intracellular compartment. Cellular invasion enables the bacteria to escape host defense mechanisms, persist in the host and invade tissues surrounding the initial site of colonization. As a result of its critical role in the infection process, bacterial adherence to host tissues represents a potential target for the development of new antimicrobial agents. Agents that can block adherence, such as specific antibodies, may prove to be effective in combating infectious disease. For example, active immunization of mice with a recombinant fragment of the collagen-binding protein from Staphylococcus aureus, or passive immunization with antibodies against this protein, protect against sepsis-induced death ŽNilsson et al., 1998.. Also, mice actively immunized with a recombinant decorin-binding protein from Borrelia burgdorferi are immune to challenge in a model of lyme borreliosis ŽHanson et al., 1998.. The adherence of bacteria involves bacterial surface components, called adhesins, that recognize and bind to host extracellular matrix ŽECM. and cell surface molecules. Host ECM components that are known to support bacterial adherence include fibronectin ŽFN., collagen, fibrinogenrfibrin, elastin, vitronectin, laminin, as well as decorin and heparan sulfate-containing proteoglycans. The subfamily of bacterial adhesins that bind to ECM molecules are collectively known as Microbial Surface Components Recognizing Adhesive Matrix Molecules ŽMSCRAMMs. ŽPatti and Hook, ¨¨ 1994; Patti et al., 1994.. Although a number of microbes have been shown to bind host ECM components, the MSCRAMMs responsible for these interactions have, in most cases, not been identified or characterized. However, a group of related FN-binding MSCRAMMs from gram-positive bacteria has been analyzed in some detail. In this review, we will discuss the bacterial MSCRAMMs that interact with FN. FN is a 440-kDa mosaic glycoprotein which is present in soluble and matrix forms in various body fluids and tissues. It is composed of three types of modules, presented in two similar disulfide-linked subunits, and contains discrete binding sites for a variety of other extracellular

molecules, including fibrin, heparin and collagen, as well as for a number of integrins, including ␣4b1, ␣ 5b1, ␣ IIbb3 and ␣ vb3.

2. FN-binding MSCRAMMs from staphylococci and streptococci Staphylococci and streptococci are clinically important gram-positive bacteria that are capable of causing a wide variety of diseases in humans and animals. The specific binding of FN to Staphylococcus aureus was first reported by Kuusela Ž1978.. Subsequently, two FN-binding proteins ŽFnbpA and B. and the corresponding genes were isolated and characterized ŽFlock et al., 1987; Froman et al., 1987; Signas ¨ ¨ et al., 1989; Jonsson et al., 1991.. Many staphylococci and ¨ streptococci have since been shown to bind FN. At least a dozen FN-binding MSCRAMMs have been identified and their corresponding genes have been sequenced ŽTable 1.. These MSCRAMMs exhibit structural features typical of other cell wall-anchored proteins of gram-positive bacteria. At the N-terminus is a putative signal sequence involved in transport of the proteins through the cytoplasmic membrane. The C-terminus is composed of: Ži. a conserved LPXTG motif which is required for accurate sorting and anchoring of the proteins to the cell wall; Žii. a hydrophobic membrane-spanning region; and Žiii. a tail of positively-charged residues which remain in the cytoplasm. Once the protein has been transported to the cell surface, the LPXTG motif is cleaved between the Thr and Gly residues. The carboxyl group of the Thr residue is then linked to a free amino group of a branch peptide within the peptidoglycan cell wall ŽSchneewind et al., 1995; Ton-Hat et al., 1997.. The enzyme responsible for catalyzing these reactions has not been identified but has been named ‘sortase’. A proline-rich stretch of residues, thought to span the peptidoglycan layer of the cell wall, is commonly found on the N-terminal side of the LPXTG motif in these proteins. The prototype FN-binding MSCRAMM, FnbpA Žalso called FnbA. from S. aureus, has a molecular mass of approximately 100 kDa. FN binds primarily to a region adjacent to the proline-rich wall-spanning region of the MSCRAMM ŽFig. 1A.. This binding region is composed of a ; 45 amino acid long unit which is tandemly repeated three times ŽD1, D2, D3 units., followed by a single incomplete repeat unit ŽD4 unit.. A fifth unit ŽDu. is located approximately 100 amino acid residues N-terminal of D1. FN can bind to each of the D units, as demonstrated using synthetic peptide mimics of the sequences. Amino

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213

Table 1 FN-binding MSCRAMMs from staphylococci and streptococci. Species

Protein

References

Staphylococcus spp. S. aureus

FnbpA & FnbpB

Signas et al. Ž1991. ¨ et al. Ž1989., Jonsson ¨

Streptococcus spp. S. saprophyticus

Hemaglutinin

Hell et al. Ž1998.

SfbIrProtein F1 SfbII Protein F2 Opacity factor ŽSof 22. Glyderaldehyde-3phosphate dehydrogenase FBP54 M1

Hanski Caparon Ž1992., Talay et al. Ž1993. Kreikemeyer et al. Ž1995. Jaffe et al. Ž1996. Rakonjac et al. Ž1995. Pancholi and Fischetti Ž1992.

S. dysgalactiae

FnbA &FnbB

Lindgren et al. Ž1992.

S. equi

FNZ

Lindmark et al. Ž1996.

S. equisimilis

FnB

Lindgren et al. Ž1994.

S. pyogenes

Courtney et al. Ž1994. Cue et al. Ž1998.

S. mitis

Sugano et al. Ž1997.

S. gordonii

McNab et al. Ž1994, 1996.

S. pneumoniae

van der Flier et al. Ž1995.

S. agalactiae

Zabel et al. Ž1996.

S. milleri

Willcox et al. Ž1995.

S. anginosus

Willcox et al. Ž1995.

Group G streptococci

GfbA

acid sequence comparison of the different D units shows that the D1᎐D4 units are highly conserved whereas the Du unit is more divergent ŽFig. 1A.. The overall arrangement of the ligand-binding region in FnbpA is also found in the staphylococcal protein FnbpB and the streptococcal proteins SfbIrprotein F1, SfbII, Sof22, protein F2, FnbA, FnbB, and FNZ, albeit with slight variations ŽFig. 1A.. Furthermore, the sequences of the tandemly repeated FN-binding units are quite highly conserved among these proteins ŽFig. 1B. ŽHanski and Caparon, 1992; McGavin et al., 1993; Lindgren et al., 1994; Talay et al., 1994; Kreikemeyer et al., 1995; Jaffe et al., 1996; Ozeri et al., 1996.. In a comparative study, we analyzed the inhibitory activities of recombinant proteins derived from the ligand-binding sites of FnbpA of S. aureus, FnbA and FnbB of Streptococcus dysgalactiae, and SfbI of Streptococcus pyogenes ŽJoh et al., 1994.. Each of the polyhistidine-tagged proteins inhibited the interaction of FN with S. aureus, S. pyogenes and S. dysgalactiae. The cross-inhibitory activities of these recombinant FN-binding units suggest that the dif-

Kline et al. Ž1996.

ferent MSCRAMMs may operate by a similar mechanism and may bind to similar or overlapping regions in FN. A kinetic analysis of the interaction between the recombinant binding domains and intact FN, using BIAcore analysis, indicated that the K d ranged from 3 to 16 nM ŽHouse-Pompeo et al., 1996.. In addition, extensive analysis of the residues involved in ligand binding suggested that a conserved core sequence, EDŽTrS.Ž9 or 10 Xs.GGŽ3 or 4 Xs.ŽIrV.DF, is important for the ligand-binding activity of these units ŽMcGavin et al., 1991, 1993.. In FNZ, the FNbinding MSCRAMM of Streptococcus equi subspp. zooepidemicus, a strong ligand-binding activity was found associated with the sequence LAGESGET which is located between the first ŽR1. and second ŽR2. repeat units of the MSCRAMM ŽLindmark et al., 1996.. This sequence is also present in the FNbinding sequence ŽUR or UFBD. located upstream of the tandem repeats in protein F1 of S. pyogenes Žsee below. ŽOzeri et al., 1996.. The primary binding site in FN for FnbpA of S. aureus is located in the 29-kDa N-terminal proteolytic

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Fig. 1. ŽA. Domain organization of FnbpA from Staphylococcus aureus, the prototype of the FN-binding MSCRAMMs from staphylococci and streptococci. S, signal peptide; A, non-repetitive region; ␦, two almost identical repeats of 30 amino acid residues with unknown function; Du-D4, FN-binding units; W, cell wall-spanning region; M, cytoplasmic membrane-spanning region; C, positively-charged intracellular residues. The cell wall-anchoring LPXTG motif is indicated between regions W and M. ŽB. Alignment of the amino acid sequences of the ligand-binding units from FnbpA of S. aureus, and FnbA and FnbB of Streptococcus dysgalactiae. Conserved residues that have been implicated as being important for FN binding are shown in bold face.

fragment of FN ŽFN-N29. ŽMosher and Proctor, 1980.. This fragment is composed of five type I modules, designated 1FI, 2FI, 3FI, 4FI and 5FI, each of which is folded tightly through hydrophobic packing and two disulfide bonds ŽPotts and Campbell, 1994.. The repetitive nature of the binding sites in both FN and the MSCRAMM represents a two-dimensional complexity in the interaction of these proteins. This raises several intriguing questions concerning the binding mechanism that investigators have attempted to answer. Experiments with recombinant FN fragments, expressed in baculovirus-infected insect cells, suggested that deletion of any of the five FI modules abolished the binding of the recombinant proteins to S. aureus ŽSottile et al., 1991.. Analysis of the interaction of FI modules with FnbpA fragments, using fluorescence polarization and affinity chromatography, revealed that the 4FI᎐5FI pair was the dominant binding site for the D3 unit of this protein ŽHuff et al., 1994.. We recently confirmed these findings and further analyzed the binding patterns of the other D units ŽJoh et al., 1998.. Each of the full-length D units ŽDu, D1, D2, and D3., expressed as GST fusion

proteins, recognized the 4FI᎐5FI pair. Additional binding to 2FI᎐3FI was observed for recombinant D1, whereas recombinant D4 only bound to 2FI᎐3FI. Analysis of the FN-binding units from other MSCRAMMs revealed different binding patterns ŽJoh et al., 1998.. The B3 unit of S. dysgalactiae FnbB, for example, bound to 1FI᎐2FI and 2FI᎐3FI, whereas the A2 unit of S. dysgalactiae FnbA bound only to 4FI᎐5FI ŽJoh et al., 1998.. According to Huff et al. Ž1994., the stoichiometry of the interaction of FN-N29 with a recombinant protein containing the D1᎐D3 units of FnbpA was 1.9:1. However, the data regarding the specificity of the individual FI modules and the ligand-binding units should be interpreted with caution. These experiments were performed using short recombinant proteins or synthetic peptides corresponding to the MSCRAMM ligand-binding units or the FI module pairs. How the units and modules interact in the context of the full-length proteins may not be directly inferred from experiments with truncated recombinant proteins. Other regions of FN may also interact with MSCRAMMs ŽKuusela et al., 1984, 1985; Bozzini et

D. Joh et al. r Matrix Biology 18 (1999) 211᎐223

al., 1992; Sakata et al., 1994.. Protein F1 from S.pyogenes and GfbA from group G streptococci, for example, possess a FN-binding unit designated UR in addition to the repeat unit region ŽOzeri et al., 1996.. The binding site in FN for UR is located in the 40-kDa collagen-binding domain, which is adjacent to the N-terminal 29-kDa region. The C-terminal heparin-binding domain of FN may also bind to the streptococcal and staphylococcal MSCRAMMs, although comparatively weakly ŽKuusela et al., 1984; Bozzini et al., 1992.. The binding site for this domain appears to lie outside the repeat units in these proteins ŽBozzini et al., 1992.. It is interesting to note that the collagen- and heparin-binding domains of FN also contain FI module pairs. However, the MSCRAMM binding sites in these domains have not yet been localized.

3. Mechanism of FN-MSCRAMM interaction: insights from immunological studies Analysis of the interactions of polyclonal and monoclonal antibodies with the FN-binding MSCRAMMs of staphylococci and streptococci has provided important insights into the mechanism of ligand-MSCRAMM binding. Interestingly, it has been noted by several investigators that antibodies raised against the FN-binding units of FnbpA of S. aureus do not effectively inhibit the binding of FN to the MSCRAMM ŽSchennings et al., 1993; Mamo et al., 1994, 1995.. In a recent study, sera were collected from more than 30 individuals who had a history of S. aureus infection. The IgG fractions were isolated and analyzed for reactivity with FnbpA ŽCasolini et al., 1998.. Essentially all of the IgG preparations reacted with the MSCRAMM, consistent with the presence of the gene encoding FnbpA in ) 90% of S. aureus clinical isolates ŽMinhas et al., 1995.. Epitope mapping, using recombinant truncates of FnbpA, revealed that the different IgG preparations reacted preferentially with the ligand-binding site ŽDu-D4., suggesting that this is the immunodominant region of the MSCRAMM. However, the IgG preparations did not effectively inhibit the binding of FN to the MSCRAMM ŽCasolini et al., 1998.. Similarly, we have found that antibodies raised against recombinant or synthetic peptide forms of the FN-binding units failed to display any significant inhibitory activity. In fact, we observed several monoclonal antibodies ŽmAbs., raised against FnbpA of S. aureus and FnbA of S.dysgalactiae, which actually enhanced the ligand-binding activities of the MSCRAMMs ŽSpeziale et al., 1996, manuscript in preparation.. For example, one mAb, 3A10, was incapable of binding FnbA by itself and only bound to the MSCRAMM if soluble FN was

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included in the assay. This antibody was shown to recognize an epitope that was formed in the MSCRAMM upon binding to FN. Subsequent epitope mapping experiments, using truncates of FnbA, revealed that the epitope for 3A10 resided within Au, one of the FN-binding units of the MSCRAMM ŽFig. 1.. Furthermore, 3A10 enhanced, rather than inhibited, the interaction between FN and the Au unit. The unexpected behavior of these antibodies has provided invaluable clues as to how the MSCRAMMs interact with FN. It appears that the FN-binding repeat units of the MSCRAMMs have a largely unorganized structure and only assume an ordered structure upon binding to FN. This hypothesis is supported by the results of biophysical studies of the MSCRAMM᎐FN interaction ŽHouse-Pompeo et al., 1996; Penkett et al., 1998.. For example, circular dichroism spectroscopy of recombinant proteins corresponding to the ligand-binding domains of FnbpA, FnbA, FnbB and SfbI has demonstrated that these proteins lack a secondary structure and undergo a structural change upon binding FN ŽHouse-Pompeo et al., 1996.. Furthermore, gel permeation chromatography indicated that the complex of recombinant D1᎐D3 and FN-N29 was more compact than free D1᎐D3 Žunpublished observations.. MAbs that react with ligand-induced binding sites ŽLIBS., such as 3A10, appear to recognize a conformation of the MSCRAMM induced upon binding to FN. The ability of 3A10 to enhance the MSCRAMM᎐ligand interaction may be the consequence of the mAb stabilizing the MSCRAMM᎐FN complex. The binding mechanism of 3A10 is reminiscent of some anti-␣ II b ␤ 3 integrin mAbs which have been reported to bind the integrin only when it is bound to fibrinogen and which also enhance fibrinogen binding ŽFrelinger et al., 1990, 1991.. Thus, these anti-MSCRAMM and anti-integrin antibodies both appear to bind to cell surface ‘receptors’ which undergo conformational changes upon ligand binding. The presence of anti-LIBS antibodies in immunized animals or infected humans is, perhaps, not surprising. When a FN-binding MSCRAMM is used as an immunogen, it is likely that the protein quickly interacts with plasma FN before the immune system has time to recognize the unoccupied form of the binding site in the MSCRAMM. However, Sun et al. Ž1997. by immunizing with short subsegments of the FN-binding units of FnbpA, were able to generate antibodies that blocked the binding of the individual binding units to FN . The rationale behind this approach was that, as the FnbpA-derived peptides were too short to bind FN, antibodies raised against them should recognize the binding units in their unoccupied form in the native protein and thus block the MSCRAMM᎐FN interaction. The success of this approach is consistent

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with the idea that rapid binding of the MSCRAMM to plasma FN hampers the formation of inhibitory antibodies specific for the unoccupied binding site of the MSCRAMM. Inhibitory antibodies have also been successfully raised in rabbits against SfbI of S. pyogenes ŽMolinari et al., 1997.. In this study, recombinant SfbI fragments, containing the FN-binding repeat units and also the upstream ‘FN-binding spacer 2’ region, were used as immunogens. The latter region is equivalent to the UR unit Župstream FN-binding sequence. of protein F1 of S. pyogenes, which appears to be the dominant FN-binding unit in this MSCRAMM ŽOzeri et al., 1996.. As the UR unit binds to the 40-kDa collagen-binding domain of FN, the ligand-induced-conformation mechanism described above may not apply to either the SfbI-FN or protein F1᎐FN interactions. Thus, it is possible that the inhibitory activities of the antibodies described by Molinari et al. may be due to antibodies specific for the upstream FN-binding spacer 2 region of SfbI.

4. FN-binding MSCRAMMs from other bacteria A variety of other important pathogenic bacteria, including Mycobacterium spp., Escherichia coli, and Borrelia burgdorferi, are also capable of binding specifically to FN. The MSCRAMMs involved in these interactions have been characterized to varying degrees ŽTable 2.. As a detailed account of all these FN-binding organisms is beyond the scope of this article, we have chosen to discuss only the interactions that have been analyzed in some detail. Mycobacterium bo¨ is BCG and Mycobacterium paratuberculosis bind soluble FN in a saturable, doseand time-dependent fashion ŽAslanzadeh et al., 1989; Valentin-Weigand and Moriarty, 1992.. The number of FN binding sites was determined to be 8000᎐15 000 and 1600 per bacterium for M. bo¨ is BCG and M. paratuberculosis, respectively. In addition, Scatchard analysis indicated the presence of a homogeneous population of FN-binding components, which bound the ligand with a K d of 1.25= 10y9 M in the case of M. paratuberculosis. Several mycobacterial species secrete a set of FN-binding proteins Ž30᎐31 kDa. into the culture medium in ¨ itro. These proteins are known as the Antigen ŽAg. 85 complex Žrelated to MPB or MPT51. ŽAbou-Zeid et al., 1988, 1991; Pessolani and Brennan, 1992; Thole et al., 1992; Ohara et al., 1995; Naito et al., 1998.. However, as these proteins are primarily extracellular, it is unclear whether they mediate the adherence of mycobacteria to FN. The Ag85 complex is composed of three proteins, Ag85A ŽMPT44., B ŽMPT59. and C ŽMPT45., which are genetically distinct but show extensive immunological cross-reactivity and amino acid sequence similarity,

both within a particular species and between different species. Recently, two separate FN-binding sites have been identified on the M. kansasii Ag85B homologue which appear to be conserved in the Ag85 proteins from this and other mycobacterial species ŽNaito et al., 1998.. Studies with synthetic peptides defined 11 residues as a minimum binding unit on Ag85B. This peptide could inhibit the binding of FN to Ag85B and to other Ag85 proteins from different species ŽNaito et al., 1998.. A sequence composed of six residues ŽFEWYYQ., which has no homology to other known bacterial FN-binding proteins, was shown to be critical for FN binding. The binding site in FN for the Ag85 complex has not been defined. The interaction of recombinant Ag85B with FN could be specifically inhibited by gelatin but not by heparin, tentatively localizing a binding site to the 40-kDa collagen-binding domain of FN ŽPeake et al., 1993.. Several other mycobacterial FN-binding proteins, distinct from the Ag85 complex, have been identified. A 50-kDa FN-binding protein, FAP, has been purified from M. ¨ accae culture supernatant ŽRatliff et al., 1993.. Polyclonal and monoclonal antibodies to this protein recognized a cell wall component in M. ¨ accae and several other mycobacterial species, including M. bo¨ is BCG, M. tuberculosis, M. kansasii and M. a¨ ium ŽRatliff et al., 1993.. Antibodies to FAP inhibited the binding of several mycobacteria to FN and to mammalian cells ŽKuroda et al., 1993.. Thus, FAP is a putative FN-binding MSCRAMM on mycobacteria. FAP homologues have also been identified in M. tuberculosis ŽFAP-TB. ŽAbou-Zeid et al., 1991., M. leprae ŽFAP-L. ŽSchorey et al., 1995. and M. a¨ ium ŽFAP-A. ŽSchorey et al., 1996.. Purified recombinant FAP-L bound to plasma FN and the C-terminal heparin-binding chymotryptic fragment of FN. A study with overlapping synthetic peptides revealed two distinct FN-binding sites conserved among FAPs from different mycobacterial species ŽSchorey et al., 1996.. These units possess little sequence similarity with FN-binding MSCRAMMs from other bacteria. A large number of Escherichia coli strains from animal and human sources have been reported to adhere to FN in solid phase assays and to attach to fibroblasts via FN ŽFroman et al., 1984; Faris et al., ¨ 1988; Ljungh et al., 1990; Yu et al., 1995.. The binding of this gram-negative bacterium to FN appears to be mediated by several types of fimbriae Žpili. on the bacterial cell surface. Fimbriae are hair-like structures that are composed of repeating major subunit proteins, often arranged in an alpha helical array ŽSmyth et al., 1996.. Minor subunit proteins are present at the tip and sometimes along the length of these organelles and often mediate the binding activities of these structures. Two distinct sites in FN are targeted by the E. coli receptors, FN-N29 and the

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Table 2 FN-binding MSCRAMMs from other bacterial species Bacterial Species

Protein

References

Mycobacterium spp.

Ag85A ŽMPT44.

Borremans et al. Ž1989., DeWit et al. Ž1990.

Ag85B ŽMPT59.

Matsuo et al. Ž1988., Thole et al. Ž1992.

Ag85C ŽMPT45.

Abou-Zeid et al. Ž1991., Peake et al. Ž1993.

MPT51 ŽMPB.

Ohara et al. Ž1995, 1997.

FAP ŽFAP-TB, FAP-L, FAP-A.

Abou-Zeid et al. Ž1991., Kuroda et al. Ž1993., Schorey et al. Ž1995, 1996.

Escherichia coli

P fimbriae ŽFsoE, FsoF. Type I fimbriae Curli

Westerlund et al. Ž1989, 1991. Sokurenko et al. Ž1992. Olsen et al. Ž1989.

Campylobacter jejuni

CadF Flagellin

Konkel et al. Ž1997. Moser et al. Ž1997.

Salmonella enteritidis

Fimbriae

Collinson et al. Ž1993.

Yersinia spp.

YadA

Tertti et al. Ž1992., Schulze-Koops et al. Ž1993.

Porphyromonas gingi¨ alis

150-kDa protein Fimbrillin

Lantz et al. Ž1991. Murakami et al. Ž1996.

Aeromonas salmonicida

A-protein

Doig et al. Ž1992.

Aeromonas hydrophila

Ascencio et al. Ž1991.

Haemophilus ducreyi

Abeck et al. Ž1992.

Propionibacterium acnes

80-kDa protein

Fusobacterium nucleatum Neisseria gonorrhoeae

Yu et al. Ž1997. Winkler et al. Ž1987., Babu et al. Ž1995.

OpaA

Neisseria meningitidis

van Putten et al. Ž1998. Eberhard et al. Ž1998.

Mycoplasma penetrans

65-kDa protein

Giron et al. Ž1996.

Treponema pallidum

P1 and P2 proteins

Peterson et al. Ž1987.

Treponema denticola

53- and 72-kDa proteins and 38-kDa axial flagellar protein

Umemoto et al. Ž1993.

Borrelia burgdorferi

Fn-BA BBK32

Probert and Johnson Ž1998. Grab et al. Ž1998.

Borrelia garinii

147-kDa protein

Kopp et al. Ž1995.

120-kDa C-terminal region ŽFroman et al., 1984; Faris ¨ et al., 1988; Westerlund et al., 1989; Visai et al., 1991.. The P-fimbriae ŽPap pili. of uropathogenic E. coli mediate adherence to immobilized intact FN and also to FN-N29 and the 120-kDa fragment of FN, but do not bind soluble FN ŽWesterlund et al., 1989.. The FsoE and FsoF Žalso called PapE and PapF, respectively. minor subunit proteins of the P-fimbriae have been implicated in this interaction ŽWesterlund et al.,

1991.. Although it has not been demonstrated that these subunit proteins bind specifically to FN-N29, it is noteworthy that they do contain sequences similar to the FN-binding repeat units of the staphylococcal and streptococcal FN-binding MSCRAMMs ŽWesterlund and Korhonen, 1993.. Campylobacter jejuni, another gram-negative pathogenic bacterium which causes gastrointestinal diseases such as diarrhea, can adhere to ECM pro-

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teins including FN ŽKuusela et al., 1989; Moser and Schroder, 1995.. Proteinase K treatment of the bacteria abolished FN binding and demonstrated that the binding components on the cell surface are proteinaceous in nature ŽMoser and Schroder, 1995, 1997.. A major outer membrane protein, CadF, has been identified as a FN-binding protein. The corresponding gene has been cloned, sequenced and identified in all isolates tested so far ŽKonkel et al., 1997.. Another major outer membrane protein, flagellin, has also been shown to bind FN and intestinal epithelial cells ŽMoser et al., 1997.. The sites in the FN molecule targeted by these putative MSCRAMMs have not been identified. The adherence of Porphyromonas gingi¨ alis to ECM components, such as FN, appears to play a role in the pathogenesis of periodontal disease caused by this organism ŽWinkler et al., 1987.. The binding of soluble FN to P. gingi¨ alis was demonstrated to be timedependent, specific, reversible, and saturable. The K d for the interaction was estimated to be in the order of 10y7 M, with approximately 3500 binding sites per cell ŽLantz et al., 1991.. Lantz et al. identified a FN-binding protein of 150-kDa in SDS-solubilized P. gingi¨ alis. Fimbrillin, a major component of the fimbriae of P. gingi¨ alis, has also been identified to be a FN-binding MSCRAMM ŽMurakami et al., 1996.. The fimbriae appeared to interact with the N-terminal heparin-binding and C-terminal cell-binding domains in FN ŽMurakami et al., 1996.. In addition, by using a series of synthetic 20 mer fimbrillin peptides, two distant regions in this protein have been implicated in binding FN ŽSojar et al., 1995.. Interestingly, Kontani et al. Ž1997. recently demonstrated that an argininespecific protease expressed by P. gingi¨ alis enhanced the binding of purified fimbriae to immobilized FN using BIAcore analysis . It was proposed that the P. gingi¨ alis protease exposes a cryptic binding site Žcryptitope. in FN, allowing the bacteria to adhere more efficiently to FN-coated surfaces. The syphilis-causing spirochete, Treponema pallidum, has been reported to bind to cultured mammalian cells via the C-terminal cell-binding domain of FN ŽThomas et al., 1985.. The binding of intact FN and its cell-binding domain to the spirochetes was saturable, with an estimated K d of 10y7 M. Intact FN and FN fragments containing the cell-binding peptide, RGDS, also supported the tip-mediated adhesion of the human oral pathogen T. denticola ŽDawson and Ellen, 1990.. These spirochetes express surface proteins of 53 and 72 kDa as well as a 38-kDa axial flagellar protein that have been shown to bind to FN ŽUmemoto et al., 1993.. Several reports have indicated that FN may play a role in the interaction of Borrelia burgdorferi and Borrelia garinii, both causative agents of lyme disease, with cells and the ECM.

Szczepanski et al. Ž1990. demonstrated that anti-FN antiserum could inhibit the binding of B. burgdorferi to a subendothelial matrix.. Recently, a surface-expressed protein ŽBBK32. with FN-binding activity was purified from B. burgdorferi and the corresponding gene was cloned ŽProbert and Johnson, 1998.. The binding site for BBK32 was localized to the gelatinrcollagen-binding domain in FN. The ability of recombinant BBK32 to bind FN and inhibit the attachment of B. burgdorferi to FN was demonstrated ŽProbert and Johnson, 1998.. A 52-kDa protein ŽFnBA. has also been identified as a FN-binding component on the surface of B. burgdorferi, however, the relationship between this protein and BBK32 is unclear ŽGrab et al., 1998.. It has been observed that B. garinii can bind FN in a specific and saturable fashion and Scatchard analysis revealed the presence of two types of receptors on the spirochetal surface, one with high and one with low affinity for FN ŽKopp et al., 1995.. A 147-kDa protein with high FN-binding activity was subsequently solubilized from the surface of B. garinii. This protein, soluble FN and anti-FN antibodies could competitively inhibit the adherence of B. garinii to epithelial cells ŽKopp et al., 1995..

5. Role of FN-binding MSCRAMMs in bacterial invasion of mammalian cells The ability to invade non-phagocytic mammalian cells may provide bacteria with a niche in which they are protected from host defense mechanisms or antimicrobial agents, which often operate in the extracellular melieu. This may enable the bacteria to persist in the host and may also provide a route for tissue invasion from a primary site of colonization. Recent observations suggest that FN-binding MSCRAMMs, host integrins and FN play important roles in cellular invasion by a variety of bacteria. Several gram-positive bacteria, such as Streptococcus spp. and S. aureus efficiently multiply in the extracellular environment but also have the ability to invade host cells ŽHamill et al., 1986; Boschwitz and Timoney, 1994; Lapenta et al., 1994; Almeida et al., 1996; Calvinho and Oliver, 1998; Winram et al., 1998.. Expression of the FN-binding MSCRAMMs SfbI or protein F1 promoted the invasion of cultured human epithelial cells by S. pyogenes and soluble forms of these proteins inhibited this process ŽMolinari et al., 1997; Jadoun et al., 1998; Okada et al., 1998.. Polystyrene beads coated with SfbI or protein F1 were also effectively internalized by these cells ŽMolinari et al., 1997; Ozeri et al., 1998.. Anti-FN antibodies completely abolished protein F1-mediated invasion, whereas increasing FN concentrations resulted in a significant enhancement of bacterial uptake, suggest-

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ing a role for FN in this process ŽJadoun et al., 1998.. It has recently been demonstrated that the host cell receptors involved in protein F1-mediated entry of S. pyogenes are the FN-binding a v ␤ 3 and ␤ 1 integrins ŽOzeri et al., 1998.. Antibodies against these integrins effectively inhibited invasion by this bacterium ŽOzeri et al., 1998.. The entry of S. pyogenes into human epithelial cells also can be mediated by protein M1, a multifunctional surface protein, in an FN-dependent manner which is inhibited by anti-␣ 5␤ 1 integrin antibodies ŽCue et al., 1998.. Our preliminary observations indicate that the FN-binding MSCRAMMs are essential for S. aureus invasion of mammalian cells. Simultaneous inactivation of the genes encoding FnbpA and FnbpB resulted in a 100-fold reduction in the invasion of HeLa cells by the mutant staphylococcal cells. In addition, soluble fragments of FnbpA and a RGD-containing peptide inhibited the invasion Žmanuscript in preparation.. Taken together, these studies suggest that FN may act as a bridging molecule between FN-binding bacterial MSCRAMMs and mammalian cell integrins. Mycobacteria are obligate intracellular pathogens which naturally infect macrophages but can also invade non-phagocytic cells such as HEp-2 cells and Schwann cells ŽBermudez et al., 1995; Schorey et al., 1995.. The ability to bind FN appears to play an important role in cellular invasion by these bacteria. Schorey et al. Ž1995.. observed that the invasion of Schwann cells was enhanced by pretreatment of the mycobacteria with FN and that this enhancement was abolished by anti-FN antibodies or antibodies against the mycobacterial FN-binding protein FAP. FN also mediates efficient entry of the facultative intracellular bacterium Neisseria gonorrhoeae into HEp-2 cells Žvan Putten et al., 1998.. The FN-induced entry of OpaA-expressing N. gonorrhoeae, which also appeared to be dependent on glycosaminoglycans, was inhibited by RGD-containing peptides and anti-␣ 5 ␤ 1 integrin antibodies Žvan Putten et al., 1998.. The N-terminal region of FN is believed to interact with OpaA, through the bridging action of glycosaminoglycans, while the type III modules of FN bind ␤ 1 integrins on the HEp-2 cell surface Žvan Putten et al., 1998.. However, studies have revealed that vitronectin and ␣ v integrins may be more important for the internalization of N. gonorrhoeae by other cell lines ŽDuensing and van Putten, 1997; Gomez-Duarte et al., 1997; Dehio et al., 1998; Duensing and van Putten, 1998.. The mechanism by which FN-binding MSCRAMMs trigger the internalization of bacteria by mammalian cells is unknown. Many bacterial surface proteins that mediate invasion interact with integrins via bridging molecules such as FN, or directly bind to integrins, as has been demonstrated by Isberg and colleagues for

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the invasin protein of Yersinia spp. ŽIsberg et al., 1987; Isberg and Leong, 1990; Isberg and Van Nhieu, 1994.. This suggests that these host receptors may play a central role in the internalization process. The binding of an integrin to immobilized FN initiates a complex cascade of cell signaling that leads to reorganization of cytoskeletal components, resulting in cell spreading and the formation of adhesion plaques. Engagement of integrins with FN bound to a bacterial cell surface may trigger a similar process but, due to the small size and curvature of the bacterial cells, the interaction may result in internalization of the bacteria. Consistent with this notion, FN-coated polystyrene beads are effectively internalized by BHK cells ŽMcAbee and Grinnell, 1983.. Furthermore, inhibitors of actin polymerization, such as cytochalasin D, inhibit the invasion of several bacterial species, suggesting that the process does involve rearrangement of cytoskeletal components of the host cell ŽYoung et al., 1992; Almeida and Oliver, 1995; Greco et al., 1995; Menzies and Kourteva, 1998.. How similar are the intracellular signaling pathways that induce mammalian cells to spread on FN-coated surfaces or to internalize FN-coated bacteria? Does the integrin-dependent internalization of various bacterial species proceed through similar or different mechanisms? These are some of the intriguing questions that warrant further investigation.

6. Are FN-binding MSCRAMMs virulence factors? Despite the general belief that FN-binding MSCRAMMs are pathogenic determinants of microorganisms, the importance of these MSCRAMMs in the infection process remains to be determined in many cases. For example, it is clear from studies with isogenic mutants lacking FnbpA and FnbpB that these proteins are responsible for mediating the attachment of S. aureus to immobilized FN in ¨ itro and contribute to the adherence of this bacterium to plasma clots and ex vivo biomaterial ŽVaudaux et al., 1993; Greene et al., 1995; Vaudaux et al., 1995.. However, data on the role of these FN-binding MSCRAMMs in animal models of staphylococcal endocarditis are conflicting ŽKuypers and Proctor, 1989; Flock et al., 1996; Greene et al., 1996.. These conflicting results may be due to the difficulties inherently associated with animal studies, such as variability among the animals used in each case. In addition, the outcomes of experimental infections may be dependent on the animal model employed. For example, if a collagen-binding MSCRAMM is responsible for colonization of cartilaginous tissues, such as joints, it may be more relevant to use an animal model of septic arthritis than a model of endocarditis to assess the role of this

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MSCRAMM in virulence. Furthermore, it is likely that multiple adhesins are involved in different stages of a particular infection and that a degree of redundancy exists in the activities of these adhesins. All of these issues should be taken into consideration when investigating the potential roles of individual MSCRAMMs in the infection process. In this review, we have tried to describe the multitude of interactions which exist between bacterial MSCRAMMs and FN which promote both bacterial adherence to host tissues and invasion of host cells. During the course of evolution, pathogenic microbes have developed elaborate ways to effectively colonize their hosts, evade host defense systems, interfere with normal cellular activities and destroy host tissues, resulting in the symptoms of disease. The expression of FN-binding MSCRAMMs is only one example of this adaptation. With the emergence of multiple drug resistance, it is critical to expand our knowledge of the molecular mechanisms of microbial pathogenesis to target the development of novel preventive and therapeutic strategies.

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