Introduction

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Mycobacterium tuberculosis remains an important cause of pulmonary disease throughout the world, and in developing countries is the leading cause of death from a single infectious disease (1). M. tuberculosis organisms are facultative intracellular pathogens that attach to alveolar macrophages (AMs), undergo phagocytosis, survive, and replicate within the AMs (2). Thus, AMs may represent a safe habitat for M. tuberculosis organisms, permitting establishment of infection within the alveolar spaces. The basic mechanisms underlying the earliest steps of M. tuberculosis infection, however, remain poorly understood.

AMs express many surface receptors that facilitate the binding of microorganisms (7). Complement receptors (CR1 and CR3), CD14 receptor (the mannose receptor), a transferrin receptor, and an unknown receptor that is inhibited by -glucan have all been proposed as mediators of M. tuberculosis cell attachment (8). Alternatively, proteins normally found in the alveolar spaces may serve as ligands between microorganisms and AMs. In this respect, surfactant protein A (SP-A) is the most abundant surfactant apoprotein present in the alveoli, possesses opsonic activity, and represents a potential ligand to mediate attachment of M. tuberculosis to AMs (12, 13).

SP-A has an N-terminal collagenlike domain and a C-terminal region with structural similarity to C-type lectins (14, 15). The collagenlike region has significant homology to mammalian lectins such as mannose-binding proteins (16). The noncollagenous C-terminal end of SP-A is a globular structure with lectinlike properties that may interact with carbohydrates on the cell surface. SP-A shares several structural features with complement factor C1q and mannose-binding protein, including the short amino-terminal domain and the collagenlike domain (17). C1q enhances the phagocytosis of opsonized erythrocytes by monocytes and macrophages, and mannose-binding protein acts as an opsonin in the phagocytosis of bacteria by phagocytes (18). SP-A has also been shown to function as an opsonin by increasing the phagocytosis of viruses (12) and immunoglobulin-coated erythrocytes (19). Although the macrophage-binding domain of SP-A has not been definitely identified, a recent study suggests that SP-A binds to AMs through the collagenlike domain of SP-A, providing a mechanism for the interaction of SP-A with AMs during phagocytosis (20).

In the present study, we hypothesized that SP-A attaches to M. tuberculosis organisms via the carbohydrate recognition domain, thus acting as a "bridge" between the organism and the macrophage. The attachment of M. tuberculosis organisms to murine AMs was determined with ⁵¹Cr-labeled M. tuberculosis organisms. The data suggest that the interaction of M. tuberculosis organisms with AMs can be mediated by SP-A. The study also demonstrates that SP-A binds to M. tuberculosis organisms in a saturable, Ca²⁺-dependent, and carbohydrate-specific manner. Because SP-A levels can be markedly increased in human immunodeficiency virus (HIV)-infected subjects (21), these data suggest that SP-A may be an easily accessible ligand, permitting the interaction of M. tuberculosis organisms with AMs in certain disease states.

	Materials and Methods

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M. tuberculosis Isolation

The H37Ra strain of M. tuberculosis was obtained as a lyophilized preparation from American Type Culture Collection (Rockville, MD). The bacteria were cultured at 37°C in dispersed form in Middlebrook 7H9 broth containing albumin, dextrose, and catalase as enrichments (Becton Dickinson, Cockeysville, MD). Bacterial cultures (10 to 14 days old) were centrifuged and washed once with saline, and the final bacterial suspension was adjusted to 1 × 10⁶ organisms/ml with a nephelometer, according to the method of McFarland (22). Routinely, samples of bacteria were also grown on plates of 7H11 Middlebrook medium (Becton Dickinson) as stock. Purity of cultures was verified with an acid-fast Kinyoun stain (Midlantic Biomedical Inc., Paulsboro, NJ).

SP-A Isolation and Purification

SP-A was isolated according to Wright and colleagues (23). Briefly, a surfactant pellet was obtained by centrifugation (10,000 × g, 60 min) of bronchoalveolar lavage fluid (BALF) from patients with alveolar proteinosis, followed by extraction with butanol. The butanol-insoluble proteins were resuspended in octylglucoside to solubilize serum proteins. SP-A purified by this method was dialyzed extensively against 5 mM Tris buffer (pH 7.4) and quantified by the method of Lowry and colleagues (24). Deglycosylated SP-A was prepared according to the method of Gaynor and associates (25). SP-A purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (26).

Rat SP-A was isolated according to Kuroki and associates (27) with minor modifications. Lavage fluid was obtained from rats that had received intratracheal injection of silica (0.25 mg/rat) 4 wk prior to killing. The lavage was centrifuged at 22,000 × g for 18 h. The pellet (4 ml from 10 rats) was injected into 200 ml of butanol and stirred for 30 min. The butanol extract was centrifuged at 15,000 × g for 30 min. The pellet was resuspended in butanol, centrifuged, resuspended in 5 mM Tris buffer (pH 7.4), and dialyzed against the same buffer. Insoluble material was removed by centrifugation at 60,000 × g for 1 h. The supernate was brought to 10 mM CaCl₂, and passed through a fucose-agarose column (Sigma Chemical Co., St. Louis, MO), and washed with approximately 10 ml of 5 mM Tris containing 10 mM CaCl₂ (pH 7.8), and SP-A was eluted with 5 mM Tris containing 2 mM ethylenediamine tetraacetic acid (EDTA), pH 7.8. 

¹²⁵I-labeling of SP-A

[¹²⁵I]SP-A and [¹²⁵I]deglycosylated SP-A were prepared according to the method of Bolton and Hunter (28). The purified human SP-A protein (1 to 2 mg) was dialyzed in 0.1 M sodium borate-carbonate buffer (pH 8.5) at 4°C. An aliquot of 1 to 1.5 ml SP-A suspension was added directly to an iced Bolton-Hunter reagent vial (Amersham Corp., Arlington Heights, IL) after the benzene was removed by evaporation under a stream of nitrogen. The reaction mixture was incubated for 30 min on ice, with occasional mixing. The reaction was terminated by the addition of excess of 0.2 M glycine. Free ¹²⁵I was removed by dialysis against 5 mM Tris buffer (pH 7.4), and by passage over two Sephadex G-10 columns (Boehringer Mannheim Inc., Indianapolis, IN). The specific activity of the [¹²⁵I]SP-A was determined to be 31 to 54 µCi/mg protein.

[¹²⁵I]SP-A Binding Assay

The [¹²⁵I]SP-A binding assay was performed as previously described by our laboratory (29). M. tuberculosis organisms grown in 7H9 Middlebrook broth were isolated, washed once with Hanks' balanced salt solution (HBSS) containing 2 mM CaCl₂ and 1% bovine serum albumin (BSA), and resuspended in Dulbecco's modified Eagle's medium (DMEM) at a final concentration of 1 × 10⁸ M. tuberculosis organisms/ml. In this assay, the specificity and saturability of SP-A binding to M. tuberculosis organisms were determined according to the method of Proctor and coworkers (30). The specific binding of 0 to 4,200 ng of [¹²⁵I]SP-A to 1 × 10⁸ M. tuberculosis organisms in 100 µl of reaction mixture (5 mM Tris-HCl, 1% BSA, pH 7.4) was determined in the presence and absence of a 10-fold excess of cold SP-A. After a 60 min incubation at 37°C, the pellet was washed once with HBSS and the counts per minute (cpm) of radioactivity in the pellet were determined in a gamma counter. The amount of bound [¹²⁵I]- SP-A was quantified (ng bound = cpm pellet/protein specific activity).

Additional binding assays were performed to examine the effects of time, Ca²⁺ concentration, -methylene-D-mannosepyranosidase (mannosyl-BSA), trypsin, and deglycosylation of SP-A on SP-A binding. M. tuberculosis organisms (1 × 10⁸) were incubated with [¹²⁵I]SP-A in the presence of increasing concentrations of Ca²⁺ (0 to 2 mM), mannosyl-BSA (1,000 µg, with a molar mannose:albumin ratio of 26:1), or type V collagen (1 mg/ml)(Sigma Chemical Co.). Similarly, [¹²⁵I]SP-A binding to M. tuberculosis organisms was assessed after pretreatment of the organisms (1 × 10⁸) with trypsin (1 mg/ml) for 30 min and subsequent washing before incubating the organisms with SP-A for 60 min. Identical studies were also conducted with [¹²⁵I]deglycosylated SP-A. The amount of [¹²⁵I]SP-A bound was quantified (% bound = 100 × cpm pellet/[cpm pellet and supernatant]).

Antibody Production and Purification

Polyclonal antibodies to SP-A were raised in rabbits by the following method (31): Four subcutaneous injections containing 200 µg of SP-A were given at 3- to 4-wk intervals. Four weeks after the last injection, blood was removed from the rabbit and dialyzed in 0.1 M phosphate-buffered saline (PBS) (pH 8.1). The rabbit immune serum was applied to a Whatman DEAE 52 column and eluted with a buffer containing increasing concentrations of NaCl. The IgG fractions were concentrated by ultrafiltration and absorbed to human serum conjugated to cyanogen bromide-activated Sepharose 4B. The immunoreactivity and specificity of these polyclonal antibodies were demonstrated earlier (32).

Identification of SP-A-binding Protein on M. tuberculosis

To identify the SP-A-binding protein on M. tuberculosis, cell-wall membrane proteins were prepared from 7- to 10-day-old cultures as previously described (33). Briefly, M. tuberculosis organisms grown in 7H9 Middlebrook broth were washed three times in PBS and the whole bacterial suspension was freeze thawed and sonicated for 10 min on ice. The sonicated material was centrifuged at 12,000 × g for 10 min, and the pellet containing cell-wall proteins was resolved with SDS-PAGE in a discontinuous buffer system (26) and stained with silver stain (BioRad Laboratories, Richmond, CA) to visualize all the protein bands associated with M. tuberculosis cell wall. For each gel, a mixture of prestained molecular weight standards (BioRad Laboratories) was used for molecular size determination of unknown proteins.

Specific [¹²⁵I]SP-A-binding proteins on M. tuberculosis were determined by autoradiography. The cell-wall proteins were electrophoretically transferred onto Immobilon membrane (Millipore Corporation, Bedford, MA) (34). The membrane was incubated with 5% milk in Tris-buffered saline (TBS), 20 mM Tris-Cl, 50 mM NaCl (pH 7.5) at room temperature for 2 h to prevent nonspecific binding. The immunoblot was incubated with ¹²⁵I-labeled SP-A (3 µg/ml) and diluted in TBS-T for 1 h and autoradiographed.

AM Isolation

Murine AMs were isolated from pathogen-free, 10-wk-old BALB/c mice (Harlan Sprague Dawley Inc., Indianapolis, IN). The mice were killed by intraperitoneal injection of Beuthanasia-D solution (Schering-Plough Animal Corp., Kenilworth, NJ). The trachea was cannulated after a midline neck incision was made, and the lungs were lavaged 10 times with 1.0 ml of 0.9% NaCl solution containing EDTA (0.6 mM), penicillin (100 U/ml), gentamicin (4 µg/ml), and amphotericin B (0.5 µg/ml). Approximately 7 to 8 ml of lavage fluid was obtained from each mouse. The lavage fluid was centrifuged (600 × g, 10 min) and cells were washed with normal saline and resuspended in DMEM (BioWhittaker, Inc., Walkersville, MD) with 10% heat-inactivated fetal bovine serum, glutamine (300 µg/ml), penicillin (100 U/ml), and 20 mM N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (Hepes) (pH 7.2). Cell preparations were shown to be 98% AMs by Diff-Quik staining (Harleco, Aguada, PR) and to be more than 95% viable by trypan blue exclusion. AMs were plated at a density of 2.5 × 10⁵/100 µl/well on rat IgG (Calbiochem, San Diego, CA)-coated 96-well tissue culture plates, incubated for 48 h at 37°C in 5% CO₂, and washed three times with 1 ml of DMEM to remove unattached cells. The plates were stored at 4°C for 1 h prior to the attachment assay.

M. tuberculosis Attachment Assay

Attachment of M. tuberculosis organisms to AMs was quantified by adapting a ⁵¹Cr-labeled attachment assay developed in our laboratory (35, 36). Freshly isolated M. tuberculosis organisms were incubated for 18 h in 1 ml of DMEM and 200 µCi of ⁵¹Cr (New England Nuclear, Boston, MA). The ⁵¹Cr-labeled M. tuberculosis organisms were centrifuged (3,800 × g, 15 min), the supernatant discarded, and the pellet washed four times to remove unincorporated ⁵¹Cr. The ⁵¹Cr-labeled M. tuberculosis organisms (2.5 × 10⁵) were added to each well of attached AMs in a 96-well tissue culture plate.

To determine the time dependence of M. tuberculosis attachment to AMs, the ⁵¹Cr-labeled M. tuberculosis organisms were incubated with the AMs at 4°C for 30, 60, 120, 240, and 480 min. After the incubation, the medium containing unattached M. tuberculosis organisms was removed, the AMs with attached M. tuberculosis were washed three times, and all of the washes were saved. The adherent AMs in the 96-well plate containing bound M. tuberculosis were disrupted with 10% Triton X-100 (Sigma Chemical Co.). ⁵¹Cr-labeled M. tuberculosis organisms were quantified in each fraction (Model 5500 gamma counter, Beckman Instruments Inc., Palo Alto, CA) and percent attachment was expressed as follows: % attachment = (A/A + B) × 100, where A = ⁵¹Cr-labeled M. tuberculosis organisms bound to the AMs and B = unattached ⁵¹Cr-labeled M. tuberculosis organisms free in the medium. Maximal attachment occurred at 4 h, and this incubation was used for all subsequent experiments. To determine the percent of injury to M. tuberculosis organisms, the percent of ⁵¹Cr release was expressed as follows: % release = C/(A + B + C) × 100, where A = ⁵¹Cr-labeled M. tuberculosis organisms bound to the AMs, B = unattached ⁵¹Cr-labeled M. tuberculosis organisms free in the medium, and C = ⁵¹Cr-released into the medium.

The effect of calcium-free conditions on M. tuberculosis attachment was determined with the specific calcium chelator agent ethylene glycol-bis-(-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) and the nonspecific calcium specific chelator EDTA. The M. tuberculosis attachment assay was conducted in the presence or absence of EGTA or EDTA, using a final concentration of 5 mM, which effectively removes all available ionized calcium in the medium.

To determine the possible mechanisms of attachment, an attachment assay was performed to examine the effect on SP-A-mediated M. tuberculosis attachment to AMs of mannosyl-BSA (1,000 µg, with a molar mannose:albumin ratio of 26:1), type V collagen (1 mg/ml), deglycosylated SP-A (10 µg/ml), or polyclonal antibodies (100 µg/ml).

Statistical Analysis

The results are expressed as mean ± SEM. For each experiment, the differences between control and experimental data were compared by using Student's t test for comparison of two data sets or a one-way analysis of variance (ANOVA) for multiple comparisons. Significance was accepted at P < 0.05 (37).

	Results

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[¹²⁵I]SP-A Binding to M. tuberculosis Organisms

[¹²⁵I]SP-A binds to M. tuberculosis organisms in a time- dependent manner, and maximal binding occurs after 60 min (Figure 1a). The calcium requirement for optimal binding of SP-A to M. tuberculosis was determined by using increasing concentrations of extracellular calcium (Figure 1b). The results indicated that [¹²⁵I]SP-A binds to M. tuberculosis in a calcium concentration-dependent manner with maximal binding occurring at approximately 2 mM calcium. Exemplifying this is that in the absence of calcium, negligible amounts of SP-A bound to 1 × 10⁸ M. tuberculosis, whereas in the presence of 2 mM calcium, 58 ng of [¹²⁵I]SP-A was bound. Therefore, extracellular calcium is required for the optimal binding of SP-A to M. tuberculosis. Magnesium and Mn²⁺ also significantly increased SP-A binding as compared to control values; however, Ca²⁺ increased SP-A binding to M. tuberculosis to a greater degree than either Mg²⁺ or Mn²⁺ (Figure 1c) (both comparisons: P < 0.001).

View larger version (10K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   View larger version (24K): [in this window] [in a new window]   Figure 1.   (a) Binding of radioiodinated SP-A to M. tuberculosis organisms. 125I-labeled SP-A binds to M. tuberculosis organisms in a time-dependent fashion, reaching a maximum after 60 min. (b) Calcium is required for binding of radioiodinated SP-A to M. tuberculosis. The binding assay was performed with increasing concentrations of extracellular calcium, and the radioactivity bound to M. tuberculosis was measured. Maximum binding of SP-A to M. tuberculosis occurred at 2 mM calcium concentration. (c) Calcium significantly increased SP-A binding to M. tuberculosis as compared with the other divalent cations such as Mg2+ and Mn2+. Results are expressed as mean ± SEM of six experiments performed in triplicate (*P < 0.05).

The specificity and concentration dependence of [¹²⁵I]- SP-A binding to M. tuberculosis was demonstrated with increasing concentrations of [¹²⁵I]SP-A in the presence and absence of excess unlabeled SP-A (Figure 2a). Addition of increasing amounts of SP-A to M. tuberculosis organisms resulted in an increase in both total and nonspecific binding of SP-A to the organisms, with a saturation of specific binding occurring at concentrations in excess of 1,200 ng of added ¹²⁵I-labeled SP-A. Scatchard plots of the specific binding data were linear (R = 0.9), suggesting that a relatively homogenous population of binding sites may exist for SP-A on M. tuberculosis (Figure 2b). The binding dissociation constant was 1.9 × 10⁹ M, with an estimated 6.3 × 10² binding sites for SP-A/M. tuberculosis organism (mass of SP-A estimated as 750 kD) (38, 39). To examine reversibility of SP-A binding to M. tuberculosis, [¹²⁵I]SP-A at 2 µg/ml was incubated with M. tuberculosis organisms (1 × 10⁸) at 37°C for 60 min in order to saturate all binding sites on M. tuberculosis. Competition for [¹²⁵I]SP-A binding to M. tuberculosis organisms was provided by the addition of unlabeled SP-A for 1 to 24 h. Significant reversibility of SP-A binding was apparent by 1 h, and appeared to be maximal at 12 h (Figure 2c). The results of these experiments suggest that SP-A binding to M. tuberculosis organisms is reversible.

View larger version (16K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Figure 2.   Saturation and specific binding of radioiodinated SP-A to M. tuberculosis. Binding assays were performed with 125I-labeled SP-A with and without a 10-fold excess of unlabeled SP-A. (a) Saturation of specific SP-A binding to M. tuberculosis organisms demonstrated in the presence of 4,200 ng (excess) of added [125I]SP-A. Values are mean ± SEM of three experiments performed in triplicate. (b) Scatchard plot of specific binding data. The slope of the line represents a Kd of 1.9 × 109 M for the binding interaction of SP-A and M. tuberculosis. The number of SP-A binding sites per organism calculated from the x intercept is 6.3 × 102. (c) Reversibility of [125I]SP-A binding to M. tuberculosis organisms occurs within 1 h and is maximal at 12 h.

[¹²⁵I]SP-A binding assays were conducted with mannosyl-BSA or trypsin to determine whether SP-A binding to M. tuberculosis might involve recognition of a mannose-rich glycoprotein. [¹²⁵I]SP-A binding to M. tuberculosis was effectively reduced from 66.5 ± 8.4 ng to 23.7 ± 3.4 ng in the presence of mannosyl-BSA to 14.0 ± 1.6 ng on trypsin-pretreated M. tuberculosis organisms (P < 0.05 in each case) (Figure 3). However, there was no significant difference in SP-A binding to M. tuberculosis in the presence or absence of type V collagen (P > 0.60) (Figure 3). This suggests that the collagenlike domain of SP-A may not interact with M. tuberculosis organisms. However, the ability of mannosyl-BSA and trypsin to inhibit [¹²⁵I]SP-A binding to M. tuberculosis suggests that a mannosylated glycoprotein expressed by the organism is a possible ligand for SP-A. Further, a deglycosylated SP-A had minimal binding to M. tuberculosis, indicating that sugar moieties are important in SP-A binding (Figure 3).

View larger version (39K): [in this window] [in a new window]   Figure 3.   SP-A bound to the surface of M. tuberculosis. The binding assay was performed with [125I]SP-A and incubated with M. tuberculosis organisms in the presence or absence of type V collagen, mannosyl-BSA, and trypsin-pretreated M. tuberculosis organisms for 60 min at 37°C, and the radioactivity bound to M. tuberculosis was measured. The binding of SP-A to M. tuberculosis decreased in the presence of mannosyl-BSA and trypsin. However, collagen had no significant effect on binding of SP-A to M. tuberculosis. Addition of deglycosylated SP-A alone had no significant effect on binding of SP-A to M. tuberculosis. Results are expressed as mean ± SEM of experiments performed in triplicate (*P < 0.05).

To verify that SP-A-binding proteins are present on the surface of M. tuberculosis organisms, cell-wall proteins were isolated and resolved on SDS-PAGE, transferred to Immobilon-P membrane, and incubated with [¹²⁵I]SP-A. Silver-stained gels revealed the presence of multiple cell-wall proteins (Figure 4a). However, autoradiography of the ¹²⁵I-labeled SP-A revealed only one protein band, with an apparent molecular weight of 60 kD (Figure 4b).

View larger version (33K): [in this window] [in a new window]   Figure 4.   Identification of SP-A-binding proteins on M.tuberculosis. Cell-wall proteins were prepared from M. tuberculosis organisms to demonstrate that SP-A-binding proteins are present on the surface of the organisms. The isolated proteins were resolved on SDS-PAGE under reducing conditions and (a) stained with silver stain or (b) transferred onto Immobilon-P membrane, incubated with 125I-labeled SP-A, and autoradiographed. The positions of the known molecular weight standards are also indicated. The results indicate that SP-A binds to a 60-kD protein on the cell wall of M. tuberculosis.

Attachment of M. tuberculosis to AMs

M. tuberculosis attachment to AMs in the absence of SP-A increased as a function of time of incubation, reaching a maximum at 4 h (12.8 ± 0.9%) (Figure 5). Attachment increased in the presence of increasing concentrations of SP-A (1 to 10 µg/ml), reaching a maximum at 5 µg SP-A/ml (38.6 ± 2.9%, P < 0.001) (Figure 6a). There was no evidence of injury to M. tuberculosis organisms for up to 8 h of incubation with AMs, as measured by increased ⁵¹Cr release (data not shown).

View larger version (10K): [in this window] [in a new window]   Figure 5.   Attachment of M. tuberculosis to murine AMs. Attachment assays were performed at 4°C, using cultured AMs with 51Cr-labeled M. tuberculosis organisms at a 1:10 ratio. The results indicate that attachment of M. tuberculosis to murine AMs is a time-dependent process, reaching a maximum after 4 h incubation with AMs. Results are expressed as mean ± SEM of six experiments performed in triplicate (*P < 0.05).

View larger version (12K): [in this window] [in a new window]   View larger version (39K): [in this window] [in a new window]   Figure 6.   Role of SP-A in M. tuberculosis attachment to AMs. (a) Attachment of M. tuberculosis to AMs was examined in the presence of increasing concentrations of SP-A, and was found to increase significantly as the amount of SP-A was increased. Maximal binding occurred in the presence of 5 µg SP-A/ml. (b) Attachment of M. tuberculosis to AMs was examined after preincubation of the AMs with anti-SP-A antibody, and the attachment of M. tuberculosis was found to decrease significantly in the presence of rabbit anti-human SP-A antibodies. Results are expressed as mean ± SEM for a minimum of four experiments performed in triplicate (*P < 0.05).

To determine the role of SP-A in the attachment of M. tuberculosis to AMs, the effect of anti-SP-A antibodies was assessed in the attachment assay. Pretreatment of AMs with polyclonal anti-SP-A antibodies for 1 h resulted in a significant decrease in M. tuberculosis attachment to AMs, from 38.6 ± 2.9% to 18.7 ± 3.3% (P < 0.05) (Figure 6b). To demonstrate that rodent-derived SP-A functioned similarly to human SP-A, rat SP-A was assayed in the attachment assay, and showed no significant difference from human SP-A in mediating the attachment of M. tuberculosis to murine AMs (data not shown). A control of normal rat serum or heat-inactivated preimmune serum had no effect on M. tuberculosis attachment to AMs (data not shown). Likewise, attachment of M. tuberculosis to control wells containing no AMs was not significant (1.4 ± 1.0% to 4.5 ± 1.2%) over an 8-h incubation period. These data suggest that SP-A may function as a ligand that promotes the attachment of M. tuberculosis to AMs.

Addition of either EGTA or EDTA resulted in a significant decrease in M. tuberculosis attachment to AMs, from 38.6 ± 2.9% to 9.5 ± 1.4% and 9.0 ± 0.9%, respectively (P < 0.01 for both comparisons) (Figure 7). Hence, the SP-A-mediated attachment of M. tuberculosis to AMs is a calcium-dependent mechanism.

View larger version (30K): [in this window] [in a new window]   Figure 7.   Effect of calcium-free conditions on M. tuberculosis attachment to AMs. Attachment of M. tuberculosis to AMs was examined after preincubation of AMs in the presence or absence of SP-A, 2 mM calcium, and with or without 5 mM EDTA or 5 mM EGTA. The attachment of M. tuberculosis decreased significantly in the presence of EDTA and EGTA. Results are expressed as mean ± SEM for six experiments performed in triplicate (*P < 0.05, control versus SP-A; **P < 0.05, SP-A versus EDTA or EGTA).

To determine the possible mechanisms of SP-A-enhanced attachment of M. tuberculosis to AMs, M. tuberculosis and AMs were incubated with SP-A in the presence and absence of mannosyl-BSA or type V collagen, respectively. SP-A-enhanced attachment of M. tuberculosis to AMs was significantly decreased, from 38.6 ± 2.9% to 16.6 ± 1.5% (P < 0.001) in the presence of mannosyl-BSA, and to 19.1 ± 1.4% (P < 0.05) in the presence of type V collagen (Figure 8). The deglycosylated SP-A had no significant effect on M. tuberculosis attachment to murine AMs (14.3 ± 0.3%) as compared with control (12.8 ± 0.9%). These results suggest that both the carbohydrate-recognition domain and the collagenlike domain of SP-A are involved in SP-A-mediated attachment of M. tuberculosis to AMs.

View larger version (36K): [in this window] [in a new window]   Figure 8.   Effect of mannosyl-BSA, type V collagen, and deglycosylated SP-A on M. tuberculosis attachment to AMs. Attachment of M. tuberculosis to AMs was examined after preincubation of the AMs with mannosyl-BSA (1,000 µg, with a 26:1 molar ratio of mannose:albumin) or type V collagen (1 mg/ml). The attachment of M. tuberculosis decreased significantly in the presence of both mannosyl-BSA and type V collagen. Results are expressed as mean ± SEM for six experiments performed in triplicate (*P < 0.05, control versus SP-A; **P < 0.05, SP-A versus man-nosyl-BSA or type V collagen).

	Discussion

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The results presented in this study suggest that the attachment of M. tuberculosis organisms to AMs can be mediated by SP-A. Further, SP-A probably mediates this  attachment by recognizing M. tuberculosis via its carbohydrate-recognition domain. SP-A appears to bind to M. tuberculosis in a specific and saturable manner. Inhibition of SP-A-mediated attachment of M. tuberculosis to AMs by type V collagen is consistent with prior findings indicating that the collagenlike domain of SP-A may bind to AMs (20). Therefore, the data in the present study are consistent with a role of SP-A as a "bridge" between the organism and the AM, facilitating an important first step in the development of infection.

M. tuberculosis organisms gain access to the intracellular compartment of the AM early in the course of infection (40). In doing so, the M. tuberculosis organisms enter a stage in which their survival and replication are permitted, and thus convert the AM from a phagocytic cell of host defense into a potentially safe habitat for the microorganism. SP-A appears to facilitate attachment of M. tuberculosis to the surface of AMs. Recently, it has been suggested that the SP-A-mediated entry of microorganisms into monocytes does not trigger the respiratory burst, a finding consistent with this hypothesis (41).

SP-A binds to M. tuberculosis in a specific and saturable manner. Scatchard plot analysis indicates that the number of binding sites per M. tuberculosis organism is approximately 6.3 × 10², with a linear specific binding suggesting a homogenous group of binding sites. SP-A is an oligomer with a reported size ranging from 650,000 (42) to 1,600,000 (27). Because of its oligomeric nature and tendency to self-associate, the calculations of its molecular weight in kildaltons and the number of specific binding sites for it on M. tuberculosis should be interpreted cautiously. It appears that the carbohydrate-recognition domain of SP-A interacts with a glycoprotein on the surface of M. tuberculosis organisms, since binding is inhibited by either deglycosylated SP-A or by competition with mannosyl-BSA. Prior studies indicate that the carbohydrate-recognition domain binds to a variety of monosaccharides including mannose, glucose, galactose, and N-acetyl sugars (43); consequently, the carbohydrate profile of this putative glycoprotein receptor on M. tuberculosis may be diverse.

The investigation of SP-A binding with M. tuberculosis cell-wall proteins in the present study indicates that SP-A binds to a cell-wall protein with an apparent molecular weight of 60 kD. Previous studies described a 60-kD protein as an antigen complex on the surface of mycobacteria (44). Another study has reported that this protein complex is highly immunogenic and contains three moieties, consisting of lipid, polysaccharide, and protein (45). Although the polysaccharide sugar moieties were not identified, it is likely that the antigen complex contains sugar moieties recognized by SP-A, such as mannose. The immunogenic property of this antigen complex suggests that the protein is a cell-wall component and plays an important role in M. tuberculosis infection.

The molecular site on SP-A that recognizes AMs during the attachment of M. tuberculosis is probably the collagenlike domain of the molecule. Our data suggest that collagen inhibits the SP-A-mediated attachment of M. tuberculosis to AMs, although we cannot exclude the possibility that an interaction between SP-A and soluble collagen is responsible for the inhibition. These findings are consistent with those of Pison and colleagues, which indicate both the protein C1q and type V collagen inhibit SP-A attachment to AMs (20). Others have shown that the carbohydrate-recognition domain of SP-A can directly interact with AMs (46). It is possible that SP-A in its multimeric form interacts with AMs either through its collagenlike domain or through its carbohydrate-recognition domain.

SP-A levels are known to be markedly increased in the BALF of subjects infected with HIV (21). Whereas this may be an appropriate nonspecific host response to pathogens in the lower respiratory tract, it is possible that increased SP-A levels paradoxically represent a risk factor for infections such as tuberculosis. For instance, a recent study suggests that SP-A-mediated attachment of M. tuberculosis may be the principal mechanism of attachment for this organism in HIV-infected subjects (47). However, the precise mechanisms underlying attachment of M. tuberculosis to AMs in vivo still need to be clarified.

It is of interest that another pulmonary disorder associated with increased levels of SP-A is silicosis (48), a condition associated with a lifelong predisposition to tuberculosis (49). The mechanisms responsible for the increase of SP-A in silicosis are unclear, but this may also represent a compensatory mechanism of the lung to facilitate clearance of silica particles from the lower respiratory tract. Regardless of the process by which this occurs, it is of interest that two disorders so different in etiology, HIV and silicosis, show the common features of increased SP-A levels and increased risk for pulmonary tuberculosis. Further insight into the mechanisms of M. tuberculosis attachment to AMs may provide important information about the earliest stages in the pathogenesis of tuberculosis, and may permit the development of novel therapeutic strategies to modulate this attachment process.