INFECTION AND IMMUNITY, Apr. 1979, 0019-9567/79/04-0261/08$02.00/0

Vol. 24, No. 1

p. 261-268

Mucopolysaccharidase of Treponema pallidum T. J. FITZGERALD* AND R. C. JOHNSON Department ofMicrobiology, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455 Received for publication 26 January 1979

Treponemapallidum (Nichols strain) exhibited mucopolysaccharidase activity. Acidic mucopolysaccharides were broken down more rapidly by viable treponemes than by heat-inactivated treponemes or membrane filtrates of treponemal suspensions. Ouchterlony immunodiffusion demonstrated the occurrence of antibodies to the hyaluronidase-like enzyme within syphilitic sera. After intratesticular inoculation of 2 107 to 6 107 treponemes, these anti-mucopolysaccharidase antibodies were detected between 9 and 35 days postinoculation. In addition, acidic mucopolysaccharides were present in the serum of infected animals 9 and 16 days postinoculation. Immune serum that contained antibodies to the mucopolysaccharidase restricted treponemal breakdown of acidic mucopolysaccharides. It has been previously demonstrated that immune rabbit serum contains a factor that blocks attachment of T. pallidum (Nichols strain) to cultured mammalian cells. This factor was effectively absorbed by prior incubation with bovine hyaluronidase. It is postulated that T. pallidum attaches to acidic mucopolysaccharides on the surface of cultured cells through the mucopolysaccharidase enzyme at the surface of the organisms. These findings are discussed in terms of the histopathogenesis of T. pallidum with applications to the healing immune X

X

response.

There is a characteristic accumulation of mucoid material within testicular and dermal lesions in syphilis (1, 7, 15, 16, 24-26, 30-34). This material has been tentatively identified as acidic mucopolysaccharide comprised of hyaluronic acid and chondroitin sulfate (7, 24, 25, 30, 32, 33). Scott and Dammin (24) initially suggested that Treponema pallidum possesses hyaluronidase activity; the accumulated mucopolysaccharides were attributed to breakdown of host tissue. There are two other observations that support the possibility that T. pallidum contains hyaluronidase. First, during infection, some treponemes attain intracellularity (2, 27, 28). Inasmuch as most tissue cells are enmeshed within ground substance material (3), it would be necessary for the organisms to penetrate the ground substance prior to entering the tissue cells. Second, the histological influences of hyaluronidase on normal tissue (4) are quite similar to the histopathology of the early stages of syphilis. The previous report demonstrated an association between T. pallidum (Nichols strain) and acidic mucopolysaccharides at the surface of cultured rabbit testicular cells (11). It was suggested that the treponemes specifically associate with acidic mucopolysaccharides within ground substance in vivo. The purpose of this report was to extend the previous findings and to de-

termine whether T. pallidum exhibits hyaluronidase activity.

MATERIALS AND METHODS T. pallidumL The organisms were maintained and harvested in tissue culture medium as previously described (9). Incubation of treponemes was performed at 30'C in an atmosphere of 2.5% oxygen-92.5% nitrogen-5% carbon dioxide (9). Tissue culture. Cultured cells derived from normal rabbit testes (13) were maintained in tissue culture medium as previously described (9) with the exclusion of glutathione, cysteine, and dithiothreitol. Mucopolysaccharide assay. Acidic mucopolysaccharides react with acidified bovine serum albumin (acid BSA) to form a precipitate (17, 18). To acidify the BSA, 1 ml of 10% BSA (wt/vol) in physiological saline was added to 0.4 ml of 2 N acetic acid (pH 4). The minimum amount of mucopolysaccharide detectable with this method should be approximately 8 jig/ ml (18). Various concentrations of commercial preparations of hyaluronic acid and chondroitin sulfate were tested, and the results were in agreement with the previous findings (18). The quantity of acidic mucopolysaccharides within a test suspension was assessed by acid BSA titration using twofold dilutions in saline until a precipitate was no longer detected. The previous dilution was then assumed to contain approximately 8 jg of mucopolysaccharide per ml. As a control for this reaction, hyaluronidase was added. The inclusion of this enzyme in the reaction mixture prevented the formation of the precipitate (18).

261

262

FITZGERALD AND JOHNSON

TF. The viscous mucoid testicular fluid (TF) that accumulated during intratesticular infection was removed by needle aspiration. The acidic mucopolysaccharide content was assayed by titration with acid BSA (17, 18). Rabbit serum. Sera were freshly prepared for the experiments listed in Tables 6 and 7. Blood was removed 1 day prior to the experiment, and the sera were isolated and heat inactivated (56°C for 30 min) on the day of the experiment. Normal rabbit sera were obtained from rabbits that were rapid-plasma-reagin nonreactive. Immunodiffusion techniques. Ouchterlony techniques were used to determine the presence of antibodies to hyaluronidase in syphilitic serum. The composition of the agar medium was 1 g of Noble agar, 1 ml of 1% thimerosol, 75 ml of distilled water, and 25 ml of Veronal buffer at pH 8.6. Serum was diluted 1:2 with physiological saline, then added to the agar wells. Bovine hyaluronidase at 10 mg/ml and streptomyces hyaluronidase at 0.01 mg/ml in physiological saline were tested with different sera and with various treponemal preparations. Incubation was performed in humidified chambers at 24°C. Precipitin lines developed within 3 to 10 days. The presence of bovine hyaluronidase and streptomyces hyaluronidase in outer wells and immune syphilitic serum in the center well resulted in one precipitin line of identity in the two outer wells. Inasmuch as the two enzymes were prepared from either bovine testicular tissue or Streptomyces hyalurolyticus, the lines of identity were presumably specific for hyaluronidase, rather than a contaminating substance within the two commercial preparations. RESULTS

Experiments were performed to determine the ability of T. pallidum to break down acidic mucopolysaccharides. The mucoid TF that accumulates within infected tissue contained large amounts of acidic mucopolysaccharides as shown by acid BSA reactions (9). Preparations of this material were used as a source of mucopolysaccharide. A freshly harvested suspension of T. pallidum in tissue culture medium was adjusted to 5 x 107 organisms per ml. One portion of this suspension was heat inactivated at 48°C for 10 min. After heating, all observable organisms were nonmotile. A second portion of the treponemal suspension was membrane filtered with a Millipore filter with a pore size of 0.20 [Lm. No organisms were detected within this filtered preparation. TF was then added to the viable preparation, the heated preparation, and the membrane filtrate. As a control, TF was added to tissue culture medium. At various times during incubation, samples were removed and assayed for mucopolysaccharide. The data in Table 1 represent a summary of eight experiments. The initial mucopolysaccharide content was 325 ,Ig/ml. The most rapid breakdown of mucopolysaccharide occurred in

INFECT. IMMUN.

the viable treponemal preparation. The degradation of mucopolysaccharide occurred at similar rates in both the membrane filtrate and the heated preparation. After 10 days, the viable preparation contained 20 ,ug of mucopolysaccharide per ml, compared to 140 ,ig of mucopolysaccharide per ml in the heated preparation and 170 ,jg of mucopolysaccharide per ml in the membrane filtrate. No mucopolysaccharide degradation was detected in the control preparation. In these eight experiments, treponemal motility in the viable preparation was retained for 5 to 6 days. These studies suggested that T. pallidum contained a mucopolysaccharidase. Ouchterlony immunodiffusion was used to assay for antibodies to mucopolysaccharidase that might develop during syphilitic infection. Sera were obtained from a number of different rabbits that had been injected intratesticularly with 2 x 107 to 6 x 107 treponemes. This inoculum produced a detectable orchitis within 9 to 11 days. Bovine hyaluronidase and streptomyces hyaluronidase (outer wells) were tested for reaction with syphilitic serum (center well). Precipitin lines to the serum developed with both enzymes, indicating the presence of antibodies to hyaluronidase within the syphilitic serum. To assay individual serum samples, streptomyces hyaluronidase was placed in the center well and the rabbit serum samples were placed in the outer wells. Table 2 presents a survey of different sera isolated from 15 rabbits that had been injected intratesticularly with 2 x 107 to 6 X 107 treponemes for periods ranging from 6 to 720 days. Precipitin antibodies that reacted with hyaluronidase were detected only between day 10 and day 35 postinoculation. Precipitin antibodies were not detected in sample 12 from an immune animal that had been infected 120 days previously and challenged with 108 treponemes on day 90, or in sample 13 from an immune animal that had been infected 180 days previously and challenged with 108 treponemes on TABLE 1. Breakdown of mucopolysaccharides by

different treponemal preparations Time

(days) 0 1 2 3 4 8 10

Mucopolysaccharide concn (tg/ml) Viable Heated Memtrepobrane fil- Medium trepocontrol nemes nemes trate

325 150 75 60 50 40 20

325 200 175 170 170 170 140

325 220 175 170 170 170 170

325 325 325 325 325 325 325

MUCOPOLYSACCHARIDASE OF T. PALLIDUM

VOL. 24, 1979

TABLE 2. Antibodies to hyaluronidase within sera obtained from different rabbits that had been injected with T. pallidum at various times Rabbit sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Days after intratestiua inetin (no. fchallenges) of challenges) 0 6 7 10

12 13 17 20 35 60 60 120 180 360 720

(1)b (1)b

. tto Antibodies hyaluronidase

No No No

Yes Yes Yes Yes Yes Yes No No No No

(10)C Yes (9)c Yes a Intratesticular infection with 2 X 107 to 6 X 107

tical manner used for infected testicular tissue also contained hyaluronidase. The data in Table 2 were obtained from 15 different animals that had been infected for various periods. Experiments were performed to evaluate the development of antibodies to hyaluronidase within consecutive serum samples from four rabbits infected intratesticularly with 6 x 107 treponemes. Two control rabbits were injected intratesticularly with either heat-inactivated (480C for 10 min) T. pallidum at 6

c

day 150. Precipitin antibodies were detected in sample 14 from a hyperimmune animal that had been infected 360 days previously and challenged nine times with 108 treponemes per challenge at 1-month intervals. Precipitin antibodies were also detected in sample 15 from a hyperimmune animal that had been infected 720 days previously and challenged 10 times with 108 treponemes per challenge at 2-month intervals. During these Ouchterlony studies, additional information was obtained. When hyperimmune (T. pallidum) serum was placed in the center well, and bovine hyaluronidase, freshly harvested treponemes, and streptomyces hyaluronidase were placed in consecutive outer wells, one line of identity occurred between the two enzymes and the treponemes. Besides indicating antibodies to the hyaluronidase in the hyperimmune serum, this also indicated the occurrence of hyaluronidase in the treponemal suspension. Similar experiments revealed the presence of hyaluronidase in a membrane filtrate of the treponemal suspension and in a treponemal suspension that had been heat inactivated. In addition, when high-speed centrifugation (12,000 x g for 30 min) was used to separate the organisms from the suspending medium, hyaluronidase was detected in both the supernatant fluid and the pelleted treponemes. Uninfected rabbit testicular tissue that was extracted in the iden-

x

107

organisms, or 2 ml of uninfected rabbit testicular extract. Serum was isolated and analyzed from each animal at 0, 5, 9, 16, 34, and 62 days postinoculation, and the results appear in Tables 3, 4, and 5. The rapid plasma reagin titers of these sera are shown in Table 3. The occurrence of this antibody parallels syphilitic infection (23, 30). The four animals injected with viable trepo-

treponemes.

Challenge was performed intradermally with approximately 105 treponemes per challenge 1 month previous to bleeding. Challenges were performed intradermally with approximately 105 treponemes per challenge at 1- to 2month intervals.

263

TABLE 3. Serum rapid plasma reagin titers Serum rapid plasma reagin titers' 6 x 107 treponemes per tests, rabbit no.:

Time (days)

Control heated trepo-

1

2

3

4

0

-

-

-

-

5

-

-

-

9

2 32 8 16

4 32 384 112

nemesh _ _

_

6 120 30 16

4 32 192 32

Control

unin-

fected testes

extract" _ _ -

a Reciprocal of highest reactive serum dilution. b Intratesticular injection of 6 X 107 treponemes that were heated at 480C for 10 min. ' Intratesticular injection of extract from uninfected rabbit testes. 16 34 62

TABLE 4. Development ofprecipitin antibodies during infection Presence of antibodies to hyaluronidase 6x

(days)

per

1

107 treponemes testis, rabbit no.:

Control heated trepo-

2

3

4

nemes

Control unin-

fected

textract

0

-

-

-

-

_

5

-

-

-

-

-

_ _

9 16 32

+ +

+ +

_

+

+ + +

_

+

+ + +

-

-

64

-

-

-

-

-

-

Intratesticular injection of 6 x 107 treponemes that were heated at 480C for 10 min. b Intratesticular injection of extract from uninfected rabbit testes. a

264

INFECT. IMMUN.

FITZGERALD AND JOHNSON

TABLE 5. Detection of serum mucopolysaccharides during infection Serum mucopolysaccharides (,ug/ml) Time (days)

6 x 107 treponemes per testis, rabbit no.:

Control heated trepo-

nemesa

Control

fuenctned

testes testes extract

3

4

-

-

-

-

-

-

24 8

_ 16 -

8 -

-

_ -

-

-

-

-

-

-

-

-

1

2

0

-

5 9 16 32 64

-

64 32 -

_

-

aIntratesticular injection of 6 x 107 treponemes that were heated at 480C for 10 min. bIntratesticular injection of extract from uninfected rabbit testes.

nemes were initially reactive on day 9 and remained reactive until day 62. Highest titers were observed on day 16 or day 32, and thereafter the titers declined. The serum samples from both control rabbits were nonreactive at each interval. The data in Table 4 show the presence of precipitin antibodies to hyaluronidase. All serum samples from each of the four rabbits injected with viable treponemes were positive on day 9, day 16, and day 34. Precipitin antibodies to hyaluronidase were not detected in the serum samples from the two control rabbits. All sera were also assayed for acidic mucopolysaccharides as demonstrated by the. acid BSA reaction (Table 5). Serum from each of the four rabbits injected with viable treponemes contained 8 to 64 1Lg of mucopolysaccharide per ml on day 9; serum from two of the four rabbits contained 32 Ag of mucopolysaccharide per ml on day 16. Mucopolysaccharide was not detected in any other serum sample, including those from the two control rabbits. The next experiments were performed to determine whether the antibodies to hyaluronidase interfered with enzyme activity. An uninfected rabbit was injected intramuscularly 10 times with 10 mg of bovine hyaluronidase per injection over a period of 1 year. This immunization protocol resulted in the development of antibodies to the enzyme as demonstrated by immunodiffusion techniques. Freshly harvested preparations of T. pallidum adjusted to 5 x 107 organisms per ml were added to serum from an uninfected rabbit (NRS), serum from a rabbit infected intratesticularly for 15 days with 3 x 107 treponemes per testis (IRS), and serum from an animal immunized with hyaluronidase (antiHase RS). The final concentration of serum was 50% (vol/vol). Mucoid testicular fluid was added

to provide a final concentration of 250 jg of mucopolysaccharide per ml. At various times, the quantity of mucopolysaccharide remaining within the preparations was assayed. The data in Table 6 indicate that the breakdown of mucopolysaccharide was most rapid in NRS and slowest in IRS. After 15 days, the mucopolysaccharide content in NRS had decreased to less than 8,ug/ml. After 22 days, the mucopolysaccharide content in IRS had only decreased to 125 tg/ml. Breakdown of mucopolysaccharide in the anti-Hase RS was intermediate between NRS and IRS. Identical results were recorded in two separate experiments using slightly higher initial concentrations of mucopolysaccharide and rabbit serum from other animals. Similar results were obtained when rabbit testicular tissue was used instead of mucoid TF as a source of mucopolysaccharide. Freshly harvested T. pallidum at 5 x 107 treponemes per ml was added to NRS, IRS (16 days post-intratesticular inoculation), and anti-Hase RS. Each preparation initially contained 125 jg of mucopolysaccharide per ml. The data are also included in Table 6. In NRS, the mucopolysaccharide content decreased to less than 8 ,g/ml in 2 days. In IRS a similar decrease required incubation for 7 days. In the anti-Hase RS a similar decrease was observed after 3 days. In these experiments the motility of the organisms was quite similar in the NRS, IRS, and anti-Hase

TABLE 6. Interference with treponemal breakdown of mucopolysaccharides by factors within immune serum and anti-hyaluronidase serum Mucopolysaccharide concn (ug/ml) Time

(days)

0 1 2 3 4 5 6 7 8 9 10 15 22

Expt. la 15AntiNRS Day Hase RS IRS

250 62 62 62 62 31 16 8 16 8 8

Mucopolysaccharidase of Treponema pallidum.

INFECTION AND IMMUNITY, Apr. 1979, 0019-9567/79/04-0261/08$02.00/0 Vol. 24, No. 1 p. 261-268 Mucopolysaccharidase of Treponema pallidum T. J. FITZG...
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