Kalfas et al.

238

dards. However, at higher dilutions of the sample, only one protein has consistently demonstrated '^'I-Lm binding. A molecular weight of 5s62 kDa was estimated for this protein (Fig. 5). The protein was detected both in the whole cell extract and outer membrane preparations. Discussion

Microorganisms that colonize periodontai spaces are able to resist removal by the flow action of the crevicular fluid. The ability of these bacteria to adhere to tissue surfaces is therefore an important determinant of their establishment in the environment of the periodontai pocket. The Lm-rich periodontai tissues (25) may be ecologically advantageous for allogcnic succession of microorganisms that can bind to this matrix protein. Certain gram-negative bacteria associated with periodontai disease demonstrate attachment to basement membrane proteins and in particular to Lm; other gram-positive microbes bind less (38). In the present study, the interaction of another gram-negative periodontitis-associated bacterium, P. in-

I

Moiarity

Fig. 3. Stability of bacteria-Lm eomplex in the presence of NaCl (•), urea (A), and KSCN ( • ) . The Ltn binding values are expressed relative to the binding in PBS, which is considered 100%.

ndin

100-

80-

5 60-

40-

IK 20 •

0

JA^f, 10

,-,,.,.-*, f, n I'l r r f r t i 20

30

40

50

60

Time (min)

Fig. 4. Thermo-sensitivity of Lm-binding cotnponent(s) in P. intermedia strain OMGS105. D; 60°C; A; 80 "C; O; 100"C.

Mr 212 — 1 ro94— "m 76 — 67— m 53— 4 3 — «W

w

-62

30 — 20 — 14 —

Fig. 5. Demonstration of the Ltn-binding protein (62 kDa) in P intermedia OMGS105. A. SDS-PAGE of molecular weight standards. B. SDS-PAGE of whole cell extract. C. Autoradiogram with '-'FLm of whole cell extract proteins (from SDS-PAGE) electroblotted to nitrocellulose membrane

termedia, with Lm is demonstrated. Most of the test strains exhibited moderate to high binding capacity and about half bound more than 30% of the protein added. The latter figure is similar to the binding values reported for E nucieatum and P. gingivalis (38) but higher than Staphyloeoecus aureus, streptococci and Eseheriehia co//(18, 21, 31, 32). However, the Lm binding of P. intermedia varied with the environmental pH, a pattern also observed with other protein-bacteria interactions (15, 22). Further characterization of the Lm interaction with a highly binding strain revealed a binding that was inhibited only by lactofcrrin atnong the different proteins and carbohydrates tested. Lactofcrrin is secreted in elevated concentrations by ncutrophilic leukocytes, the predominant inflammatory cell population in the crevicular space, during periodontai diseases (3, 26). Thus, interaction of lactofcrrin with P. intermedia lnight play a protective role by interfering with the bacterial adhesion to the Lm-rich periodontai tissues. Unlabeled Lm in excess failed to inhibit '~'I-Lm binding. This is probably due to the unsaturation of the binding sites or to the coaggregation of the unlabeled Lm molecules with the labeled Lm on the

bacterial surface. It may also be possible that the affinity for labeled Lm is higher than for the unlabeled Lm. Such irreversible matrix protein binding has also been reported for other bacteria (10, 21). The thermolability of the interaction and its sensitivity to broad-spectrum proteases suggest a proteinaceous nature of the binding sites, as also reported for other bacterial Lm-binding cotnponents (7, 18, 32). However, proteolytic enzymes of P. intermedia do not appear to interfere with the interaction, since the Lm binding was not affected by the presence of protease inhibitors. The interaction seems to be dependent on the structural conformation of the interacting molecules since the binding was dissociated by the chaotropic agent KSCN. The results of the competitive experiments with carbohydrates did not provide support for a lectin-mcdiated interaction. The Lm binding cotnponent of strain OMGS105 had a molecular weight of «62 kDa, as revealed by SDS-PAGE and Western blot analysis. However, when the sample load was increased, additional protein bands, one at about 95 kDa and four in the range of 20-35 kDa, also interacted with Lm (not shown). A 62 kDa Lm-binding protein has been reported for C. albieans and murine EHS tumor cells (7, 13). This protein was suggested as a degradation product of the 68 kDa Lm receptor on these cells. It is possible that in P. intermedia the low-tnolecular-wcight proteins are degradation products of the 62 kDa, compoticnt and the 95 kDa band probably is an aggregate. Another possibly is that these additional bands are separate Lm-binding proteins. Taken together, the results of the interaction at different Lm conccn- , trations, the competitive experiments and the Western blot analysis rather indicate that the Lm binding to P. intermedia is a nonspecific, probably hydrophobic interaction with certain cell surface proteins. A majority of P. intermedia strains isolated from periodontai pockets expresses such Lm-binding cell surface structures. This bacterial property may enable colonization of the periodontai tissue by P. intermedia.

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