Quantitative demonstration of hyaluronidase activity in cercariae of Schistosoma mansoni by the streptococcal decapsulation test

Quantitative Demonstration of Hyaluronidase Activity in Cercariae of Schistosoma mansoni Streptococcal Decapsulation Test’ A. s. Il:vans’

by the

418

k. s. EV.\iiS

HY.\I,1.1ZONID.\Sl’

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IS

S. M.\NSONI

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F‘rom olx3erv:~tiott in wet l~rc~lt:~r:~tiotts, it appf’:ws that thus cal)sul:~r material of the st,rcptococri is :wcretctl in cqutd cIuant~itir3 I:ctf~tdly from t ho orgattism5. crctttittg 2, soliri of revolution \\hirh \v:ts thought originally to t)fl cssrttti:tll>. :t proi:rlc~ sl)hcroid with the lctigt,h of the strf~l~tococc:tl chain 3s t hrl itttf~rfoc;rl dist:~ttw. To t~ccomplish t,he cdcuiations. cnrf~full>~ culihratd l)hotogr:~phs \v~~I’c t :tlic~tt itt \vhic-11, If.1 tiug tlw line of t ho chain of organisms It? t hr s-axis, the CUM swtion matIc 1)~ atty 1ti:tnft ltf~tpnflif~ul:t,r to t ha1 xsis w’:ts taken to bra :L circle. lxtlittg fcl, .r,, 1’2, ... s,, rr~~rcscti( succcssivc equally spaced vducs of .i‘. :t~ttl y,& 1.l~: fii:tmf:trr of t.hf, c:tqsule :It thf. eorrf:sl~ottding wluc of .r. ii !/II. !I!. !I?, is at ottw ol)vioux that. for :~ttj. tliff’rrrtttixl of s. th(t volume (41’) of c’t~psuI:tt m:ttrri:tl is

To obtain t,he volume of cal~sul:~ materid in cultif* microns per micron of strep tococc:ll chain, this result was cliviticd 1)~ t,he length of thr chain of org:tttisms in micrclns. The vduc thus c:tlculatetl was correctd to climimttc the influrttcc of thus volume of the contained streptococci in the smdlrr c:tpsulrs I)>- sul)tractin~ the rolumc (O.!G pa) of 1 p of strcptococcal chaitt similarly comltutctl. For Itrf~vit!-, this finttl rcsull will he referred to 3s t hr unit volume (I*,,). The unit volutnr of 408 c:~l~ulrs t,hroughout the rxttye of both length antI dixmrlcr 14x8 c:~icul:~tetl t)J, the :thovc mcthocl, using not less than nine 0rfiitt:ltc:s. Whc~tt t hc Iogttrit,hm of thft unit, volutnr is plotted against the logarithm of t.hc mirl-c:tIxul:tr tiiamet.er [IS = g xt $h(.rU + x,A)l. it is rt:dily seen to lx :I lineat func-t ion (Fig. 2). The lint oht:~itted I)>- the meth rqu:~tion

Ctilizing t,ltc: c’onstants of this line, the volume of sulbstrate pfar unit of chain fwulti lx f~stimatd with less than &IO’% t’rror from thcl single mrasuwmettt, thr mittc:~j~sultw fli:tmf>tcr.

The unit volun~ea of these sa,me capsules ohLined by presuming t~hcm to IN? prolate spheroids wit.h the length of the chain of organisms as the interfocal distance were not, romparsble lvit,h those obtained hy approximat~e integration throughout the size range. In the larger capsules t,he values were similar, but as capsular dcslruction frrogressed the longitudinal planes were apparently no longer

FIG. 2. Volume of capsular material per micron of streptococcal chain obtained by approximate integration plotted as a function of the mid-capsular diameter. The scales are logarithmic.

ellipses and the length of the streptococcal chain did not approximate the interfoea distance of an ellipsoid with equivalent major and minor axes. The resuIts of these studies are therefore based on those values obtained by approximate integration, since only the assumption that the capsules are solids of revolution is necessary and no arbitrary equation for the longitudinal section nor length of streptococcal chain is presumed. The unit volume of as many as 300 capsules was determined on some slides, but the uniformity was such that the mean unit volume of 50 capsules selected at random was sufficient to estimate the mean of the population at each time interval

422

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S. EVhiXS

with an average st:tntl:w~ error of 0~1lg&A%. lk tl:lta. were plotted ill t.ermsof percentage reduction of unit capsular volume as compared to the specific streptococcal control.

The reduction of substrate material by graduated concentrations of cercarial extract at, 5 minute intervals for t,he 30 minut,e experiment’al

- Symbol 2,

(thousands) 85.2 i;:: 21.3 9.8

0 0 . I 20

I 25

1 30

FIG. 3. Reduction in streptococcal capsular volume as a function of time by indicated cercarial concentrations.

period is shown graphically in Fig. 3. These are raw data, and the variation normally encountered in biological material is readily apparent. While no attempt has been made to fit theoretical curves, certain aspects of the mode of presentation must be considered. The lines are shown as radiating from the origin, since the time lapse from mixing the cercarial extract with substrate suspension and its t’ransfer to t’he 56°C water bath for the zero time points was less t,hat 5 seconds, during which time capsular reduction was found to be negligible. It is probable, however, that in the higher concentrations, straight lines from the origin to the 5 minute points are not the best representation of the early course of

the reactions. Had a shorter time int,erval been feasible, the activity of such highly concentrated extracts during t’his period would probably have been curvilinear with slightly greater initial slopes than those of the lines shown. This statement. is substant,iated by consideration of the overall course of capsular reduction by successive levels of cercarial concentrat’ions. Rit,h a cwncentrat’ion of 9500 cercariae per ml, t’he rate of t’he react’ion is essentially linear tShroughout’ the 30 minute period indicating probably that suMrate is present in excess during t,he whole time. The gradual slope of the trend and t,he comparat,i\-ely low terminal value, 49.9 %, indicate ill addition that t,his conrent.rat,ion is probably at the lwt-er limit of sensitivity of the test. In the middle range, 21,300 and 42,500 cercariae per ml, the rat,e of capsular dissolution is slowed markedly after t,he initial five minutes and the trend is characterized by a slow climb to the terminal value. Highly concentrat’ed extracts, representing G3,800 and 85,200 cercariae per ml reach their limit’ in the very early t)ime periods; the former at 10 minutes and the latter by the elld of the first 5 minut,e interval, and no furt’her reduction occurs for the rernaillder of t,he 30 minutes. It is interesting to note that even in the highest collcentration, total decapsula(.iotl of the organisms was ilot achieved. In other experimenk, using as high as 100,000 cercariae per ml, the limit remained at 90-95 y0 reduction. This finding may reflect a difference in the density or even stjructllre of the “core” of t’he capsule or merely anot,her case of differences in reaction of hyaluronidases from different’ sources and of varying degrees of “purity” (McClean, 1941; Meyer, 1947; Ikicicome, 1953). At the S-minute interval, the reduction in unit capsular volume by graduated roncentrat,ions of cercarial extract’s was found to be directly proportional to the number of cercariae represented by the extract (Fig. 4), thus substantiating quant,itatively the suggestion made in t’he qualitati,c \vork @tire\\-alt and Evans, 1952) that, the degree of capsular dissolutiotl by this method of assay is directly referable to the density of the larval suspensioll. Two points are seen to be inconsistent with the order of progression. The points representing the activity of 21,300 and 31,000 and 42,500 and 52,200 cercariae taken by pairs, mn.y indicate t)hat, for quant,itative assay of t hc enzymatic: nc*tivity of this crude t+&raot , ilwrcment,s of grcatw fllan 10,000 cwcwk per ml should be used. In addition, nlltl pr~)td)ly of mow import :Lll(‘e, the cnzyniat ic* fact or is apparently nol itl:wi ivatccl immctlintcl~y in the 5fiY: water bath once the reaction has

424

A. S. EVANS

attained its maximum velocity, and in the lower concentrations, less than 25,000 cercariae per ml, capsular degradation proceeds to a point higher than that predicted by the coefficient of proportionality. On the ot’her hand, the higher concentrations have nearly reached their limit and the reaction is leveling off so that t’he lag is not apparent. Further dat,a from ot,her experiments substantiate this latter suggestion. At the subsequent time intervals, volumetric degradation of the substrate material as a function of the cercarial concentration is no longer

Final cercarial (thousands

FIG. 4. relationship

Accumulated of capsular

data from degradation

concentration per ml.)

different experiments illustrating the linear as a function of cercarial concentration.

linear throughout the range, indicating either that, after the first rapid catalysis, the substrate does not remain in excess, or that inhibitory products are formed, and the reaction is not proceeding at maximum velocity. The ready availabilit,y and widespread use of bull-testicular hyaluronidase have become such as to form somewhat of a base line with which to compare the activity of enzymes from other sources which degrade hyaluronic acid. Therefore, an experimental series, employing serial dilutions of t.his t)esticular enzyme, was carried out under identical conditions as with the cercarial extra& to determine whether such experimental design would permit. expression of the n&vit,y of the latter in terms of t,he former.

Capsular degradation at’ the 5-minute interval by serial dilutions of commercial butl-test,ieular hyaluronidase Mrated against, the same substrate concentration as t#hat,used in t’he wrcarial assays is shown graphically in Fig. 5. Obviously, the activity of the crude cercarial extract (Fig. 4) and the purified testicular enzyme cannot be directly compared at the five minut,e interval since, at that t,ime, the relationship of capsular reduction to enzyme concentration is IIO~,linear in the latter case. This suggests that’, while t,he activity of the crude extract, remains linear for the first, 5

Final concentrotlon of bull-iestlcular hyoluronidase (Schering) in terms of TRU per ml.

FIG. 5. Cnpsulsr degradation by a commercial hyaluronidase of enzyme concentrntion at the 5-minute interval (raw data).

a3 a function

minutes but is no longer SO at the 10 minute and subsequent intervals, the purified extract reacts much faster and by the end of the first time period the curve has begun to flatten in the higher concentrations. Repetition of the experiment, using earlier time intervals in an attempt to obtain both reactions in linear form solely for the purpose of comparing the activity of one system in terms of the other was considered beyond the scope of these studies. It is assumed however, from its action on the specific substrate, the hyaluronic acid capsules of group C mucoid beta hemolytic streptococci, that the cercarial enzyme factor is of the “hyaluronidase” complex. Whether the obviously minute amount of hyaluronidase present per cercaria is a factor in either initial skin penetration or early migration remains an open question.

426

A. S. EV.4NS SUMMARY

The streptococcal decapsulatjion test has been adapted to afford an extremely sensitive method for the quantitative assay of the activity of enzymes of the hyaluronidase complex. By this method, the activity of an enzymatic factor extracted from cercariae of Schistosoma mansoni has been quantitat’ively measured in t,erms of volumetric degradation of the hyaluronic acid capsules of a strain of group C, mucoid bet.a hemolytic streptococci as a function of cercarial concentrat)ion. CONCLUSIONS

From these studies, the following conclusions have been reached: 1. Reduction in capsular volume of the streptococci as a function of the concentration of the cercarial extract is a linear function throughout the interval 0 to 85,000 cercariae per ml, the react,ion time being held constant at 5 minutes. 2. Concentrations above 85,000 cercariae per ml give no further capsular reduction, the limit remaining at 90-95 % even after reaction times up to and including 30 minutes. 3. The lower limit of sensit’ivity of the test appears to be in t,he neighborhood of 10,000 cercariae per ml, and increments of greater t’han 10,000 cercariae per ml should be used in assay. 4. From its action on the specific substrate, the cercarial enzyme is complex. However, its activity presumed to be of the “hyaluronidase” cannot be stated in terms of bull-testicular hyaluronidase from these experiments, since the 5 minute interval is past the period of linerarity of the reaction of the testicular enzyme. ACKNOWLEDGEMENTS The author gratefully acknowledges the aid of Dr. D. R. Lincicome, of the Stine Laboratory, Newark, Delaware, who originally suggested the use of encapsulated streptococci as a substrate in these studies; the generosity of Dr. C. V. Seastone of the Department of Medical Microbiology, University of Wisconsin, who supplied a suitable strain of streptococci, and the invaluable criticisms of Dr. Osamu Hayaishi of the National Institutes of Health, Bethesda, Maryland, and of Drs. M. A. Stirewalt and T. 1,. Hill of the Naval Medical Research Institute, Bethesda, Maryland. REFERENCES BUTT, E. M., BONYNGE, C. W., AND JOYCE, R. L. 1936. The demonstration of capsules about hemolytic streptococci with India ink or azo blue. J. Infectious Diseases 68, 59.

HODGMAN, C. D. (Editor)

1945. Handbook of Chemistry and Physics, 29th ed. Chemical Rubber Publishing Co., Cleveland, Ohio, p. 1368. LEVINE, M. D., GAR~OLI, R. F., KUNTZ, R. E., and Killough, J. H. 1948. On the demonstration of hyaluronidase in cercariae of Schistosoma mansoni. J. ParaSW. 34, 158-161. LIXCICOME, D, R. 1953. A streptococcal decapsulntion test for detection of hyaluronidase activit,y in animal parasites. E.cptl. Parasitol. 2, 333-320. MCCLEAN, D. 1941. The cnpsulation of streptococci and its relation to diffusion factor (hyaluronidase). J. I’alhol. Huctf/Yol. 53, 13-27. MEYER, K. 1947. The biological significance of hysluronic acid and hyaluronidase. Physiol: Revs. 27, 335-359. MORISON, J. 15. 1941. The physiology of capsulated streptococci. J. Patho/. Bncteriol. 63(l), 1-12. SEASTONE, C. 5’. 1939. The virulence of group C hemolytic streptococci of animal origin. ,I. E.zptl. Med. 70, 361-378. STIRWALT, AI. A., AND EVANS, A. S. 1952. Demonstrnt,ion of an enzymatic factor in ccrcariae of Schistosomn mansoni by the strcptococcal decapsulntion test. J. Znfectio~ts Diseases 91(2), 191-197.

Sx.kIL, L. L. 1949. CalcuIus. .4pplcton~Celltury-Crofts,

New York, pp. 333-335.