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Infection and Immunity, May 2002, p. 2670-2675, Vol. 70, No. 5
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.5.2670-2675.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Veterinary Pathobiology, Purdue University, West Lafayette, Indiana 47907
Received 3 August 2001/ Returned for modification 27 September 2001/ Accepted 22 January 2002
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The ability of a microorganism to bind fibronectin (FN) may potentially facilitate its colonization of the host through attachment to the extracellular matrix in areas of epithelial damage. Furthermore, because several host cell integrins have binding sites for FN, the ability of a microorganism to bind soluble FN establishes a bridge between the organism and the host cell cytoskeleton, a condition necessary for the internalization of the microbe by the cell (3, 6). Fibronectin attachment proteins (FAPs) comprise a family of FN-binding glycoproteins that are expressed by several species of mycobacteria (16-19, 24). Expression of FAP is critical for the attachment of Mycobacterium bovis BCG and M. avium subsp. avium to tissue in vivo and ex vivo (11, 24). Moreover, the internalization of M. bovis BCG and Mycobacterium leprae by cultured epithelial cells was shown to be a FAP-dependent process (10, 18).
M. avium subsp. paratuberculosis also expresses a FAP (designated FAP-P) which mediates soluble FN binding (19). However, unlike the FAPs of other mycobacteria, FAP-P is not present on the surface of the organism (19). Thus, the manner in which FAP-P engages FN may sequester the cell binding domain from host cell receptors. As a result, binding and ingestion of M. avium subsp. paratuberculosis by host cells would be FN independent.
To examine the effect of the interaction of FAP-P and FN on the ability of M. avium subsp. paratuberculosis to attach to and invade epithelial cells, FN-opsonized and nonopsonized organisms were used to infect T-24 human bladder carcinoma cells (a gift from J. S. Schorey, University of Notre Dame, South Bend, Ind.) and Caco-2 human intestinal adenocarcinoma cells (supplied by M. Popielarczyk, Purdue University, West Lafayette, Ind.). T-24 cells were propagated as described previously (18). Caco-2 cells were propagated in minimum essential medium with Earle's salts, L-glutamine, and nonessential amino acids (Life Technologies) supplemented with 20% fetal bovine serum (FBS) and antibiotic-antimycotic solution. M. avium subsp. paratuberculosis strain 5781 (19) was propagated in Middlebrook 7H9 broth (Becton Dickinson, Cockeysville, Md.) supplemented with 10% oleic acid-albumin-dextrose-catalase (Becton Dickinson), 0.05% Tween 80 (Sigma, St. Louis, Mo.), and 2 µg of mycobactin J (Allied Monitor, Fayetteville, Mo.) per ml in tissue culture flasks.
M. avium subsp. paratuberculosis cultures were centrifuged for 15 min at 1,600 x g. The bacterial pellet was mixed thoroughly by vigorous pipetting and vortexing in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (Sigma) to generate a single-cell suspension and resuspended in a volume of PBS containing 0.05% Tween 20 sufficient to yield 5 x 106 CFU/ml. Five milliliters of bacterial suspension was centrifuged as described above, and the pellet was resuspended in 5 ml of ANT buffer (10 mM ammonium acetate, 0.85% sodium chloride, 0.05% Tween 20; pH 3) After incubation for 5 min at room temperature, the suspension was centrifuged as before. The pellet was resuspended in 5 ml of ANT buffer (pH 6) and split into four 1-ml units. Twenty micrograms of bovine FN (Biomedical Technologies, Stoughton, Mass.) was added to each of two tubes. After incubation for 1 h at 37°C, the bacterial suspensions were diluted 1:1 with either Iscove’s modified Dulbecco’s medium or minimum essential medium basal medium (no FBS or antibiotics) and used to infect T-24 or Caco-2 cells, respectively.
T-24 and Caco-2 cells (5 x 104) in complete media were seeded into each well in the top and bottom rows, respectively, of an eight-well chamber slide (Lab-Tek II; Nunc, Napierville, Ill.). The wells were precoated with 100 µg of murine laminin (Sigma)/ml. After being seeded, cells were allowed to attach for 9 h at 37°C in 5% CO2. The wells were washed twice with prewarmed PBS, and 0.2 ml of FN-opsonized or nonopsonized bacteria, prepared as outlined above, was added to the appropriate wells to yield a multiplicity of infection of 10. After incubation for 3 h at 37°C in 5% CO2, the wells were washed twice with PBS and bacterial attachment and invasion were assessed by the double immunofluorescence assay described previously (10). Briefly, the wells were blocked with 5% FBS in PBS followed by incubation with a 1:100 dilution of rabbit anti-M. bovis BCG immunoglobulin G (IgG; Dako, Carpinteria, Calif.). After being washed in PBS, the slides were incubated with a 1:200 dilution of tetramethyl rhodamine isothiocyanate-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) to label extracellular bacteria. Host cell membranes were rendered permeable by exposure to methanol for 5 min, and the staining procedure was repeated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Antibodies, Inc., Davis, Calif.). Slides were examined with an Optiphot-2 epifluorescence microscope (Nikon, Mellville, N.Y.). Attached bacteria fluoresced both red and green, whereas internalized bacteria stained green only. For most experiments, 200 cells were counted per treatment, and the number of cells having attached or internalized mycobacteria was recorded. Three hundred cells were counted for each antisense strain and for vector controls. Cells with both attached and internalized mycobacteria were scored as ingesting cells.
Opsonization of bacteria with FN resulted in a nearly 2-fold increase in the number of T-24 cells binding mycobacteria and a 1.5-fold enhancement in bacterial attachment to Caco-2 cells (Fig. 1A). FN-opsonized bacteria were also more readily internalized, with 2.5- and 3-fold increases in ingestion of organisms by T-24 and Caco-2 cells, respectively (Fig. 1B). These observations indicate that opsonization of M. avium subsp. paratuberculosis with FN enhances the ability of the organism to adhere to and invade epithelial cells. In this and all other experiments, no more than 10% of the cells binding mycobacteria that were untreated or treated only with FN had three or more organisms attached. Similarly, no more than 12% of the ingesting cells counted in any experiment contained more than two mycobacteria.
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One M. avium subsp. paratuberculosis clone transformed with pTS026 (designated strain 5781/26A) and two clones transformed with pTS028 (designated strains 5781/28F and 5781/28H) were chosen for further study following screening of kanamycin-resistant colonies for their FN binding capacities (19). The presence of antisense FAP-P vectors in each strain was confirmed by Southern hybridization, and the vectors were estimated by densitometry to be present at roughly 60 copies per organism in strain 5781/28H and approximately 40 copies per organism in the other strains (data not shown). Soluble FN binding by strains 5781/26A and 5781/28F was reduced by approximately 10% relative to that of the vector control strain (designated 5781/W4C), and that of strain 5781/28H was reduced by approximately 30% (data not shown).
Equal amounts of total protein from antisense mutant lysates were separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis, electroblotted to nitrocellulose, probed with anti-FAP IgG (purified by protein A chromatography from rabbit anti-FAP serum supplied by J. S. Schorey), and subjected to densitometric analysis (Scion Image; Scion Corp., Frederick, Md.). Different patterns of FAP-P expression were revealed in each of the three mycobacterial strains harboring antisense FAP-P vectors (Fig. 3). Cell-associated FAP-P from strain 5781/W4C migrated in sodium dodecyl sulfate-12% polyacrylamide gels as two bands: a minor band at 54 kDa and a major band at 49 kDa. This banding pattern is consistent with that previously reported for wild-type M. avium subsp. paratuberculosis (19), and it has been speculated that the low-Mr form of FAP is generated by C-terminal cleavage of the high-Mr form (5). Both bands were present in sonicates of strain 5781/26A and had essentially the same proportionate intensities as those of the vector control strain, but total FAP-P expression was reduced by 10 to 15%. Strain 5781/28H also expressed both forms of FAP-P in the same proportion as that seen for strain 5781/W4C. Two additional anti-FAP-reactive species migrating as 52- and 48.5-kDa bands were also observed. These probably represent truncated variants of the 54- and 49-kDa forms of FAP-P, respectively. Given this, FAP-P expression in strain 5781/28H was approximately 72% of that observed for strain 5781/W4C. The reduction in FAP-P expression by strains 5781/26A and 5781/28H appeared to correlate with the reduction in soluble FN binding observed for these strains (r2 = 0.93). The FAP-P banding pattern was reversed in strain 5781/28F, with the intensity of the 54-kDa form being twice that of the vector control and the 49-kDa form appearing as a faint band. Total FAP-P production by this strain was 31% lower than that by strain 5781/W4C.
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To determine the effect of attenuated FAP-P expression on the attachment and internalization of M. avium subsp. paratuberculosis by epithelial cells, infections of T-24 cells and Caco-2 cells with FN-treated FAP-P mutant mycobacteria and with FN-opsonized strain 5781/W4C were compared. In preliminary experiments, strain 5781/W4C bound to T-24 and Caco-2 cells at levels similar to those of the parent strain (20 to 25% and 25 to 30%, respectively; data not shown). However, in the experiments shown in Fig. 4, FN-opsonized strain 5781/W4C attached to 40% of the T-24 cells counted. The reason for this observation was not immediately clear. The attachment of the vector control strain to Caco-2 cells and the invasion of both cell lines by this strain (5 to 7% of cells counted) were similar to those previously noted for strain 5781. The binding of strain 5781/26A by both cell lines was approximately 35% lower than that observed for the vector control strain; however, this difference was not significant (Fig. 4A). The attachment of strain 5781/28F to T-24 and Caco-2 cells was diminished by almost 50%, and this reduction was significant for Caco-2 cells. Strain 5781/28H bound to 55% fewer T-24 cells and 57% fewer Caco-2 cells than did the vector control strain.
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In a previous study, M. avium subsp. paratuberculosis bound FN most efficiently at pH 3 (19), suggesting that FN binding is activated by passage through the ruminant digestive tract, allowing the organism to efficiently bind FN that is secreted into the duodenum (23). The present observation that bacterial attachment to and ingestion by both T-24 and Caco-2 cell lines are markedly enhanced by FN imparts additional significance to FN binding as a potential virulence trait of M. avium subsp. paratuberculosis.
Because treatment with FAP-A peptide did not interfere with M. avium subsp. paratuberculosis attachment to Caco-2 cells and actually enhanced its attachment to T-24 cells regardless of the presence of FN, these results suggest that this organism may also attach to these cells in an FN-independent manner. If that is the case, these unidentified cellular receptors are not likely to be associated with the cytoskeleton, since internalization was abolished in FAP-A peptide-treated replicates. It is also possible that FAP-A peptide may have served to nonspecifically bridge mycobacteria to the cells.
The reduction in FN binding observed for antisense FAP-P mutant strains was disappointingly low. This was not surprising, given that the antisense fragments used were very G+C rich (75 and 77% G+C for the fapP inserts present in pTS026 and pTS028, respectively) and were therefore likely to be able to form stable secondary structures. Nevertheless, FAP-P expression was clearly attenuated in all strains carrying antisense expression cassettes, and the number of antisense plasmid copies present in these strains may have driven this effect.
The molecular basis for the origin of the additional anti-FAP-reactive bands in strain 5781/28H is unclear. Antisense RNA may have mediated the truncation of FAP-P in strain 5781/28H, possibly through latent nuclease activity. The large difference in the number of antisense plasmid copies between strains 5781/28F and 5781/28H may explain why these additional bands were not observed in the former strain. Alternatively, the presence of these additional anti-FAP-reactive bands may indicate the presence of a persistent subpopulation of organisms that contain a fapP mutation other than that observed in strain 5781/28F.
Studies with neonatal bovine calves and goat kids have shown that like many other invasive enteropathogens, M. avium subsp. paratuberculosis enters Peyer's patches through M cells, which are specialized antigen-sampling cells present in the dome epithelium (12, 20). The mechanism by which nonprofessional phagocytes such as M cells ingest these organisms is presently unknown. T-24 cells have been shown to bind and ingest mycobacteria via 
5ß1 integrin in a manner that requires the interaction of FN and FAP (10, 18). However, this cell line may not be representative of intestinal epithelial cells. The means by which Caco-2 cells, which also express 5ß1 integrin (4), bind and ingest organisms is likely to be more relevant to Johne's disease, particularly because these cells can be induced to develop an M-cell-like phenotype in vitro (9). The fact that disruption of the interaction of FAP-P and FN inhibits bacterial ingestion by Caco-2 cells implies that 5ß1 integrin serves as the receptor for FN-opsonized M. avium subsp. paratuberculosis in these cells, as well. The results of the present study indicate that FN-bound FAP-P in the cell envelope is a significant adhesin and the dominant invasin of M. avium subsp. paratuberculosis for Caco-2 cells as well as T-24 cells. M cells, unlike other intestinal epithelial cells, express ß1 integrins on their apical surfaces (1). Therefore, the mechanism described for the attachment and invasion of the cell lines used herein may be relevant in vivo.
The incubation time used to assess attachment and ingestion of M. avium subsp. paratuberculosis in this study was identical to that used for M. leprae in a previous investigation (18) but considerably longer than that typically used to study these events with other invasive pathogens. Despite this extended time period, the frequencies of internalization of M. avium subsp. paratuberculosis strains 5781 and 5781/W4C by T-24 cells were approximately half those reported for M. bovis BCG and M. leprae (10, 18). This may reflect differences in the subcellular location of FAP in these species: FAP-B and FAP-L are surface exposed in M. bovis BCG and M. leprae, respectively, whereas FAP-P is not (10, 16, 18, 19). This, in turn, may affect the way in which FAP-P-bound FN is able to engage host cell receptors. Indeed, the proportionate reductions in attachment and internalization of the antisense FAP-P mutants to epithelial cells were greater than those that could be predicted from the soluble-FN binding capacity of these strains. The effect of antisense FAP-P mutations on ingestion by epithelial cells was particularly acute. The effect of the manner in which FAP-P binds FN in the mycobacterial cell envelope may have combined with that of diminished FAP-P expression in antisense mutants to interfere with receptor clustering, which is necessary to initiate integrin-mediated internalization of microorganisms (2, 6).
The relatively slow penetration of cells observed for M. avium subsp. paratuberculosis in vitro is consistent with earlier in vivo observations. Although M. avium subsp. paratuberculosis was detected in the dome epithelium covering Peyer's patches within 30 min of inoculation into caprine gut loops (20), very few acid-fast bacilli were observed in cross sections of ileal domes 5 h after injection into bovine calf gut loops (12).
M. avium subsp. paratuberculosis may express additional colonization and penetration factors that are not apparent in the present in vitro system. Reducing the influence of FAP-P through mutation may aid in the identification of factors other than FAP-P produced by this organism that may participate in attachment and internalization processes. Defining the nature of the initial host-pathogen interface will allow the design of adhesin or receptor analogues that can be added to milk replacer or feed to competitively block the attachment of M. avium subsp. paratuberculosis to the host. These approaches have been successfully employed against human mucosal pathogens in animal model systems (7, 8). The development of the FAP-P mutants described herein will facilitate the investigation of the role of FAP-P in the attachment and internalization of M. avium subsp. paratuberculosis by M cells in vivo and provide insight into the pathobiology of this organism and the pathogenesis of Johne's disease.
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ACKNOWLEDGMENTS |
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This work was supported by a Purdue University School of Veterinary Medicine Internal Grant.
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FOOTNOTES |
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