Inhibition of Bacterial Aggregation by Serum- and Blood-Derived

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INFECTION AND IMMUNITY, Jan. 1984, p. 386-39 Vol. 43, No. 1 19-9567/84/1386-5$2.

1 INFECTION AND IMMUNITY, Jan. 1984, p Vol. 43, No /84/1386-5$2./ Copyright 1984, Amerian Soiety for Mirobiology Inhibition of Baterial Aggregation by Serum- and Blood-Derived Proteins DANIEL MALAMUD,* CAROLYN BROWN, AND RON GOLDMAN Department of Biohemistry, University of Pennsylvania, Shool of Dental Mediine, Philadelphia, Pennsylvania 1914 Reeived 19 May 1983/Aepted 13 Otober 1983 Human and animal sera ontain potent inhibitors of saliva-mediated aggregation of oral streptooi. The inhibitors onsist of a high-moleular-weight heat-labile fator and a lower-moleular-weight heat-ativated fator. The latter appears to be serum albumin. Analyses of purified blood-derived proteins indiated that several high-moleular-weight proteins (fibrinogen, fibronetin, and ferritin) were able to inhibit aggregation at low onentrations. These data suggest that high-moleular-weight proteins may modulate the aggregation proess. Saliva-mediated baterial aggregation involves a sequene of steps initiated by the binding of agglutinins to the ell surfae and followed by a temperature-dependent agglutination of bateria (9). Aggregation demonstrates baterial speies and strain speifiity and alium dependene (7-9). The salivary agglutinins appear to be high-moleular-weight glyoproteins sereted from both parotid and submandibular glands (4). To investigate the moleular properties of the aggregation proess, we have been studying a variety of inhibitory moleules, most of whih appear to interat with the salivary agglutinins rather than with the bateria. For example, streptooal aggregation mediated by salivaryderived agglutinins an be inhibited by extrats from human polymorphonulear leukoytes and platelets (8a). In the present report, we demonstrate that serum ontains at least two inhibitory ativities-one is heat labile and has a high moleular weight (>16,), whereas the other is heat ativated and appears similar or idential to serum albumin. The inhibitory ativities in serum are ompared with a number of known serum and blood ell-derived proteins. Of these proteins, fibrinogen is the most potent inhibitor of saliva-mediated aggregation. MATERIALS AND METHODS Stimulated human parotid saliva was olleted with a modified Carlson-Crittenden up plaed over the opening of the parotid dut. Streptoous sanguis M5 and Streptoous mutans KPSK2 were grown in brain heart infusion (Difo Laboratories), washed in phosphate-buffered saline (PBS;.1 M K2HPO4 + KH2PO4,.15 M NaCl [ph 7]), and frozen in portions ontaining 11 bateria per ml. Human fibronetin was obtained from Collaborative Researh. Histones and serum proteins were obtained from Sigma Chemial Co. Aggregation assay. Saliva-mediated baterial aggregation an be monitored either by the entrifugation assay with 3Hlabeled bateria (6) or by following the derease in turbidity with unlabeled bateria (2). We have previously reported that the turbidimetri assay is more sensitive for analysis of inhibitory moleules (8a). For this assay, uvettes ontained.1 to.2 ml of parotid saliva plus.2 ml of unlabeled bateria with PBS or inhibitors added to a total volume of 1. ml. Samples were mixed and inubated at 37 C, and the derease in optial density at 675 nm was monitored for several hours. The perent aggregation was determined from * Corresponding author. 386 the linear portion of the dose-response urve when 3 to 5% of the bateria in the saliva ontrols were aggregated. Results are expressed as perent of the total bateria aggregated after subtration of a blank ontaining bateria and PBS only. Serum was frationated on Affi-Gel blue (Bio-Rad Laboratories) with a small olumn (bed volume 1 ml) prewashed - with.2 M phosphate buffer (ph 7). Adsorbed proteins were eluted with 1.4 M NaCl. Gel exlusion hromatography was arried out on a olumn (1.5 by 54 m) of Sepharyl 2 SF (Pharmaia Fine Chemials) with PBS as eluent. Protein was monitored at an optial density of 22 nm or by the Lowry method with bovine serum albumin (BSA) as a standard. Serum proteins were also separated by isoeletri fousing in 4% polyarylamide gels ontaining 2% Ampholines (3). Gels were foused to equilibrium, setioned, and dialyzed overnight against PBS. After dialysis, the eluted frations were tested for aggregation inhibitory ativity. Repliate gels were used for determination of the ph gradient and for protein staining with Coomassie blue. RESULTS Human serum was tested as an inhibitor of saliva-mediated baterial aggregation by the turbidimetri assay. In a standard 1-ml assay, serum inhibited the aggregation of both S. sanguis M5 (5% inhibition [151 =.31 RIl) and S. mutans KPSK2 (Io = 3.8,Il) in a dose-dependent reation (Fig. 1). Owing to the inreased sensitivity of S. sanguis, this speies was used for the remaining studies. As shown in Table 1, sera obtained from humans, rabbits, hikens, horses, and mie all inhibited baterial aggregation mediated by human salivary agglutinins. Serum alone had no effet on baterial aggregation in the absene of agglutinins. The serum inhibitory ativity was stable after storage at 4 C, freezing, or overnight dialysis against PBS. To determine whether inhibitory ativity was heat labile, serum samples were heated for 3 min at 37 to 1 C and then tested for the ability to inhibit saliva-mediated baterial aggregation. Preheating of serum above 8 C inreased the inhibitory ativity. A dose-response urve of heated (85 C) versus unheated serum indiated that the I5o was dereased from.3 to.8,ul; i.e., heated serum was approximately four times more potent as an aggregation inhibitor (Fig. 2). Heat ativation was seen within 5 min at 85 C and reahed a maximum value after 15 min (Fig. 3). Serum (.8 ml) was dialyzed against.2 M phosphate

2 VOL. 43, 1984 SERUM INHIBITS BACTERIAL AGGREGATION 387 sonquis M5 _J S. mutans KPSK2. 4-.) U ) PI // / I5z3.8A Human serum, Al/mI FIG. 1. Inhibition of saliva-mediated aggregation of S. sangiuis M5 and S.,nttans KPSK2 by human serum. Portions of S. sangiis M5 () or S. mutans KPSK2 () were inubated with parotid saliva and inreasing onentrations of human serum in a total volume of 1. ml. Results are expressed as perent inhibition relative to a ontrol sample with bateria and saliva only. The Io onentration of serum is indiated. buffer and then frationated on an Affi-Gel blue olumn. Unadsorbed proteins were eluted with 2 ml of phosphate buffer. The adsorbed proteins were eluted with 1.4 M NaCl. Aggregation inhibition ativity was obtained in both the unadsorbed and adsorbed frations but only the adsorbed ativity demonstrated heat ativation (data not shown). Serum proteins were separated on polyarylamide gels by isoeletri fousing. After fousing overnight, gels were setioned into.5-mm piees and dialyzed against PBS for 24 h to elute protein from the gels and remove Ampholines. Frations were tested for inhibition of saliva-mediated aggregation. As shown in Fig. 4, aggregation inhibitory ativity was found between pls of 6. to 7.2 and at the top of the gel surfae. To resolve the aggregation inhibitory ativity by moleular size, serum was loaded onto a Sepharyl 2 SF olumn and eluted with PBS. Protein was monitored by following the optial density at 22 nm, and frations were tested for inhibitory ativity. As shown in Fig. 5, most of the inhibitory ativity had moleular weights of 16, to 5,. Samples from three regions (A, B, and C) were tested for inhibitory ativity before and after heating at 85 C for 3 min. Fration A was heat labile, fration B was unaffeted Ii by heating, and only fration C demonstrated heat ativation of inhibitory ativity. Sine results with Affi-Gel and Sepharyl frationation suggested that the heat-ativated inhibitor in serum might be albumin, we tested Cohn fration V and purified bovine albumin (Sigma). Both of these preparations inhibited baterial aggregation, and inhibitory ativity was inreased 8- to 1-fold by heating at 85 C. Analysis of albumin by sodium dodeyl sulfate-polyarylamide gel eletrophoresis demonstrated that after heating to 85 C, there was a derease in the 56, omponent and the generation of a series of highermoleular-weight bands (data not shown). To gain insight into the nature of the inhibitory moleules, we began to study purified ompounds as inhibitors of baterial aggregation. Initial studies were direted at highly harged moleules, sine Ginsburg and Quie (5) have impliated harged moleules as modulators of inflammation. A series of aidi ompounds inluding heparin and DNA were found not to influene saliva-mediated baterial aggregation. A survey of the histones, however, revealed that some were inhibitors, whereas others atually indued baterial aggregation in the absene of salivary agglutinins. As shown in Table 2, there appears to be a orrelation of stimulatory or TABLE 1. Inhibition of baterial aggregation by human and animal sera" Serum ( Il/ml) '4 Inhibition Human (.2) Human (1.)... 1 Rabbit (.2) Rabbit (1.)... 1 Chiken (.2) Chiken ( Horse (.2) Horse (1.)... 1 Mouse (.2) Mouse (1.) " Eah serum sample was tested at.2 and 1. p.1 in a 1-ml reation volume as inhibitors of saliva-mediated aggregation of S. sangguis M5 by the turbidimetri assay. Results are expressed as perent inhibition ompared with ontrols with saliva and bateria only for tripliate determinations. 6-4 / 2 / Human serum, Al/ml FIG. 2. Inhibition of baterial aggregation by heated versus unheated serum. A titer of serum () or serum preheated to 85 C for 3 min () was determined by the turbidimetri assay with S. sanguis MS.

3 388 MALAMUD, BROWN, AND GOLDMAN INFECT. IMMUN. t) _, 4- '4-4 a Time at 85, min. FIG. 3. Heat ativation of serum inhibitory ativity. Serum was heated to 85 C for 5 to 6 min and then tested for inhibitory ativity for S. sanguis aggregation. inhibitory ativity with the relative ontent of lysine and arginine. Histones ontaining high amounts of arginine-indued baterial aggregation, whereas lysine-rih histones inhibited saliva-mediated aggregation. Complete dose-response studies for the inhibition of ba- 18-.o 5,C I 4 U Fration number Fration number FIG. 4. Frationation of aggregation inhibitory ativity by isoeletri fousing. Serum was applied to the top (basi) end of gels ontaining 4% polyarylamide and 2% Ampholines. Gels were run overnight at 25 V at 4 C, setioned into.5-mm piees, and dialyzed overnight against PBS. Portions of the eluted frations were tested for aggregation inhibition ativity with S. sanguis M5. The ph was determined on a repliate gel. This experiment was arried out three times with similar results. terial aggregation were arried out with a series of bloodderived proteins. The data are expressed as the '5 for the inhibition of saliva-mediated baterial aggregation in Table 3. There is a wide range of inhibitory potential, with fibrinogen being the most potent substane tested as an inhibitor of 65, 158,.5 B~~~~~~~~~~~~~' 3.. o m a....6 S * S2 1 FIG. 5. Frationation of inhibitory ativity on Sepharyl 2 SF. Serum was loaded onto a Sepharyl 2 SF olumn, and protein( ) and aggregation inhibitory ativity () was monitored. Three regions, as indiated, were tested, dialyzed against 1:1 PBS, and lyophilized. Eah of these frations was tested for inhibitory ativity before and after heating to 85 C for 3 min. The results shown are from one of three olumn runs, eah giving onsistent inhibitory patterns.

4 VOL. 43, 1984 saliva-mediated aggregation. As an be seen in Table 3, egg white albumin, like BSA, shows heat-stimulated inhibitory ativity. Analysis of egg white albumin by sodium dodeyl sulfate-polyarylamide gel eletrophoresis revealed the appearane of higher-moleular-weight forms as a result of heating to 85 C as had been seen with BSA. The inhibitory ativity of Cohn fration II was heat labile. DISCUSSION Serum ontains potent inhibitors of saliva-mediated strepnulear leukoytes and platelet extrats (8a), it appears that the inhibition involves an interation of the serum fators with salivary agglutinins, rather than a binding of the inhibitor to the baterial surfae. For example, pretreatment of bateria with serum followed by washing does not lead to inhibition of aggregation (data not shown). Dose-response studies with sera from different animal speies showed similar 15 values, with the exeption of mouse sera whih was a less potent inhibitor. The presene of inhibitory ativity in sera from diverse animal speies against human saliva suggests that the inhibitor is a ubiquitous serum omponent. Erison et al. (1) reported that anti-immunoglobulin antisera and high onentrations of human serum albumin more effetively inhibited saliva-mediated aggregation of S. sanguis than of S. mutans. As in the present study, the inhibition appeared to be on the salivary agglutinin rather than on the baterial omponent. A surprising finding was that the preheating of serum led to a marked inrease in inhibitory ativity. Frationation studies of human serum revealed at least two inhibitory frations-a high-moleular-weight (>16,) heat-labile omponent and a low-moleular-weight (-6,) omponent that was heat ativated within 5 min at temperatures above 8 C. This heat-ativated omponent appears idential to serum albumin, and indeed, purified BSA inhibits baterial aggregation. Heat ativation appears to involve the formation of high-moleular-weight aggregates of albumina phenomenon observed with serum, purified BSA, and egg white albumin. The seond inhibitory fration in serum has a moleular weight of >16, and a pl of 6. to 7.2. These moleular harateristis resemble immunoglobulin G (IgG), however, we were unable to deplete inhibitory ativity from human serum with protein A or anti-human IgG. There is no obvious orrelation between protein pl and inhibition of TABLE 2. Histone Histones as aggregators and inhibitors of baterial aggregation' Lys-Arg % Aggregation 5 for saliva- (1 p.g of aggregation histone/mi) (,ug/ml) without saliva H H H2A H2B Hl a Eah histone was tested at a series of onentrations as a stimulator (without saliva) or inhibitor (with saliva) of S. sanguis M5 aggregation. The perent aggregation shows results obtained with 1 jg of eah histone per ml. The 15 indiates the onentration of histone giving 5% inhibition of saliva-mediated aggregation. Histones H3, H4, and H2A were not tested as inhibitors sine they demonstrated high endogenous aggregation ativity. SERUM INHIBITS BACTERIAL AGGREGATION 389 TABLE 3. Inhibition of saliva-mediated aggregation of S. sanguis M5 by purified proteins' Substane SLg/ml nm Fibrinogen Fibronetin Ferritin 4 8 Latoferrin 9 12 Cohn fration II 4 27 Cohn fration II, heated to 85 C Egg white albumin Egg white albumin, heated to 85 C BSA 9 14,5 BSA heated to 85 C 1 15 a Eah substane was tested as an inhibitor of saliva-mediated aggregation at six onentrations. The 15 results are given in mirograms of inhibitor per milliliter of reation mixture and as nanomolar onentration. Cohn fration II, egg white albumin, and BSA were tested before and after heating (85 C for 3 min). -, No inhibitory ativity. baterial aggregation. However, most of the potent inhibitors are high-moleular-weight proteins (fibrinogen, 341,; fibronetin, 44,; ferritin, 45,). Coupled with the observation that heating bovine and egg white albumin generates high-moleular-weight aggregates with inreased inhibitory potential, this suggests the possibility that highmoleular-weight ompounds inhibit aggregation by physially bloking the binding of agglutinins to the baterial surfae. The biologial role of serum inhibitors of baterial aggregation is not lear. Although serum albumin is present in saliva, the requirement for heat ativation suggests that this moleule does not play an important role in situ. If baterial aggregation is viewed as a means of learing miroorganisms from the oral avity (1), then inhibition of this aggregation ould serve to promote baterial olonization. It is possible that in the ourse of gingival inflammation and periodontal disease, the inreased serum omponents released into the gingival revie ould serve to impede salivary antibaterial ativities and exaerbate the disease proess. Sine the olonization of bateria within the oral avity represents an interplay of learane and adherene mehanisms (1), seletive inhibitors an failitate the identifiation of the salivary moleules involved in these two distint proesses (11). ACKNOWLEDGMENTS These studies were supported by Publi Health Servie grants DE-3995, DE-5642, and RR-1224 from the National Institutes of Health. LITERATURE CITED 1. Erison, T., D. Bratthall, and J. Rundegren Baterial agglutination indued by saliva. Inhibition studies with anti-ig antisera and serum omponents, p In I. Kleinberg, S. A. Ellison, and I. D. Mandel (ed.), Saliva and dental aries (a speial supplement to Mirobiology Abstrats) Information Retrieval, In., Washington, D.C. 2. Erison, T., K. Pruitt, and H. Wedel The reation of salivary substanes with bateria. J. Oral Pathol. 4: Franks, D. J., and D. Malamud Isoeletri fousing of brain adenylate ylase. Anal. Biohem. 73: Gibbons, R. J., and J. van Houte Baterial adherene in oral mirobial eology, Annu. Rev. Mirobiol. 29: Ginsburg, I., and P. G. Quie Modulation of human I5

5 39 MALAMUD, BROWN, AND GOLDMAN polymorphonulear leuoyte hemotaxis by leuoyte extrats, baterial produts, inflammatory exudates, and polyeletrolytes. Inflammation 4: Golub, E. E., M. Thaler, C. Davis, and D. Malamud Baterial aggregating ativity in human saliva: simultaneous determination of free and bound ells. Infet. Immun. 26: Kashket, S., and C. G. Donaldson Saliva-indued aggregation of oral streptooi. J. Bateriol. 112: Malamud, D., B. Appelbaum, R. Kline, and E. E. Golub Baterial aggregating ativity in human saliva: omparisons of INFECT. IMMUN. baterial speies and strains. Infet. Immun. 31: a.Malamud, D., R. Goldman, and N. S. Taihman Modulation of baterial aggregation by PMN and platelet extrats. Inflammation 7: Malamud, D., and E. E. Golub A model for saliva mediated baterial aggregation. Adv. Physiol. Si. 28: Mandel, I. D Immunologi aspets of aries resistane. J. Dent. Res. 55:C22-C Rosan, B., D. Malamud, B. Appelbaum, and E. Golub Charateristi differenes between saliva-dependent aggregation and adhesion of streptooi. Infet. Immun. 35:86-9.

Release and Uptake of Gene Transfer Agent by

JOURNAL OF BACTERIOLOGY, Aug. 1975, p. 651-657 Vol. 123, No. 2 Copyright 1975 Amerian Soiety for Mirobiology Printed in U.S.A. Release and Uptake of Gene Transfer Agent by Rhodopseudomonas apsulata MARC