Adherence of Brucella to human epithelial cells by Jose Carrasquero-Diaz

Blackwell Science, LtdOxford, UKCMICellular Microbiology1462-5814Blackwell Publishing Ltd, 200465435445Original ArticleE. I. Castañeda-Roldán et al.Brucella adherence and fibronectin binding

Cellular Microbiology (2004) 6(5), 435–445

doi:10.1111/j.1462-5822.2004.00372.x

Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues Elsa I. Castañeda-Roldán,1 Fabiola Avelino-Flores,1 Monique Dall’Agnol,1 Enrique Freer,2 Lilia Cedillo,1 Jacques Dornand3 and Jorge A. Girón1* 1 Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edificio 76, Complejo de Ciencias, Puebla, México. 2 Unidad de Microscopía Electrónica, Universidad de Costa Rica, San José, Costa Rica. 3 INSERM U431, Université Montpellier II, Place Eugène Bataillon, c.c. 100, 34095 Montpellier, Cedex 05 Montpellier, France. Summary The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and Nacetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new Received 5 August, 2003; revised 8 December, 2003; accepted 9 December, 2003. *For correspondence. E-mail jagiron@yahoo.com; Tel. (+52) 222 233 2010; Fax (+52) 222 244 4518. © 2004 Blackwell Publishing Ltd

insights into understanding the interplay between Brucella and host cells. Introduction Adherence to host tissues is an essential and complex stage for bacterial colonization and, consequently, the establishment of bacterial infectious disease. In many cases, adherence is mediated by one or more adhesins that can act simultaneously or in distinct steps of an infectious process (Finlay and Falkow, 1997). Thus, adherence is considered an important virulence trait, because it enables bacterial pathogens to deliver toxins efficiently to host tissues, to interact closely with the cell membrane favouring intracellular penetration, to overcome peristaltic clearance and to establish microbial communities in biological niches. Adhesins in the form of pili or outer membrane proteins may mediate direct or indirect binding to host cells. For intracellular pathogens such as Salmonella, Shigella and Yersinia, penetration and survival within eukaryotic cells are key traits that determine their pathogenic scheme. Much has been learned in recent years about the molecular mechanisms of entry of these invasive organisms (Finlay and Falkow, 1997). Brucella is a Gram-negative facultative intracellular pathogen that causes brucellosis in humans and is responsible for a widely distributed zoonosis that affects a broad range of mammals, ranging from sea mammals to domestic animals (Young, 1983; Enrigh, 1990; Brincker et al., 2000; Boschiroli et al., 2001; Foster et al., 2002). In the host, Brucella abortus enters and survives inside phagocytes and cultured non-phagocytic epithelial cells (Detilleux et al., 1990a,b; Baldwin and Winter, 1994; SolaLanda et al., 1998; Pizarro et al., 2000; Gorvel and Moreno, 2002; Kohler et al., 2003). The microorganisms exploit the autophagic machinery of the host cells and replicate within membrane-bound compartments that resemble endoplasmic reticulum structures (Pizarro et al., 1998a,b; 2000; Gorvel and Moreno, 2002). Owing to the large pathogenic spectrum of Brucella and its capacity to invade many different cell types and tissues (Enrigh, 1990), it is reasonable to presume that the bacteria are able to express bacterial surface molecules devoted to the specific recognition of unique or common receptor components present on numerous tissues. A great deal of information is available in terms of the interaction and

436 E. I. Castañeda-Roldán et al. trafficking of Brucella within cells of the immune system and epithelial cells (Pizarro et al., 1998b; 2000; Arenas et al., 2000; Rittig et al., 2001; Letesson et al., 2002). It has been shown that the ability of smooth Brucella to adsorb onto murine lymphocytes by specific lectin-like interactions can be inhibited by a-methyl-mannose-amine and lipopolysaccharide (LPS) preparations (Lee et al., 1983). Nevertheless, the mechanisms underlying the adherence properties of Brucella to the first line of epithelial cells before interacting with professional phagocytes are practically unknown. Analysis of the genome sequence of Brucella has revealed various genes coding for putative adhesins and invasins involved in attachment and actin recruitment in cells, yet no fimbrial or nonfimbrial adhesins have been described in Brucella (DelVecchio et al., 2002). The aim of this study was to further our knowledge of the extracellular interaction of the brucellae with epithelial cells and macrophages. Towards this aim, we investigated the adherence properties and dynamics of the interaction of B. abortus and Brucella melitensis with cultured nonphagocytic epithelial cells and macrophages supported by cell adherence assays and high-resolution ultrastructural studies. The nature of the host cell receptor responsible for the interaction of the bacteria with epithelial cells was explored in competition assays using several putative inhibitory compounds and determining the effect of chemical and enzymatic treatments of cultured cells on bacterial adherence. Our data strongly suggest the participation of sialic acid-containing and/or sulphated molecules as

receptors involved in the attachment of Brucella to host cells. Extracellular matrix (ECM) proteins such as fibronectin, collagen and vitronectin act as interlinking molecules in connective tissues and are ideal microbial adhesion targets for colonization of host tissues (Mckeown-Longo, 1987; Proctor, 1987; Schorey et al., 1996; Espitia et al., 1999; Gilot et al., 1999; Peacock et al., 1999; Sinha et al., 1999). We report here that B. abortus binds to immobilized human ECM, in particular to fibronectin and vitronectin. We hypothesize that adherence to eukaryotic cells and the ECM-binding properties described here are important factors for colonization and dissemination of the bacteria within host tissues. Results Brucella adheres to and colonizes the surface of epithelial cells It is well-established that Brucella invades phagocytic and cultured non-phagocytic cells (Boschiroli et al., 2001; Letesson et al., 2002; Kohler et al., 2003). However, little is known in terms of the interaction of the brucellae with the surface of epithelial and professional phagocytic cells. To further our knowledge on the adherence properties of Brucella, we compared the ratios of adherence and invasion of B. abortus and B. melitensis to HeLa and HEp-2 cells and to THP-1 phagocytic cells. For quantification of extracellular bacteria (adherence assay) (Fig. 1B), we subtracted the number of intracellular bacteria [determined by colony-forming units per ml (cfu ml-1)] obtained

Fig. 1. Kinetics of adherence and intracellular replication of Brucella strains. A. Quantification of total bacterial cfu ml-1 (extracellular and intracellular) after infection of HeLa cells for the times indicated. The cells were lysed with 0.1% Triton X-100 and plated on tryptic soy agar plates for bacterial counting. B. Determination of intracellular bacteria by the gentamicin protection assay. Monolayers of HeLa cells were infected with Brucella strains for different time intervals and then treated with gentamicin before plating out the bacteria as before. Estimation of adherent extracellular bacteria was done after subtracting the number of intracellular bacteria (B) from the total cell-associated bacteria (A). These experiments were done in triplicate and repeated at least three times. Values are averages ± standard errors for triplicate samples and are expressed as 104 cfu ml-1. © 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

Brucella adherence and fibronectin binding 437 after killing extracellular bacteria with gentamicin (invasion assay) (Fig. 1B) from the total bacteria recovered in the absence of gentamicin (Fig. 1A). A time-dependent binding profile was observed, confirming the selective interaction between Brucella strains and eukaryotic cells. The bacteria associated with cultured cells as early as 1 h after infection. Extracellular bacteria reached a peak between 12 and 36 h, whereas invasive bacteria reached a peak at 36 h. The results depicted in Fig. 1 strongly suggest that the bacteria associate with the cell surface in higher numbers while significantly less bacteria are localized in the intracellular space. Beyond 36 h of infection, fewer bacteria were counted, probably due to the fact that the cell monolayers become detached. Detachment could be the result of the presence of an increased number of extracellular and intracellular bacteria exerting a deleterious effect on the cells. When the adherence properties of different species and strains were compared, we found that clinical Brucella isolates (B. abortus A01 and B. melitensis H3) bound more efficiently to cultured cells than reference strains B. abortus S19 and B. melitensis 2308 (data not shown). The results obtained with HeLa cells were reproduced with HEp-2 cells (data not shown), suggesting that the adherence property may not be restricted to these human cell lines. Further, adherence assays using THP-1-derived macrophages demonstrated that the brucellae bound significantly to these cells and, in fact, the number of intracellular bacteria was less than the number of adhering extracellular bacteria (Fig. 2). These experiments strongly suggest that the brucellae bind to the surface of phagocytic cell and non-phagocytic epithelial cells.

We then visualized the kinetics of adhesion by Giemsa staining and light microscopy. At 4 h of infection, the bacteria were seen associated with the HeLa cells as single organisms and forming tight microcolonies localized on specific areas of the cell (Fig. 3A–F). The size of these microcolonies increased with extended times (8–72 h) of infection. We considered 36 h of infection to be the optimal time point to study further the effect on adherence of potential inhibitors. The identity of the organisms in the adhering microcolonies was confirmed by indirect immunofluorescence using anti-B. abortus antibodies (Fig. 3G– L). The fluorescence-localized bacteria seen represent bacteria associated with the cell surface, as we did not use any permeabilization technique, and the number of bacteria increased with the time of infection. A normal rabbit antiserum revealed no fluorescent clusters (data not shown). The data obtained by these staining techniques provide clues about the degree of total bacteria interacting with cultured cells. In order to visualize bacteria on the surface of infected epithelial cells, we monitored the time course of infection by scanning electron microscopy (Fig. 4). Consistent with our previous observations, B. abortus organisms initially attached as single organisms (Fig. 4B) and, 8 h after infection, the brucellae began to form small clusters, which subsequently grew into larger bacterial aggregates. Extended infection periods (72 h) led the bacteria to cover the surface of HeLa cells almost entirely. Interestingly, the adhering brucellae appeared to undergo previously unseen morphological changes. For example, at 24–36 h after infection, elongated organisms are seen on the cell surface. As the infection proceeds, numerous adhering elongated organisms folded up to

Fig. 2. Comparison of adherence and intracellular replication of Brucella strains using THP-1-derived macrophages. A. Quantification of total bacterial cfu ml-1 (extracellular and intracellular) after infection of THP-1 cells for the times indicated. The cells were lysed with 0.1% Triton X-100 and plated on tryptic soy agar plates for bacterial counting. B. Determination of intracellular bacteria by the gentamicin protection assay. Monolayers of THP-1 cells were infected with Brucella strains for different time intervals and then treated with gentamicin before plating out the bacteria as before. Estimation of adherent extracellular bacteria was done after subtracting the number of intracellular bacteria (B) from the total cell-associated bacteria (A). These experiments were done in triplicate and repeated at least three times. Values are averages ± standard errors for triplicate samples and are expressed as 104 cfu ml-1. © 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

438 E. I. Castañeda-Roldán et al.

Fig. 3. Demonstration of time-dependent adherence of B. abortus to HeLa cells. Micrographs showing HeLa cells infected with B. abortus A01 for 0 (A and G), 4 (B and H), 8 (C and I), 12 (D and J), 24 (E and K) and 36 h (F and L) at 37∞C in 5% CO2. A–F. The bacteria were stained with Giemsa. G–L. The surface-associated bacteria were immunolabelled with anti-B. abortus antiserum and goat anti-rabbit FITC conjugate. Magnification ¥100. Note the formation of localized clusters or microcolonies that grow in size and number with the time of infection.

adopt rounded or doughnut shapes (Fig. 4I and K). To summarize, we have shown compelling data by several approaches that suggest that B. abortus adheres to and colonizes the surface of eukaryotic cells. Identification of a receptor moiety In an attempt to identify the nature of receptor molecules involved in the adhesion process, a number of potential

inhibitors and chemical and enzymatic treatments were tested in the adherence assays described above and in Experimental procedures. Adherence was reduced by 82% and 59% (P < 0.05) when HeLa (Fig. 5A) and HEp2 cells (data not shown) were pretreated with neuraminidase and sodium periodate respectively. Treatment of both cell lines with trypsin increased bacterial adherence by 58% (Table 1 and Fig. 5A; data not shown for HEp-2 cells). These data suggest that a receptor, hypothetically © 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

Brucella adherence and fibronectin binding 439 Fig. 4. Demonstration of extracellular association of B. abortus with HeLa cells. Scanning electron micrographs showing B. abortus clustering on to target cells after 0 (A), 1 (B), 2 (C), 4 (D), 8 (E), 10 (F), 12 (G), 24 (H and I) and 36 h (J–L) of infection. Some of the brucellae are seen as elongated organisms that fold up to acquire a doughnut shape (I–L).

Table 1. Effect of chemical and enzymatic treatments of HeLa cells on the adherence of B. abortus. Treatment

cfu ml-1 (± SD)*

Without treatment Neuraminidase Sodium periodate Trypsin

53 10 22 84

(10.20)b (4.78)c (2.20)c (9.72)a

% Adherence 100 18 41 158

Neuraminidase and sodium periodate treatments produced an inhibitory effect while trypsin treatment caused increased adherence. *Values are for 105 cfu ml-1, and each value shown represents the mean ± SD of three different determinations. Differences between values bearing different letters are significant (P £ 0.05), whereas those between values bearing the same letter are not significant (P ≥ 0.05).

a sialic acid-containing molecule, was unmasked by trypsinization, allowing the bacteria to interact more efficiently with the cell surface membrane. In a set of parallel inhibition experiments, we performed Giemsa staining to determine any effect on cluster formation by B. abortus. In agreement with the results shown in Table 1, we confirmed the inhibitory effect of neuraminidase and the enhanced effect of trypsin treatments on the interaction of B. abortus A01 with HeLa cells (Fig. 5B). In addition, we investigated the effect of the same chemical and enzymatic treatments on the invasion of the brucellae as described above. Our data showed that intracellular replication was inhibited by 63% by pretreatment of HeLa cells with sodium periodate but more strongly (70%) with neuraminidase (data not shown). Trypsinization did not show any effect on intracellular replication of B. abortus A01, suggesting that the bacterial surface proteins

440 E. I. Castañeda-Roldán et al. Fig. 5. Effect of chemical and enzymatic treatments of HeLa cells and putative inhibitors on adherence of B. abortus to HeLa cells. A. HeLa cells were preincubated with neuraminidase (bar B), sodium periodate (bar C) and trypsin (bar D) before infection with AO1. The percentage adherence was compared with untreated HeLa cells (bar A) infected with AO1. Values are averages ± standard errors for triplicate samples of experiments performed at least three times. B. Micrographs of B. abortus A01 adhering to untreated or treated HeLa cells after 36 h of infection. A, infection of untreated HeLa cells; B, cultured cell monolayers pretreated with 1 U ml-1 neuraminidase or 100 mg ml-1 trypsin (C) before infection with B. abortus A01. D, HeLa cell adherence of B. abortus preincubated with 25 mg ml-1 heparan sulphate.

involved in attachment and invasion are different (P ≥ 0.05). The role of sialic acid residues as receptors for brucellae on THP-1-derived macrophages was investigated by neuraminidase treatment of these cell lines. Similar to epithelial cells, neuraminidase-treated macrophages bound significantly fewer bacteria than untreated macrophages (Fig. 2). To investigate the involvement of certain carbohydratecontaining receptors in the interaction between HeLa cells and B. abortus, we tested the dose effect of a number of potential putative inhibitors at 36 h after infection. Except for heparan sulphate and N-acetylneuraminic acid, which reduced the adherence of B. abortus by 50% and 40%, respectively, we were unable to show an inhibitory dose effect on adherence or cluster formation by B. abortus by the other compounds tested (Table 2, Figs 5A and 6). The attachment of B. abortus was highly reduced in the pres-

ence of sulphated polysaccharides such as heparan sulphate and chondroitin sulphate but not by non-sulphated polysaccharides (Table 2, Fig. 6). This effect could be attributed to negative charge effects of sulphated residues or to the competitive action between chondroitin sulphate and the proteoglycans present on the surface of the endothelial cells (Nadanaka, 1999). In sum, these findings strongly suggest the participation of sialic acid-containing molecules and/or sulphated polysaccharides in the interaction of B. abortus with HeLa cells and possibly with eukaryotic cells in general. Brucella abortus binds to human extracellular matrix proteins Several microbial pathogens bind extracellular matrix proteins (ECM) (Mckeown-Longo, 1987; Proctor, 1987; West© 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

Brucella adherence and fibronectin binding 441 Table 2. Effect of putative inhibitors on adherence of B. abortus to HeLa cells. Compound

cfu ml-1 (± SD)*

Without inhibitor Heparan sulphate Heparin Chondroitin sulphate Chondroitin Dextran Methyl a-D-mannopyranosyl Acetyl a-D-mannopyranosyl N-acetylneuraminic acid N-acetylneuramin-lactose

85 42 65 66 74 91 87 84 51 63

(10.20)a (3.10)d (3.10)bc (6.70)bc (4.42)b (2.90)a (7.90)a (2.10)a (3.50)d (1.40)c

% Adherence 100 50 77 78 87 107 102 98 60 75

*Values are expressed as 105 cfu ml-1, and each value represents the mean ± SD of three different determinations. Differences between values bearing different letters are significant (P £ 0.05), whereas those between values bearing the same letter are not significant (P ≥ 0.05). L-fucose, D-arabinose, D-xylose, Dglucose, D-mannose and D-lactose showed no inhibitory effect when used at 100 mg ml-1.

Fig. 6. Effect of putative inhibitors on adherence of B. abortus AO1 to HeLa cells. HeLa cells were preincubated for 30 min at 37∞C with heparan sulphate (B), heparin (C), chondroitin sulphate (D), chondroitin (E), dextran (F), methyl a-D-mannopyranosyl (G), acetyl a-D-mannopyranosyl (H), N-acetylneuraminic acid (I) and Nacetylneuramin lactose (J). Note that the highest inhibitory activities were shown by (B) and (I). Each value represents the mean ± standard deviation of three different determinations.

erlund and Korhonen, 1993; Schorey et al., 1996; Espitia et al., 1999; Gilot et al., 1999; Peacock et al., 1999; Sinha et al., 1999). The ability of Brucella to bind ECM was determined by enzyme-linked immunosorbent assay (ELISA) using immobilized human fibronectin, vitronectin, collagen and laminin (Fig. 7). B. abortus A01 bound in a dose-dependent manner to fibronectin and vitronectin and to a lesser extent to collagen and laminin. The brucellae also bound to chondroitin sulphate but not to bovine serum albumin (BSA) used as a negative control. In view of these results, we investigated the binding of B. abortus A01 to the 70 kDa fibronectin fragment containing a heparin gelatin-binding domain and to an arginine–glycine– aspartic acid (RGD) tripeptide. We found that B. abortus A01 bound weakly to the 70 kDa fibronectin fragment in a dose-independent manner and did not bind to the RGD

tripeptide (data not shown). Similar results were obtained with all the Brucella strains tested (data not shown). To characterize further the interaction of B. abortus A01 with immobilized fibronectin, we performed competition experiments using soluble human fibronectin and the 70 kDa fibronectin fragment. BSA was used as a negative control. Our results show that soluble fibronectin and the 70 kDa fibronectin fragment inhibited the interaction between the brucellae and immobilized fibronectin by 70% and 53% respectively (Fig. 8). No inhibition was obtained when BSA was used alone. Discussion The identity of adhesins and receptor molecules involved in the interplay between Brucella and host epithelial cells,

Fig. 7. Binding of B. abortus A01 to immobilized ECM proteins. Increasing concentrations of human ECM, a 70 kDa fibronectin fragment, chondroitin sulphate and bovine serum albumin were immobilized on a microtitre plate and incubated for 1 h with a suspension of B. abortus A01. Bacteria bound to ECM were detected by ELISA with anti-B. abortus antiserum diluted 1:1000 and with goat anti-rabbit IgG alkaline phosphatase conjugate. Each measure represents the average of three separate determinations after subtraction of the background value obtained in the absence of ECMs. Blank OD values obtained when similar experiments were done in the absence of B. abortus cells were lower than 0.08. Bars indicate standard errors presented as the means ± the standard deviations for three determinations in triplicate. © 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

442 E. I. Castañeda-Roldán et al.

Fig. 8. Inhibition of binding of B. abortus to human fibronectin. Human fibronectin (bar 2) and a 70 kDa fibronectin fragment (bar 3) at 25 mg ml-1 (bar 2) were preincubated with the bacteria before incubation with immobilized human fibronectin on a microtitre plate. After washings, bacterial attachment to fibronectin was quantified by ELISA. Bar 1 shows binding to fibronectin without treatment. Binding data are presented as the means ± the standard deviations for three determinations performed in triplicate.

especially before intracellular penetration and migration within polymorphonuclear leucocytes, remains largely unknown. In this study, we showed that both B. abortus and B. melitensis bind readily to the surface of HeLa and HEp-2 cells, forming large localized bacterial clusters. Interestingly, the brucellae also adhered to THP-1-derived macrophages, suggesting that the adherence property is not unique to epithelial cells. It is tempting to speculate that the adherent bacteria serve as a source for invasive infectious units. Clinical isolate strains adhered and invaded more efficiently than the reference strains used. In our hands, the clinical virulent strains used were more adherent than B. abortus S19 natural smooth attenuated vaccine strain. It is well known that expression of virulence factors in laboratory strains is affected by repeated passage, prolonged storage and growth conditions. The dynamics of B. abortus adherence to epithelial cells showed that this organism adheres and forms large aggregates that localize on the surface of host cells, and these events were inhibited by antibodies against B. abortus. To demonstrate further that the brucellae associated extracellularly with HeLa cells, we used scanning electron microscopy. Ultrastructural analysis of HeLa cells infected with brucellae revealed that, as early as 1 h after infection, the organisms were found associated with the eukaryotic cell surface. Extended times of infection rendered epithelial cells hosting large numbers of closely associated bacteria that formed microcolonies on the cell surface. Interestingly, after 24 h of infection the brucellae appeared to undergo morphological changes. At early stages of infection, the adhering brucellae exhibited an elongated morphology but, as the infection proceeded, the bacteria rounded up and folded to acquire a doughnut shape. Between 60 and 72 h of infection, the cells underwent

obvious cytopathic changes, resulting in destruction of the cell monolayer. Analysis of the basic mechanisms of morphogenesis and their relationship to the ability of B. abortus to infect host cells requires further investigation. We showed that pretreatment of cultured epithelial and THP-1-derived macrophages with neuraminidase (also called sialidase) and sodium periodate had a significant reducing effect on bacterial adherence, suggesting the involvement of sialic acid-containing molecules and carbohydrate moieties in the interaction between B. abortus and eukaryotic cells (Kelm and Schauer, 1997; Traving and Schauer, 1998). In favour of this hypothesis, we recently reported that Brucella bind to sialic acid residues on human and animal red blood cells (Rocha-Gracia et al., 2002). This interaction was inhibited by pretreatment of red blood cells with neuraminidase. Trypsinization of epithelial cells produced a high degree of adherence, suggesting the removal of masking proteins and exposure of sialic acid-containing molecules that could participate as receptor molecules for B. abortus. As sialic acids are commonly present as terminal residues of extracellular proteins (Ausubel et al., 1995; Kelm and Schauer, 1997), it is possible that these proteins are less sensitive to trypsinization or that the receptor mediating brucellae adherence is a glycolipid containing sialic acid residues. Although no experimental data are provided here to demonstrate either of these two possibilities, the reduction in adherence resulting from neuraminidase treatment and preincubation of bacteria with neuraminic acid strongly suggests the involvement of sialic acid in the interaction of the brucellae with epithelial and nonepithelial cells (Rocha-Gracia et al., 2002). In a series of competition experiments, we showed that, after preincubation of the bacteria with heparin, heparan sulphate, chondroitin and chondroitin sulphate, adherence of B. abortus to the surface of epithelial cells was reduced by heparan sulphate. Heparan sulphate has a lower density of sulphate esters than heparin and forms part of ECM proteoglycans called syndecan (integral membrane proteins). Proteoglycans are major components of connective tissue such as cartilage and are essential in the response of cells to certain extracellular growth factors (Rostand and Esko, 1997; Nelson and Cox, 2000). Syndecans have been proposed to act as adhesion and internalization receptors for pathogenic microorganisms through the action of their heparan sulphate chains (Grassmé et al., 1997; Rostand and Esko, 1997). Neisseria gonorrhoeae, also an intracellular pathogen, uses an outer membrane protein to bind cell surface heparan sulphate proteoglycans of epithelial cells resulting in tight bacterial adherence (Dehio et al., 1998a). Thus, there is a precedent for the interaction of intracellular pathogens with cell surface heparan sulphate. In this context, our results suggest that B. abortus could alternatively use heparan sulphate mol© 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

Brucella adherence and fibronectin binding 443 ecules present in ECM proteoglycans to bind to host target cells. In all, these are compelling data that suggest that B. abortus binds to sialic acid-containing and/or sulphated molecules on the surface of epithelial cells. Fibronectin is a well-characterized, multifunctional adhesive protein present in mammalian ECM and in a soluble form in plasma, cerebrospinal fluid, synovial fluid, amniotic fluid, seminal fluid and inflammatory exudates. Soluble fibronectin can be secreted and coat mucosal surfaces. Bacteria–fibronectin interactions are based on either protein–protein or protein–carbohydrate interactions and are targeted to different domains of the fibronectin molecule (Mckeown-Longo, 1987). We found that Brucella binds several ECM proteins, in particular fibronectin and vitronectin, an attribute that is widespread among other pathogenic organisms (Ryoslahti, 1988; Dehio et al., 1998b). It was reported that invasion of HeLa cells by Opa-expressing N. gonorrhoeae is mediated by binding to vitronectin (Gómez-Duarte et al., 1997). Our hypothesis is that the interactions of Brucella with ECM contribute to the spread of the bacteria through tissue barriers into the circulation and secondary infection sites as well as to the survival outside host cells and colonization of tissues. Based on the present data, we speculate that B. abortus uses different cellular surface components to adhere to and penetrate mammalian cells. The data suggest that B. abortus adheres avidly to mammalian cells through sialic acid-containing and/or sulphated molecules. The significance of these properties within the context of the interaction of B. abortus and the host target cell in infections in animals and humans is an important question to address. Our findings open new avenues to study and understand the pathogenic strategies of this microorganism. Experimental procedures Bacterial strains and growth conditions We used B. abortus S19, a smooth attenuated vaccine strain, B. abortus 2308, a smooth virulent strain, B. abortus A01 isolated from an aborted bovine fetus and B. melitensis H3 isolated from a bone marrow culture obtained from a patient with acute brucellosis. The strains were grown routinely in tryptic soy agar (Difco Laboratories) at 37∞C for 48 h under a 5% CO2 atmosphere.

Kinetics of adherence and invasion The kinetics of adherence of Brucella strains to monolayers of HeLa and HEp-2 epithelial cells was performed on glass cover slips as described previously (Cravioto et al., 1979). Before the assay, the brucellae were washed in 0.1 M phosphate-buffered saline (PBS), pH 7.4, and suspended in Dulbecco’s minimal essential medium (DMEM; Gibco) without fetal bovine serum © 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

(FBS). THP-1-derived macrophages were obtained from the ATCC. Cells were maintained at 37∞C in 5% CO2 in complete medium: RPMI-1640 medium supplemented with 5 mM glutamine (Life Technologies) and 10% (v/v) heat-inactivated fetal calf serum (FCS; Sigma-Aldrich) (Gross et al., 2003). The cells were checked regularly for the absence of mycoplasma by 4,6diamino-2-phenylindole (DAPI) fluorescence. THP-1 cells were differentiated in macrophage-like cells by a 3 day treatment with 10-7 M 1,25-dihydroxyvitamin D3 (Hoffman-LaRoche) (Gross et al., 2003). Adherent VD3-THP-1 cells were then scraped, harvested, washed and cultured overnight in 24-well plates at a cell density of 106 cells per well in 1 ml of RPMI-FCS before infection. The cell monolayers were infected with 109 (HeLa and HEp-2) or 5 ¥ 108 (VD3-THP-1) cfu ml-1 bacteria (as determined by colony count). They were then incubated at 37∞C for 0, 15 min, 1, 2, 4, 8, 12, 24, 36, 48, 60 and 72 h under a 5% CO2 atmosphere. For quantification of intracellular bacteria, the infected monolayers were incubated for 2 h in the presence of 100 mg ml-1 gentamicin (Sigma) to kill extracellular bacteria (Goldhar, 1994; Pizarro et al., 1998a). The infected cells were then washed three times with PBS and treated for 5 min with 1 ml of 0.1% Triton X-100 (Sigma) in deionized water (Goldhar, 1994). The lysates were serially diluted and plated on tryptic soy agar dishes for quantification of total (extracellular and intracellular) brucellae. The number of adherent bacteria was obtained by subtracting the number of intracellular bacteria from the total bacteria number obtained in the absence of gentamicin (Goldhar, 1994). In addition, the kinetics of adherence to eukaryotic cells seeded on glass coverslips was monitored by Giemsa staining and light microscopy, immunofluorescence using anti-brucellae antibodies and scanning electronic microscopy (Girón et al., 1996; 2002).

Immunofluorescence microscopy For visualization of the brucellae by immunofluorescence microscopy, the infected cells were washed extensively to remove nonadherent bacteria and then fixed with 3% paraformaldehyde in PBS for 30 min at room temperature. The cells were washed and incubated for 1 h with a 1:1000 dilution of anti-B. abortus antibodies, followed by washing and the addition of goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC; Sigma) and incubated for 1 h in the dark. The coverslips were mounted and examined under oil immersion with a Zeiss Axioscope microscope (Girón et al., 1996; 2002).

Scanning electron microscopy To visualize bacteria adhering on the surface of HeLa cells, the infected cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in 1% aqueous osmium tetroxide and dehydrated in 2,2-dimethoxypropane. The specimens were transferred to absolute ethanol, critical point dried using liquid carbon dioxide in an Emitech K850 apparatus, coated with goldpalladium using a Polaron E5100 sputter coater and viewed at 30 kV in a Hitachi scanning electron microscope (Girón et al., 1996).

Interaction of B. abortus A01 with pretreated host cells To investigate the chemical nature of the receptor(s) recognized

444 E. I. Castañeda-Roldán et al. by brucellae on the surface of epithelial cells, we pretreated HeLa and HEp-2 cells with neuraminidase, sodium periodate and trypsin. THP-1-derived macrophages were treated only with neuraminidase. Briefly, a semi-confluent monolayer of both cell lines was treated separately with 1 U ml-1 neuraminidase from Clostridium perfringens type V (Sigma) in 0.1 M citrate buffer (pH 5.5) (Ausubel et al., 1995), 100 mg ml-1 sodium periodate in 0.1 M sodium acetate buffer (pH 4.5) (Sigma) or 100 mg ml-1 trypsin in 10 mM CaCl2 and 20 mM Tris-HCl, pH 7.4 (Sigma), for 30 min at 37∞C, as described previously (Goldhar, 1994). After washing, the cells were used for adherence and invasion assays. The degree of adherence and invasion was compared with that obtained with non-treated cells.

Inhibition experiments The effect of putative inhibitors: heparin, heparan sulphate, chondroitin, chondroitin sulphate, dextran, methyl a-D-mannopyranosyl and acetyl a-D-mannopyranosyl, all at 25 mg ml-1; L-fucose, D-arabinose, D-xylose, D-glucose, D-mannose and D-lactose at 100 mg ml-1; and N-acetylneuraminic acid and N-acetylneuraminlactose (both at 30 mg ml-1) (Sigma), was tested in adherence inhibition assays (Goldhar, 1994; Girón et al., 1996). A 109 cfu ml-1 dilution of B. abortus A01 was preincubated for 30 min at 37∞C with each compound, and the HeLa and HEp-2 cells monolayers were incubated with the pretreated bacteria. In other experiments, a bacterial suspension (109 cfu ml-1) was incubated for 30 min at 37∞C with a 1:250 dilution of anti-Brucella antibodies and then added to cell monolayers containing the same dilution of antibodies (Goldhar, 1994; Girón et al., 1996). Normal rabbit serum was used as a negative control. After 36 h of infection, the cells were washed, lysed and plated out for quantification of bacterial adherence as described above. All inhibition experiments were performed at least three times to ensure reproducibility of the results.

Binding of B. abortus to ECM Binding of B. abortus A01 to immobilized human ECM was studied by a standard ELISA. Briefly, 96-well polystyrene microplates (Nunc) were coated overnight at 4∞C with 10-fold dilutions of human fibronectin, collagen, vitronectin and laminin (100 mg ml1 ; Sigma), 70 kDa fibronectin fragment (100 mg ml-1; Sigma), RGD tripeptide (50 mg ml-1; Sigma) (Kuusela et al., 1985; Girón et al., 1996) and BSA in carbonate buffer, pH 9.6. After washing with PBST, the wells were blocked with 2% BSA-PBS solution for 2 h at 37∞C. An inoculum of 105 cfu per well of B. abortus A01 was added and incubated for 2 h at 37∞C. After washing, anti-B. abortus antibodies were added followed by the addition of goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma). After addition of the phosphatase substrate, the reaction was stopped with 0.1 M EDTA, and the colour was measured by optical density at 405 nm in an ELISA plate reader (Kuusela et al., 1985; Girón et al., 1996; Gilot et al., 1999). To study further the interaction of the brucellae with fibronectin, B. abortus A01 were first preincubated for 30 min at 37∞C with 25 mg of fibronectin, 10-fold dilutions of sodium periodate (1– 100 mM), trypsin (1–100 mg ml-1) or the 70 kDa fibronectin fragment (5 mg ml-1) (Kuusela et al., 1985; Virkola et al., 2000). The bacteria were washed by centrifugation and then tested for binding to immobilized human fibronectin (25 mg ml-1) by ELISA.

Statistical analysis Statistical analysis of variances between the putative inhibitors was done using the Bartlett test. The effect of inhibitors, chemical and enzymatic treatments of bacteria or epithelial cells was analysed using the Mann–Whitney statistical test (Zar, 1999).

Acknowledgements This work was supported by Sistema de Investigación Regional Ignacio Zaragoza (SIZA) (no. 960202003), INSERM-CONACYT, ECOS-ANUIES-CONACYT M99-501. We thank the Unidad de Microscopía Electrónica, Universidad de Costa Rica and Dan Guerrero (University of Texas at San Antonio) for electron microscopy assistance, Jean Pierre Gorvel, Edgardo Moreno and Jean Favero for helpful discussions.

References Arenas, N.G., Staskevich, A.S., Aballay, A., and Mayorga, L.S. (2000) Intracellular trafficking of Brucella abortus. J774 macrophages. Infect Immun 68: 4255–4263. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1995) Sialidases. In Current Protocols in Molecular Biology, Vol. 3. New York: John Wiley Sons, pp. 17.12.2. Baldwin, C.L., and Winter, A.J. (1994) Macrophages and Brucella. Immunol Sem 60: 363–380. Boschiroli, M.L., Foulonge, V., and O’Callaghan, D. (2001) Brucellosis: a worldwide zoonosis. Curr Opin Microbiol 4: 58–64. Brincker, B.J., Ewalt, D.R., MacMillan, A.P., Foster, G., and Brew, S. (2000) Molecular characterization of Brucella strains isolated from marine mammals. J Clin Microbiol 38: 1258–1262. Cravioto, A., Gross, R., Scotland, S., and Rowe, B. (1979) An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotypes. Curr Microbiol 3: 95–99. Dehio, C., Freissler, E., Lanz, C., Gómez-Duarte, O.G., David, G., and Meyer, T.M. (1998a) Ligation of cell surface heparan sulphate proteoglycans by antibody-coated beads stimulates phagocytic uptake into epithelial cells: a model for cellular invasion by Neisseria gonorrhoeae. Exp Cell Res 242: 529–539. Dehio, M., Gómez-Duarte, O.G., Dehio, C., and Meyer, T.M. (1998b) Vitronectin-dependent invasion of epithelial cells by Neisseria gonorrhoeae involves av integrin receptors. FEBS Lett 424: 84–88. DelVecchio, V.G., Kapatral, V., Redkar, R.J., Patra, G., Mujer, C., Los, T., et al. (2002) The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci USA 90: 587–592. Detilleux, P.G., Deyoe, B.L., and Cheville, N.F. (1990a) Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro. Infect Immun 58: 2320–2328. Detilleux, P.G., Deyoe, B.L., and Cheville, N.F. (1990b) Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy. Vet Pathol 27: 317– 328. Enrigh, F.M. (1990) The pathogenesis and pathobiology of Brucella infection in domestic animals. In Animal Brucello© 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

Brucella adherence and fibronectin binding 445 sis. Nielsen, K., and Duncan, B. (eds). Boca Raton, FL: CRC Press, pp. 301–320. Espitia, C., Laclette, J.P., Mondragon-Palomino, M., Amador, A., Campuzano, J., Martens, A., et al. (1999) The PEPGRS glycine-rich proteins of Mycobacterium tuberculosis: a new family of fibronectin-binding proteins? Microbiology 12: 3487–3495. Finlay, B.B., and Falkow, S. (1997) Common themes in microbial pathogenicity: revisited. Microbiol Mol Biol Rev 61: 136–169. Foster, G., MacMillan, A.P., Godfroid, J., Howie, F., Ross, H.M., Cloeckaert, A., et al. (2002) A review of Brucella sp infection of sea mammals with particular emphasis on isolates from Scotland. Vet Microbiol 90: 563–580. Gilot, P., André, P., and Content, J. (1999) Listeria monocytogenes possesses adhesins for fibronectin. Infect Immun 67: 6698–6701. Girón, J.A., Lange, M., and Baseman, J.B. (1996) Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans. Infect Immun 64: 197–208. Girón, J.A., Torres, A.G., Freer, E., and Kaper, J.B. (2002) The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol Microbiol 42: 361–379. Goldhar, J. (1994) Bacterial lectin-like adhesins: determination and specificity. In Methods in Enzymology. Bacterial Pathogenesis, Part B. Interaction of Pathogenic Bacteria with Host Cells, Vol. 236. Clark, V.L., and Baviol, P.M. (eds). San Diego: Academic Press, pp. 211–231. Gómez-Duarte, O.G., Dehio, M., Guzmán, C.A., Chhatwal, G.S., Dehio, C., and Meyer, T.M. (1997) Binding of vitronectin to Opa-expressing Neisseria gonorrhoeae mediates invasion of HeLa cells. Infect Immun 65: 3857–3866. Gorvel, J.P., and Moreno, E. (2002) Brucella intracellular life: from invasion to intracellular replication. Vet Microbiol 90: 281–297. Grassmé, H., Gulbins, E., Brenner, B., Ferlinz, K., Sandhoff, K., Harzer, K., et al. (1997) Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells. Cell 91: 605–615. Gross, A., Bouaboula, M., Casellas, P., Liautard, J.P., and Dornand, J. (2003) Subversion and utilization of the host cell cyclic adenosine 5¢-monophosphate/protein kinase A pathway by Brucella during macrophage infection. J Immunol 170: 5607–5614. Kelm, S., and Schauer, R. (1997) Sialic acids in molecular and cellular interactions. Int Rev Cytol 175: 137–240. Kohler, S., Michaux-Charachon, S., Porte, F., Ramuz, M., and Liautard, J.P. (2003) What is the nature of the replicative niche of a stealthy bug named Brucella? Trends Microbiol 11: 215–219. Kuusela, P., Vartio, T., Vuento, M., and Myhre, E.B. (1985) Attachment of staphylococci and streptococci on fibronectin, fibronectin fragments, and fibrinogen bound on a solid phase. Infect Immun 50: 77–81. Lee, C.M., Mayer, E.P., Molnar, J., and Teodorescu, M. (1983) The mechanism of natural binding of bacteria to human lymphocyte subpopulations. J Clin Lab Immunol 11: 87–94. Letesson, J.J., Lestrate, P., Delrue, R.M., Danese, I., Bellefontaine, F., Fretin, D., et al. (2002) Fun stories about Brucella: the ‘furtive nasty bug’. Vet Microbiol 90: 317–328. Mckeown-Longo, P.J. (1987) Fibronectin–cell surface interactions. Rev Infect Dis 9: S322–S334. Nadanaka, S. (1999) Chondroitin sulphate: Structure, func© 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 435–445

tion, and biosynthesis. Trends Glycosci Glycotechnol 11: 233–238. Nelson, D.I., and Cox, M.M. (2000) Carbohydrates and glycobiology. In Lehninger, Principles of Biochemistry, 3rd edn. New York: Worth Publishers, pp. 293–324. Peacock, S.J., Lina, G., Etienne, J., and Foster, T.J. (1999) Staphylococcus schleiferi subsp. Schleiferi expresses a fibronectin-binding protein. Infect Immun 67: 4272–4275. Pizarro-Cerdá, J., Moreno, E., Sanguedolce, V., Mége, J.L., and Gorvel, J.P. (1998a) Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosomelike compartments. Infect Immun 66: 2387–2392. Pizarro-Cerdá, J., Méresse, S., Parton, R.G., van der Goot, F.G., Sola-Landa, A., López-Goñi, I., et al. (1998b) B. abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of non-professional phagocytes. Infect Immun 66: 1–14. Pizarro-Cerdá, J., Moreno, E., and Gorvel, J.P. (2000) Invasion and intracellular trafficking of Brucella abortus in nonphagocytic cells. Microb Infect 2: 829–835. Proctor, R.A. (1987) Fibronectin: a brief overview of its structure, function and physiology. Rev Infect Dis 9: S317– S321. Rittig, G.M., Alvarez-Martínez, M.T., Porte, F., Liutard, J.P., and Rouot, B. (2001) Intracellular survival of Brucella spp. in human monocytes involves conventional uptake but special phagosomes. Infect Immun 69: 3995–4006. Rocha-Gracia, R.C., Castañeda-Roldán, E.I., Giono-Cerezo, S., and Girón, J.A. (2002) Hemagglutination properties of Brucella: identification of a hemagglutinin that binds sulphate- and sialic acid-containing molecules. FEMS Microbiol Lett 213: 219–224. Rostand, K.S., and Esko, J.D. (1997) Microbial adherence to and invasion through proteoglycans. Infect Immun 65: 1–8. Ryoslahti, E. (1988) Fibronectin and its receptors. Annu Rev Biochem 57: 375–413. Schorey, J.S., Holsti, M.A., Ratliff, T.L., Allen, P.M., and Brown, E.J. (1996) Characterization of the fibronectinattachment protein of Mycobacterium avium reveals a fibronectin-binding motif common among mycobacteria. Mol Microbiol 21: 321–329. Sinha, B., Francois, P.P., Nübe, O., Foti, M., Hartford, O.M., Vaudaux, P., et al. (1999) Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin a5b2. Cell Microbiol 1: 101–117. Sola-Landa, A., Pizarro-Cerd, J., Grill, M.J., Moreno, E., Moriyon, E., Blasco, J.M., Gorvel, J.P., López-Goni, I. (1998) A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence. Mol. Microbiol 29: 125–138. Traving, C., and Schauer, R. (1998) Structure, function and metabolism of sialic acids. Cell Mol Life Sci 54: 1330–1349. Virkola, R., Brummer, M., Rauvala, H., Van Alphen, L., and Horhonen, T.K. (2000) Interaction of fimbriae of Haemophilus influenzae type b with heparin-binding extracellular matrix proteins. Infect Immun 68: 5696–5701. Westerlund, B., and Korhonen, T.K. (1993) Bacterial proteins binding to the mammalian extracellular matrix. Mol Microbiol 9: 687–694. Young, E.J. (1983) Human brucellosis. Rev Infect Dis 5: 821– 842. Zar, J.H. (1999) Biostatistical Analysis, 4th edn. Upper Saddle River, NJ: Prentice Hall, pp. 146–155 and 196– 200.