ROLE OF HYALURONIDASE AND THE HYALURONIC ACID CAPSULE IN

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1 ROLE OF HYALURONIDASE AND THE HYALURONIC ACID CAPSULE IN THE SURVIVAL AND DISSEMINATION OF GROUP A STREPTOCOCCI IN THE HAMSTER CHEEK POUCH' ROBERT I. KRASNER,2 AND GENEVIEVE YOUNG Department of Biology, Boston University, Boston, Massachusetts Received for publication March 24, 1958 The effect of hyaluronidase on host connective tissue and the protective action of the hyaluronic acid capsule against phagocytosis have been the basis for a number of studies on the relation of these products to the virulence of streptococci. Duran-Reynals (1933) demonstrated that the areas of dermal lesions produced by several strains of streptococci were largely a function of the amount of hyaluronidase produced by these strains. Sallman and Birkeland (1950) found that streptococcal strains isolated from patients produced higher hyaluronidase titers than strains isolated from normal people, and furthermore, the higher titers were associated with the more clinically severe infections. These workers also demonstrated a correlation between hyaluronidase production in vitro and virulence for the chick embryo. Other investigators, however, (Crowley, 1944; Russell and Sherwood, 1949; Sherwood et al., 1952) have indicated a lack of correlation between hyaluronidase production in vitro and virulence for mouse, chick, or man. Warren (1950) studied the influence of high concentrations of hyaluronidase on the course of experimental infections in the flanks of rabbits and reported no enhancement of the infections nor increase in the size of the lesions. The resistance of streptococci of groups A and C to phagocytosis has been shown to be related in part to the presence of the hyaluronic acid capsule (Seastone, 1934; Ward and Lyons, 1935; Kass and Seastone, 1944; Rothbard, 1948; Morris and Seastone, 1955). Hirst (1941) successfully protected mice infected with group C encapsu- 1 This work was done under the tenure of a fellowship from the National Heart Institute, U. S. Public Health Service, and in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2 Present address: 406th Medical General Laboratory, APO 343 (Japan), San Francisco, California. lated streptococci by treatment with leech extract containing hyaluronidase, but similar treatment did not afford protection against group A streptococci. McClean and Hale (1941) and McClean (1942) failed to protect mice against either group A or group C by the use of testicular extract. However, Kass and Seastone (1944) demonstrated a strong protective effect in mice against group A encapsulated streptococci by suspending the cells in hyaluronidase and administering a total of 19 injections of hyaluronidase in a 4 day postinfection period. Rothbard (1948), using the multiple hyaluronidase injection schedule of Kass and Seastone, obtained protection in mice against both groups A and C streptococci and further demonstrated that such protection resulted only when hyaluronidase is given concomitantly with the streptococci and followed by additional treatment with the enzyme. In view of the discrepancies noted in the literature, the present study was undertaken to reinvestigate the roles of hyaluronidase and the hyaluronic acid capsule as "virulence factors" by studying the influence of these substances on the survival and dissemination of group A,3-hemolytic streptococci in the hamster cheek pouch. The pouch is a relatively dense layer of highly vascularized fibrous connective tissue lined with stratified squamous epithelium (Fulton and Jackson, 1947). The epithelium consists of two to five layers of cells which become progressively flatter towards the surface on which a slight amount of cornification is present. Longitudinally arranged skeletal muscle fibers are numerous at the edge of the layer near the open end of the pouch but absent at the blind end. The pouch is easily everted by gentle traction with forceps and is a convenient site to follow the development and subsequent localization of bacterial infections (Young, 1954). This structure offers a new approach to the evaluation of hyaluronidase and the hyaluronic acid capsule of streptococci. 349

2 ,350 KRASNER AND YOUNG [VOL. 76 MATERIALS AND METHODS Cultures. A type 1,B-hemolytic streptococcus was obtained through the courtesy of Dr. Rebecca Lancefield of the Rockefeller Institute for Medical Research; a smooth glossy variant was picked from a blood plate streaked with the parent strain and labeled Ti-S. A type 4 f,- hemolytic streptococcus was obtained from the Rammelkamp Collection at the 406th Medical General Laboratory. Cells from 15 to 18 hr cultures (Difco brain-heart infusion broth) were suspended in 0.5 per cent saline and standardized by turbidimetric methods. One ml of the final suspension was found by plate count to contain 1.5 X 10'D colonies. Hyaluronidase. Commercial hyaluronidase was used (Searle or Wyeth Pharmaceutical Co.) and resuspended to contain 500 viscosity units per ml. Decapsulation of streptococci. Well-encapsulated streptococci were prepared by adding one part of a 12 hr culture to 9 parts of fresh broth and incubating in a water bath at 37 C for approximately 3 hr (Rothbard, 1948). The supernatant was removed by centrifugation; 500 viscosity units of hyaluronidase (1 ml) were added to the cells and the suspension incubated for 20 min. The decapsulated cells were washed, resuspended in saline, and standardized as above. Hamster cheek pouch techniques. Male hamsters, approximately 100 g body weight, were anesthetized (pentobarbital sodium. Abbott) and their cheek pouches everted. The bacterial suspensions or hyaluronidase were injected with a 27-gauge sterile needle under the dissecting microscope. The material was routinely introduced into the lower left pouch quadrant. At stated intervals an attempt was made to recover organisms from the four quadrants of the pouch designated as lower left (L.L.), lower right (L.R.), upper left (U.L.), and upper right (U.R.). Each quadrant was aspirated with a separate moist sterile syringe and needle and the contents streaked onto a blood-agar plate marked off into four corresponding quadrants; plates were incubated for 24 hr. In the lower left cheek pouch quadrant (site of injection), material was aspirated directly from the lesion. To minimize recovery of contaminating bacteria (chiefly Pseudomonas aeruginosa) from the surface of the pouches, zephiran chloride (aqueous, 1:1000) was applied topically and removed with sterile saline before aspiration was attempted. Evaluation of the effects of hyaluronidase pretreatment or decapsulation. The degree of recovery per quadrant was recorded on a +6 to a +1 scale. To determine the extent of dissemination from the site of injection (lower left quadrant), the plus signs in the three other quadrants were totaled. Also, the percentage of quadrants yielding streptococcal cells, regardless of the amount, was determined. RESULTS Hyaluronidase. Pouches of the test animals were injected with 0.1 ml of hyaluronidase (500 viscosity units per ml); controls were injected with 0.1 ml of sterile 0.9 per cent saline. Approximately 3 hr later, 0.05 ml of standardized streptococcal suspension was used to infect pouches of both groups. Pouches were aspirated 6 hr and 1 day later. The Ti-S and the T4 strains of streptococci produced local suppurative infections in the cheek pouch. At the 6 hr stage, pouches of both the hyaluronidase pretreated and the saline pretreated animals exhibited edema, general vasodilation, and, at the injection site, a flat uncircumscribed lesion which, in a few cases, contained a small amount of pus. In both groups petechiae were numerous about and overlying the lesions, but were more widespread in the hyaluronidase pretreated pouches. By 24 hr welldefined, raised lesions containing pus were formed and measured approximately 3 by 4 mm. Petechiae had diminished greatly and were absent in many of the animals, whereas all animals now showed hemorrhagic areas about the abscesses. In the hyaluronidase pretreated animals, lesions appeared slightly more diffuse and hemorrhagic areas covered a wider range. The connective tissue of pouches pretreated with hyaluronidase was more permeable to the dissemination of streptococcal cells (tables 1 and 2). In these pouches 6 hr and 1 day after introducing the bacterial cells, more organisms were recovered from regions outside the area of injection, and a greater percentage of quadrants contained viable streptococci than in the controls. At 6 hr, hyaluronidase pretreated pouches infected with the Ti-S strain yielded almost twice as many organisms and showed a 13 per cent increase in the number of quadrants positive for streptococci. With the T4 strain at the 1 day stage there was almost a 3-fold increase in the

3 1958] HYALURONIDASE IN DISSEMINATION OF STREPTOCOCCI 351 TABLE 1 Influence of hyaluronidase on invasiveness of streptococci (strain Ti -S) in the hamster cheek pouch Organisms Recovered 6 hr after Organisms Recovered 1 Day after Infection* Infection L.L.t L.R. U.L. U.R. L.L. L.R. U.L. U.R. Saline injected (0.1 ml) 3 hr before streptococci Total recovery (other than L.L.) Quadrants yielding streptococci 75% 75% Hyaluronidase injected (0.1 ml) 3 hr before streptococci (hyaluronidase, viscosity units/ml) Total recovery (other than L.L.) Quadrants yielding streptococci... 88% 92% * Growth on blood plate rated on +6 to + scale; - indicates no growth t Quadrant-site of injection: L.L., lower left; L.R., lower right; U.L., upper left; and U.R., upper right. TABLE 2 Influence of hyaluronidase on invasiveness of streptococci (strain T4) in the hamster cheek pouch Organisms Recovered 6 hr after Organisms Recovered 1 Day after Infection* Infection L.L.t L.R. U.I.. U.R. L.L. L.R. U.L. U.R. Saline injected (0.1 ml) 3 hr before streptococci Total recovery (other than L.L.) Quadrants yielding streptococci 75% 56% Hyaluronidase injected (0.1 ml) 3 hr before streptococci (hyaluronidase, viscosity units/ml) Total recovery (other than L.L.) Quadrants yielding streptococci 100% 88% * Growth on blood plate rated on +6 to + scale; - indicates no growth. t Quadrant-site of injection: L.L., lower left; L.R., lower right; U.L., upper left; and U.R., upper right.

4 352 KRASNER AND YOUNG [VOL. 76 TABLE 3 Influence of decapsulation of streptococcal cells (strain T1) on their survival in the hamster cheek pouch Organisms Recovered 6 hr after Infection* L.L.t L.R. U.L. U.R. Saline controls Total recovery (other than L.L.) Quadrants yielding streptococci... 92% Cells treated with hy aluronidase (500 vis cosity units/ml) before injection Total recovery (other than L.L.) Quadrants yielding streptococci... 63% * Growth on blood plate rated on +6 to + scale; - indicates no growth. t Quadrant-site of injection: L.L., lower left; L.R., lower right; U.L., upper left; and U.R., upper right. number of organisms recovered and a 32 per cent increase in the number of quadrants yielding streptococci. Aspiration of pouches was not attempted beyond 1 day, although animals were examined grossly at later stages. No difference was noted in the course of the infections between the two groups. At 4 days, the infections appeared to have reached their peak as evidenced by the size of the abscesses (4 by 5 mm), and the degree of erythema, tortuosity of the blood vessels, and hemorrhage. The infections gradually subsided in subsequent stages until at about 12 days the majority of pouches showed a slight erythema, small areas of almost completely resorbed hemorrhage, and traces of a lesion. No deaths resulted in either group. Hyaluronidase, in concentrations of 250 and 500 viscosity units, was applied topically to pouch sites in which a small area of epithelial and connective tissue had been dissected away exposing the blood vessels in the underlying membrane. Pouches of 6 animals were injected with 0.1 ml of hyaluronidase (500 viscosity units per ml). Whether applied topically to blood vessels or injected into the pouch, the enzyme produced no observable toxic effects. Hyaluronic acid capsule. Six pouches were infected with decapsulated streptococcal cells and examined and aspirated 6 hr later; control animals were infected with encapsulated streptococcal cells and similarly examined and aspirated. Gross examination of infected pouches through 12 days showed local suppurative infections as described above. No difference was noted in the severity or duration of the infections resulting from the decapsulated streptococcal cells as compared to encapsulated cells, nor did deaths occur in either group. However, at 6 hr, the number of organisms recovered from the outlying quadrants in the test animals was reduced to almost half the control value (table 3). Furthermore, 29 per cent less of the pouch quadrants of the test animals yielded streptococci. Smears made from material aspirated from 6 hr lesions showed a predominance of encapsulated cells, and for this reason aspiration was not done at later stages. DISCUSSION The increased invasiveness of streptococci in pouches pretreated with hyaluronidase is assumed to be due to the enzymatic depolymerization of the hyaluronic acid in the connective tissue of this structure. Certain investigators (Crowley, 1944; Russell and Sherwood, 1949; Warren, 1950; Sherwood et al., 1952) have failed to substantiate hyaluronidase as a significant bacterial weapon. In most of these studies an attempt has been made to correlate hyaluronidase production in vitro with virulence for mouse, chick, or man. Hyaluronidase production in vitro and its quantitative measurement is dependent upon many experimental conditions such as periods of incubation, assay techniques, filtration methods, and types of media. In vivo, however, these factors can not be evaluated. Furthermore, the terms "virulence" and "invasiveness" are frequently used interchangeably, but should

5 19581 HYALURONIDASE IN DISSEMINATION OF STREPTOCOCCI 353 be considered as independent factors contributing to pathogenicity (Duran-Reynals, 1942). In the present investigation invasiveness is regarded as the active dissemination of microorganisms from their site of entry. The present experiments demonstrate an increased invasiveness of streptococcal cells in tissues pretreated with hyaluronidase, and yet an increase in deaths or "virulence" did not result from this single preliminary dose of the enzyme. The observation that vascular damage, as indicated by petechial formation, was more widespread in hyaluronidase pretreated pouches is in accord with that of Arendt (1955), who reported that a combination of hyaluronidase and moccasin venom increased the rate and area of petechial formation in the cheek pouch of the hamster, as compared with moccasin venom alone. Decapsulation of streptococci has been shown to decrease the resistance of such cells to ingestion by phagocytes (Seastone, 1934; Ward and Lyons, 1935; Kass and Seastone, 1944; Rothbard, 1948; Morris and Seastone, 1955). Pike (1948) expressed the belief that encapsulation and associated hyaluronic acid production are determinants of virulence among group A streptococci, as indicated by the observed frequency of encapsulated strains in acute infections and also by the mouse protection experiments reported by others. The present work substantiates this by a different approach. Streptococcal cells previously decapsulated by exposure to hyaluronidase were recovered from regions outside the site of injection in substantially reduced numbers as compared with encapsulated controls. It would seem that decapsulated cells that do reach outlying quadrants are more readily phagocytized than encapsulated cells. Smears made from material aspirated from 6 hr lesions showed encapsulated cells and demonstrated that the original decapsulated organisms multiplied to produce encapsulated progeny. The recovery of streptococci from the area of injection of both groups in approximately the same numbers is probably due to the fact that so many bacteria are present at the site of injection that many of the decapsulated cocci are able to resist phagocytosis long enough to undergo fission and produce encapsulated cells. SUMMARY Hamster cheek pouches pretreated with hyaluronidase showed greater dissemination of streptococci from their site of injection than control pouches, as indicated by the recovery of the organisms from various regions of the pouch at 6 and 24 hr. This single preliminary dose of hyaluronidase also increased early petechial formation and resulted in slightly larger lesions at 24 hr, but had no effect on the final healing of the local infection in about 12 days. Decapsulated streptococci showed a marked decrease in ability to survive in areas of the pouch outside the site of injection. The continued recovery of organisms from the latter area seemed to be due to the rapid reproduction of some of the cocci in this region, where they were highly concentrated, with the development of encapsulated forms. REFERENCES ARENDT, K. A Moccasin venom as a test for petechial susceptibility in the cheek pouch and mesoappendix of the hamster. Ph.D. Dissertation, Boston University. CROWLEY, N Hyaluronidase production by haemolytic streptococci of human origin. J. Pathol. Bacteriol., 56, DURAN-REYNALS, F Studies on a certain spreading factor existing in bacteria and its significance for bacterial invasiveness. J. Exptl. Med., 58, DURAN-REYNALS, F Tissue permeability and the spreading factors in infection. Bacteriol. Revs., 6, FULTON, G. P. AND JACKSON, R. G Cinephotomicroscopy of normal blood circulation in the cheek pouch of the hamster. Science, 105, HIRST, G. K The effect of a polysaccharide-splitting enzyme on streptococcal infections. J. Exptl. Med., 73, KASS, E. H. AND SEASTONE, C. V The role of the mucoid polysaccharide (hyaluronic acid) in the virulence of group A hemolytic streptococci. J. Exptl. Med., 79, MCCLEAN, D The in vivo decapsulation of streptococci by hyaluronidase. J. Pathol. Bacteriol., 54, MCCLEAN, D. AND HALE, C. W Studies on diffusing factors; hyaluronidase activity of testicular extracts, bacterial culture filtrates, and other agents that increase tissue permeability. Biochem. J. (London), 35, MORRIS, M. and SEASTONE, C. V The relationship of M protein and resistance to phagocytosis in the beta hemolytic streptococci. J. Bacteriol., 69, PIKE, R. M Streptococcal hyaluronic acid

6 354 KRASNER AND YOUNG [VOL. 76 and hyaluronidase. III. Virulence of group A streptococci for mice in relation to the production and destruction of hyaluronic acid. J. Infectious Diseases, 83, ROTHBARD, S Protective effect of hyaluronidase and type-specific anti-m serum on experimental group A streptococcus infections in mice. J. Exptl. Med., 88, RUSSELL, B. E. AND SHERWOOD, N. P Studies on streptococci. II. The role of hyaluronidase in experimental streptococcal infection. J. Infectious Diseases, 84, SALLMAN, B. AND BIRKELAND, J. M The role of hyaluronidase in hemolytic streptococcal infection. Ann. N. Y. Acad. Sci., 52, SEASTONE, C. V Capsules in young cultures of Streptococcus hemolyticus. J. Bacteriol., 28, SHERWOOD, N. P., RUSSELL, B., BOWMAN, K., AND OTT, J Studies on streptococci. IV. A study of the relationship of certain virulence factors in streptococcal infections to the LD50 dose of streptococci. J. Infectious Diseases, 91, WARD, H. K. AND LYONS, C Studies on hemolytic streptococcus of human origin; observations on virulent, attentuated, and avirulent variants. J. Exptl. Med., 61, WARREN, G. H The influence of hyaluronidase on the course of experimental infections with certain bacteria and viruses. Ann. N. Y. Acad. Sci., 52, YOUNG, G Experimental staphylococcus infection in the hamster cheek pouch: the process of localization. J. Exptl. Med., 99, Downloaded from on November 20, 2018 by guest