Characterization of the interaction of bovine plasmin with Streptococcus uberis

Journal of Applied Microbiology 1998, 84, 1104–1110

Characterization of the interaction of bovine plasmin withStreptococcus uberis

R.A. Lincoln and J.A. LeighInstitute for Animal Health, Compton Laboratory, Compton, Newbury, UK

6355/08/97: received 1 August 1997, revised 10 November 1997 and accepted 12 November 1997

R.A. LINCOLN AND J.A. LEIGH. 1998. The binding of plasmin to Streptococcus uberis strain 0140 Jwas optimal in the pH range 5·0–5·5. Plasmin binding decreased exponentially with increasingNaCl concentration (0–0·8 mol l−1), reaching a minimum at NaCl concentrations exceeding0·55 mol l−1. Neither K¦, Mg2¦ nor the metal chelator EDTA had any effect on theinteraction. Plasmin binding was prevented, in a concentration-dependent manner, by theamino acids lysine, arginine and o-aminocaproic acid. Bound plasmin was also eluted fromthe bacterial cell using the same amino acids. Bound plasmin was lost from the bacterium ina time- and temperature-dependent fashion, the rate of plasmin loss increased with increasingtemperature over the range 4–55 °C, and the elution of plasmin from live and heat-killedbacteria was similar. Cell-bound plasmin was only partially inhibited by the physiologicalinhibitor a2-antiplasmin whereas the serine protease inhibitor aprotinin, and the active sitetitrant p-nitrophenyl-p-guanidiniobenzoate, inhibited the activity of the cell-bound plasminby more than 95%.

INTRODUCTION

Bovine mastitis, or inflammation of the bovine mammarygland, commonly arises as the result of a bacterial infectionof the udder. Streptococcus uberis, one of the major causativeorganisms, is responsible for 33% of all cases of clinicalbovine mastitis (Hillerton et al. 1993). The ability of thisbacterium to infect the mammary gland is dependent on theability of the bacterium to grow in milk and resist phago-cytosis by bovine neutrophils (Leigh et al. 1991). Virulencefactors associated with the initiation and pathogenesis ofStrep. uberis mastitis are not well defined. It has been pos-tulated that growth of auxotrophic bacteria such as Strep.uberis within the mammary gland may be severely limited bylack of free amino acids and dipeptides (Leigh 1993), as mostnitrogen in milk occurs in the form of milk proteins (Aston1975). Consequently, proteolysis may constitute an essentialprerequisite for growth. Streptococcus uberis exhibits nodetectable protease activity (Leigh 1993). However, milk con-tains bovine plasminogen (Benslimane et al. 1990), the inac-tive precursor of the serine protease plasmin. It has beenshown that Strep. uberis may derive essential amino acidsfrom plasmin-derived casein peptides (Kitt and Leigh 1997),

Correspondence to: James A. Leigh, Institute for Animal Health, ComptonLaboratory, Compton, Newbury, Berkshire RG20 7NN, UK (e-mail:[email protected]).

© 1998 The Society for Applied Microbiology

that the bacterium produces an extracellular protein capableof activating bovine, equine and ovine plasminogen to plasmin(Leigh 1993, 1994; Lincoln and Leigh 1997), and that Strep.uberis may bind plasmin generated by the action of this plas-minogen activator (Leigh and Lincoln 1997). The abilityto bind plasmin has previously been shown for group A(Lottenberg et al. 1987; Broder et al. 1991), group C (Ullberget al. 1989) and group G (Ullberg et al. 1989, 1992) strep-tococci. Once bound to group A streptococci, plasmin wasresistant to regulation by a2-antiplasmin (Lottenberg et al.1987), its physiological inhibitor. Acquisition of such uncon-trolled plasmin activity may alter the dynamics of the host–bacterium relationship to favour infection, as unregulatedproteolytic activity may promote degradation of host proteinsto facilitate bacterial growth, prevent opsonization via cleav-age of immunoglobulins, or promote tissue invasion byhydrolysis of matrix proteins and activation of latent pro-enzymes such as collagenases. The aim of these studies wasto determine the factors affecting the interaction betweenStrep. uberis and plasmin.

MATERIALS AND METHODS

Reagents

Bovine plasmin, aprotinin, p-nitrophenyl-p-guanidinio-benzoate hydrochloride (pNpGB) and the synthetic plasmin

INTERACTION OF PLASMIN AND STREP. UBERIS 1105

substrate, tosyl-gly-pro-lys-p-nitroanilide, were obtainedfrom the Sigma Chemical Company; a2-antiplasmin wasobtained from Boehringer Mannheim.

Bacteria

Streptococcus uberis strain 0140 J was isolated from a case ofclinical bovine mastitis at the National Institute of DairyResearch (Shinfield, UK), and obtained from the Strep-tococcal Culture Collection at the Institute for Animal Health(Compton, UK). The bacterium was stored at −20 °C inTodd Hewitt Broth (THB) containing 25% glycerol.

Plasmin:bacterium interaction

Bacteria were grown overnight at 37 °C in Brain HeartInfusion broth (BHI; 10ml), and the bacterial cell pelletrecovered by centrifugation (8000 g for 10min). Bacteria werewashed three times with phosphate-buffered saline (composedof (g l−1): KH2PO4, 0·25; Na2PO4, 1·43; KCl, 0·25; NaCl, 10)supplemented with 0·1% (w/v) gelatin and 0·05% (v/v)Tween-20 (PBSGT), and resuspended in the same buffer.Following adjustment to an O.D. of 1·0 at 550 nm (7·8× 108

cells ml−1), 1ml aliquots of bacterial suspension were trans-ferred to Eppendorf tubes and the bacteria recovered bycentrifugation (8000 g for 10min). The cells were resus-pended in 475ml of PBSGT to which either 25ml of bovineplasmin (0·5U ml−1) or 25ml PBSGT had been added. Themixture was then incubated at 37 °C for 15min and thebacteria recovered by centrifugation (8000 g for 10min),washed three times in PBSGT (1ml) and finally resuspendedin PBSGT (250ml). Bound plasmin activity was detectedusing this suspension as described below.

Detection of cell-bound plasmin

Following the addition of 20ml of the chromogenic substrate,tosyl-gly-pro-lys-p-nitroanilide (5mg ml−1), the reactionmixture was vortexed, and incubated at 37 °C for 1 h. Plasminactivity was quenched by the addition of 250ml acetic acid(10% v/v) and the bacterial cells removed by centrifugation(8000 g for 10min). The amount of p-nitroanilide releasedinto the cell supernatant fluid was then determined spec-trophotometrically at 405 nm.

Effect of pH on plasmin:bacterium interaction

This was determined by incubation of bacteria with plasminin PBSGT as described previously. In this case, the PBSGTwas adjusted to an appropriate pH (5·0–8·0) by alteration ofthe relative proportions of NaH2PO4 and Na2PO4. Binding ateach pH was determined in triplicate on three occasions.

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 1104–1110

Negative controls consisting of bacteria incubated in PBSGTat the appropriate pH were included on each occasion.

Effect of ionic strength on plasmin:bacteriuminteraction

This was determined as described previously. However,modified PBSGT (containing PBS (g l−1): KH2PO4, 0·25;NaHPO4, 1·43; KCl, 0·25) supplemented with different con-centrations of sodium chloride was used as the assay buffer.Negative controls consisting of Strep. uberis incubated witheach buffer alone were also included in each experiment. Theeffect of each salt concentration on plasmin binding wasdetermined in triplicate on at least two occasions.

Effect of Mg 2+ and K + on plasmin:bacterium interaction

This was determined by incubating Strep. uberis with bovineplasmin in PBSGT supplemented with 10mmol l−1 of theappropriate cation. The percentage change in binding wascalculated by comparison with binding in buffer alone. Theeffect of each cation on plasmin binding was assayed in trip-licate on at least two occasions.

Effect of EDTA on plasmin:bacterium interaction

The effect of EDTA on plasmin binding was determined asdescribed above. However, PBSGT could not be used as theassay buffer as chelation of the salt within this buffer resultedin a significant decrease in its ionic strength which alteredplasmin binding. Consequently, this was determined in13·2mmol l−1 phosphate buffer containing gelatin and Tween(pH 7·0), and in 13·2mmol l−1 phosphate buffer (pH 7·0)supplemented with 0·3mol l−1 sucrose.

Negative controls consisting of bacteria incubated withbuffer alone were included in each experiment. The effect ofEDTA on plasmin binding was assayed in triplicate on atleast two occasions.

Inhibition of plasmin:bacterium interaction by oo-aminocaproic acid, arginine and lysine

This was determined by incubating Strep. uberis with bovineplasmin in PBSGT, as described previously. In this case, theassay buffer was supplemented with varying concentrations(0–0·475mol l−1) of o-aminocaproic acid, lysine or arginine.The percentage binding was calculated by comparing cell-bound plasmin activity in the presence of the amino acid withthat in PBSGT alone. The data presented are the mean ofnine determinations.

1106 R.A. LINCOLN AND J.A. LEIGH

Elution of bound plasmin by oo-aminocaproic acid,arginine and lysine

The ability of o-aminocaproic acid, arginine and lysine todissociate bound plasmin from Strep. uberis was determinedby incubating 7·8× 108 bacteria with plasmin in PBSGT (asdescribed above). The plasmin-coated bacteria were washedtwice with PBSGT (1ml), resuspended in 500ml of the samebuffer supplemented with different concentrations of o-aminocaproic acid, arginine or lysine, respectively, and incu-bated for 15min at 37 °C. The cells were then washed twicein PBSGT (1ml) supplemented with the appropriate con-centration of amino acid, resuspended in 250ml of PBSGT,and cell-bound plasmin activity detected by incubation withtosyl-gly-pro-lys-p-nitroanilide. The percentage binding wascalculated by comparison of the activity obtained in the pres-ence of the amino acid with that obtained with an identicallytreated sample in the absence of the amino acid. The resultsconstitute the mean of at least five determinations at eachamino acid concentration.

Effect of temperature on temporal plasmin loss fromthe surface of Streptococcus uberis

Bacteria were incubated with plasmin as described previously,recovered by centrifugation (8000 g for 6min), washed twicewith an equal volume of PBSGT, and resuspended in halfthe original volume of PBSGT which had been pre-incubatedat 4, 22, 37, 45 or 55 °C. Bacterial suspensions were incubatedat these temperatures for 75min during which samples(0·5ml) were removed at 15min intervals. Following cen-trifugation (8000 g for 6min), the cell supernatant fluid wasremoved and 250ml aliquots assayed for the presence of plas-min. The cell pellets were washed twice in ice-cold PBSGT(1ml), resuspended in 250ml of the same buffer and assayedfor cell-associated plasmin.

Temporal plasmin loss from heat-killed Streptococcusuberis

An experiment similar to that described above was performedusing live and heat-killed (60 °C for 60min) bacterial sus-pensions. The heat-treated cells were cooled on ice prior touse and the loss of pre-bound plasmin from both suspensionswas monitored at 37 °C, as described above.

Inhibition of cell-bound plasmin by aa2-antiplasmin,aprotinin and pNpGB

A bacterial cell suspension was incubated with plasmin, asdescribed previously. The cell pellet from 1ml was recoveredby centrifugation (8000 g for 6min), and the bacteria washedtwice with an equal volume of PBSGT prior to resuspension


in 0·5ml of the same buffer containing a2-antiplasmin (106mgml−1), aprotinin (10·6mg ml−1) and pNpGB (0·552mg ml−1).These mixtures were incubated at room temperature for15min. The bacteria were pelleted by centrifugation (8000 gfor 6min), washed with 1ml PBSGT, resuspended in 250mlof the same buffer and assayed for cell-bound plasminactivity.

RESULTS

The interaction of Strep. uberis 0140 J with plasmin isdescribed below.

The effect of pH on plasmin:bacterium interaction wasdetermined by incubating Strep. uberis with plasmin inPBSGT of differing pH (5·0–8·0). The plasmin:bacteriuminteraction was maximal between pH5·0 and 5·5 and minimalat pH 8·0 (Fig. 1). Binding decreased linearly between pH5·5and pH7·5, and 50% maximal plasmin binding occurredaround pH6·5.

Similar studies were carried out to determine the effectof ionic strength on plasmin binding. Plasmin binding wasdependent on ionic strength (Fig. 2) and decreased expo-nentially with increasing ionic strength between 0 and 0·8moll−1 NaCl. Plasmin binding was minimal at salt concentrationsexceeding 0·55mol l−1 NaCl. Plasmin binding was unalteredby inclusion of 10mmol l−1 K¦, Mg2¦ or EDTA (data notshown).

Plasmin(ogen) kringle domains often mediate the inter-action of this protein with substrates and inhibitors via lysinebinding sites (Ponting et al. 1992). In order to determine theinvolvement of these sites in the interaction of plasmin andStrep. uberis, the bacterium was incubated with plasmin inPBSGT supplemented with different concentrations of lysineor the analogous amino acids o-aminocaproic acid and argi-

Fig. 1 Binding of plasmin to Streptococcus uberis strain 0140 J asa function of pH. Each data point represents the mean 2standard deviation of nine determinations


Fig. 2 Plasmin binding to Streptococcus uberis strain 0140 J as afunction of ionic strength. Each data point represents the mean 2standard deviation of six determinations

nine. The inhibition of binding was determined by com-parison of the level of plasmin activity detected in associationwith the bacteria following incubation with plasmin in thepresence/absence of the amino acids. The binding of plasminto Strep. uberis was inhibited in a concentration-dependentmanner by each of the three amino acids (Fig. 3). A 50%inhibition of the interaction was observed at an o-amino-caproic acid concentration of 0·14mmol l−1, a lysine con-centration of 3mmol l−1 and an arginine concentration of30mmol l−1. Additional studies to determine whether theseamino acids could elute bound plasmin showed that the con-centration of amino acids required to elute 50% of the activity

Fig. 3 Inhibition of plasmin binding to Streptococcus uberis strain0140 J by o-aminocaproic acid, lysine and arginine. The percentageinhibition of binding was calculated by comparison of the activitydetected in the presence of o-aminocaproic acid (Ž), lysine (�) andarginine (R) with that in PBSGT alone. Each data pointrepresents the mean 2 standard deviation of ninedeterminations


was approximately the same as that required to inhibit theinteraction by 50% (Fig. 4). However, approximately 15% ofthe cell-bound plasmin activity was resistant to elution usingthese amino acids (Fig. 4) at concentrations equivalent tothose required to inhibit plasmin binding by 100% (Fig. 3).While carrying out investigations on the elution of boundplasmin, inclusion of a buffer control was essential to com-pensate for an apparent temporal loss/release of activity. Inorder to determine whether the temporal loss of cell-boundplasmin was temperature-dependent, the level of cell-boundplasmin was monitored over a range of temperatures (4–55 °C) for a duration of 75min. This showed that the rate ofplasmin loss increased with increasing temperature up to55 °C, and that the loss of plasmin from the bacterial cellcorrelated with detection of the activity in the cell supernatantfluid (Fig. 5). In order to define this event further, plasminloss from live and heat-killed Strep. uberis was compared at atemperature of 37 °C. The loss of cell-bound plasmin andconcurrent appearance of activity in the cell supernatant fluidwas similar from both live and heat-killed cells (Fig. 6).

The ability of bound plasmin to resist inhibition was alsostudied using a2-antiplasmin (a selective physiological inhibi-tor of plasmin), aprotinin (a low molecular weight serineprotease inhibitor) and pNpGB (an active site titrant). Strep-tococcus uberis was incubated with plasmin, washed, and thenincubated with each of the inhibitors at a concentration rep-resenting a 10-fold molar excess relative to plasmin. Additionof aprotinin inhibited bound plasmin activity by more than95%, and addition of pNpGB inhibited bound plasminactivity completely (Fig. 7). However, addition of a2-anti-plasmin inhibited plasmin activity by approximately 70%.

Fig. 4 Elution of bound plasmin from Streptococcus uberis strain0140 J by o-aminocaproic acid, lysine and arginine. Thepercentage elution of activity was calculated by comparison ofthe activity detected in the presence of o-aminocaproic acid (Ž),lysine (�) and arginine (R) with that in PBSGT alone. Eachdata point represents the mean 2 standard deviation of fivedeterminations


Fig. 5 The effect of temperature on temporal loss of plasminfrom Streptococcus uberis strain 0140 J. Plasmin lost from thesurface of Strep. uberis (a and b) was detected in the supernatantfluid (c and d), respectively. Temperatures are given in °C (aand c: (�), 4, (r), 22, (�), 37; b and d: (�), 37, (r), 45, (�),55). Each data point represents the mean 2 standarddeviation of two determinations

Fig. 6 The effect of bacterial viability on the loss of plasminfrom Streptococcus uberis strain 0140 J. Viable bacterial suspensions(r, R) and heat-killed (60 °C for 60 min) bacterial suspensions(�, Ž), were used to detect cell-bound (closed symbols) andreleased (open symbols) plasmin activity. Each data pointrepresents the mean 2 standard deviation of threedeterminations


Fig. 7 The effect of protease inhibitors; a2-antiplasmin, aprotininand pNpGB on cell-bound and free plasmin activity. Percentageactivity was calculated by comparison with that observed in theabsence of inhibitor (100%). (Ž), Cell bound activity; (�), fluidphase activity. Each data set represents the mean 2 standarddeviation of three determinations

All the inhibitors reduced the activity from the original con-centration of plasmin by more than 95% in the absence ofStrep. uberis.

DISCUSSION

Characterization of the plasmin:bacterium interaction showedapproximately 50% of plasmin was bound at pH 6·5, the pHof milk. However, maximum plasmin binding was observedbetween pH5·0 and 5·5. Broeseker et al. (1988) characterizedthe interaction of human plasmin with its specific receptoron a group A streptococcal isolate (64/14). In contrast toStrep. uberis, plasmin binding by this isolate was maintainedat a constant level over the pH range 5·0–8·0.

The effect of ionic strength on plasmin binding by Strep.uberis also appeared to differ from that reported for strep-tococcal isolate 64/14 (Broeseker et al. 1988). Binding ofhuman plasmin to the group A streptococcal isolate wasoptimal between 0·1 and 0·4 mol l−1 NaCl, and decreasedsignificantly at salt concentrations above 0·5mol l−1 (Broe-seker et al. 1988). In contrast, binding of bovine plasminto Strep. uberis decreased exponentially in the presence ofincreasing NaCl concentrations and the plasmin:bacteriuminteraction was minimal at NaCl concentrations in excess of0·55mol l−1.

The involvement of the lysine binding sites in mediatingthe plasmin:bacterium interaction was demonstrated by theability of o-aminocaproic acid, lysine and arginine to inhibitthis interaction and elute bound plasmin from the cell in a


concentration-dependent manner. The displacement of theinhibition/elution curves in each case reflects the generallyacknowledged affinity of these amino acids for the lysinebinding sites of plasmin and plasminogen (Winn et al. 1980).The data obtained for Strep. uberis are comparable with thosereported by Broeseker et al. (1988) that similar concentrationsof each amino acid were required for 50% inhibition ofplasmin binding and the elution of 50% of the bound plasminfrom the Group A streptococcus 64/14. Similar results werealso reported by Ullberg et al. (1992) who showed that0·1mmol l−1 o-aminocaproic acid decreased the binding of125I-labelled human plasminogen to Strep. pyogenes strain A-2898, and a human group G strain (G-975), by 50%. Inter-estingly, they also reported that the corresponding con-centrations of o-aminocaproic acid required to decrease thebinding of 125I-labelled human plasminogen to two bovinegroup G streptococcal isolates (DG-23 and DG-26) wasapproximately 100-fold higher in each case than that requiredfor the group A streptococcus.

The a2-antiplasmin was able to inhibit plasmin activityassociated with Strep. uberis by approximately 70%. This isin contrast to the effect of the same inhibitor, at similarconcentration, on plasmin bound to the group A streptococcalisolate, 64/14 (Lottenberg et al. 1987), in which no inhibitionwas observed. It has been suggested that the failure of a2-antiplasmin to inhibit activity bound to group A streptococciresults from competition between the specific plasmin recep-tor and this inhibitor for similar regions of the plasmin mol-ecule (Lottenberg et al. 1994). Thus, once plasmin is boundto group A streptococci, it is resistant to inhibition by a2-antiplasmin (Lottenberg et al. 1987). The failure of Strep.uberis to totally protect plasmin from inhibition by a2-anti-plasmin may infer that this bacterium binds plasmin via adifferent mechanism, and/or that only the non-inhibitablefraction 30% was attached via a mechanism similar to thatseen for group A streptococci. An alternative multistep pro-cess for the acquisition of plasmin-like activity dependent onthe presence of fibrinogen, streptokinase and plasminogenhas also been demonstrated in group A streptococci (Wanget al. 1995; Christner et al. 1997). However, since the experi-ments in this investigation have been carried out in buffer inthe absence of fibrinogen and a source of plasminogen acti-vator, it is unlikely that this alternate system constitutes themechanism of plasmin binding observed for Strep. uberis. Theability of aprotinin (which binds to the active site of serineproteases) and pNpGB (a small active site titrant) to inhibitthe activity of cell-bound plasmin infers that the active siteis not involved in mediating plasmin:bacterium interaction;this was similar to the observations using the streptococcalisolate 64/14 (Lottenberg et al. 1987).

A time-dependent loss of plasmin from the bacterium wasobserved. In order to determine whether this loss was attribu-table to a physiological mechanism (which may show an


optimal temperature) or a chemical mechanism (and thusaccelerated at high temperature), the temperature sensitivityof plasmin loss was investigated. The rate of plasmin lossincreased with increasing temperatures, inferring that thisprotein was not lost via a physiological process. This was alsoinferred by comparing the loss of plasmin from live and heat-killed Strep. uberis. Loss of plasmin from both live and deadbacterial cells was identical. If a physiological mechanism wasinvolved in mediating plasmin release, it might be anticipatedthat plasmin loss from the heat-killed cells would be altered.

Despite much speculation (Lottenberg et al. 1994), theprecise role of cell-bound plasmin(ogen) in disease patho-genesis has not been elucidated, nor has there been a dem-onstration that plasmin binds to streptococci during infection.However, the observation that Strep. uberis has a host-specificplasminogen activator and a mechanism for acquiring cell-bound plasmin (Leigh and Lincoln 1997) suggests that cap-ture of this protein may have a role in the pathogenesis ofintramammary infection. Acquisition of plasmin activity maypromote the ability of this bacterium to cause infection viadegradation of host proteins to peptides and amino acids tofacilitate bacterial growth (Kitt and Leigh 1997), cleavageof immunoglobulins or hydrolysis of matrix proteins, andactivation of latent proenzymes such as collagenases. Conse-quently, prevention of plasmin acquisition may potentiallyafford a mechanism for reducing the incidence of Strep. uberisinfection.

We have shown that Strep. uberis is capable of bindingplasmin and that such an interaction is possible under theconditions prevailing in vivo. We have also shown that plas-min:bacterium interaction is temporal, and mediated via thelysine binding sites of plasmin in a manner which leaves theactive site of plasmin accessible for interaction with sub-strates.

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Characterization of the interaction of bovine plasmin with Streptococcus uberis