Extracellular neuraminidase production by group B streptococci

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Apr 18, 1977 - x g for 30 min at 40C and filtered through a 0.20-,um sterile Nalgene filter ... M sodium acetate buffer (pH 6.5) and dialysis against. 100 to 200 ...
INFECTION

AND

IMMUNITY, Oct. 1977, p. 189-195

Vol. 18, No. 1 Printed in U.S.A.

Copyright © 1977 American Society for Microbiology

Extracellular Neuraminidase Production by Group B Streptococci THOMAS W. MILLIGAN, DAVID C. STRAUS, AND STEPHEN J. MATTINGLY* Department of Microbiology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 Received for publication 18 April 1977

Neuraminidase (sialidase) activity in concentrated culture filtrates of group B streptococci was measured with bovine submaxillary mucin as substrate. Group B streptococcal neuraminidase was not active on human alpha-i acid glycoprotein and did not show increased activity on bovine submaxillary mucin that had been O-deacetylated by alkaline treatment. The enzyme was produced in a variety of media, including a chemically defined medium (FMC; Terleckyj et al., Infect. Immun. 11:649-655, 1975) supplemented with bovine serum albumin or human serum albumin. Maximal levels of activity were present in filtrates from cells grown in a dialyzable fraction of Todd-Hewitt broth harvested during the late exponential phase of growth. Dramatic decreases were seen when filtrates from the late stationary phase were assayed. The decrease in specific activity during the stationary phase was shown to be due to proteolytic digestion of neuraminidase and not to the elaboration of an extracellular neuraminic acid aldolase.

Renewed interest in the group B streptococci aminidase in several strains of group B streptohas occurred recently due to the increased cocci, no attempt was made to examine the awareness of the etiological role of the group B conditions under which maximal enzyme levels streptococci in serious iflfections of newborns cqguld be achieved. "The objective of the present study, therefore, (23). Several workers have attempted to characterize potentially pathogenic extracellular ;was to examine several strains of group B strepproducts produced by these microbes. For in- tococci, including a laboratory-passaged strain stance, as early as 1934, Todd (26) described and a fresh clinical isolate, for the production the production of an oxygen-stable, non-immu- of extracellular neuraminidase, using cell filnogenic hemolysin produced by the group B trates prepared at the time of maximal enzyme streptococci, which was destroyed when kept at expression. 60°C for 30 min and was somewhat sensitive to MATERIALS AND METHODS acid. Although Hare (12) was not able to show the production of a streptokinase, McClean (20) Bacterial strains. Group B streptococcus strain demonstrated that the group B streptococci H36B (type Ib) was kindly supplied by Hazel W. elaborated a hyaluronidase, but, unlike the Wlkinson, Center for Disease Control, Atlanta, Ga. group A streptococci, did not produce a hyalu- Strain 110 (type III), isolated from a case of neonatal ronic acid capsule. Deibel (9) examined six suppurative meningitis (3),of was obtained from Carol Baker, Baylor College Medicine, Houston, Tex. strains of group B streptococci for the presence J.Multiple of each strain were lyophilized in of extracellular protease and deoxyribonuclease. 1.0% skimportions milk and/or frozen at -70°C in Todd-HewAlthough proteolytic activity against gelatin was itt broth for long-term storage. To obtain cells for not detected, three of the strains elaborated an experiments, samples from the frozen or lyophilized enzyme capable of degrading deoxyribonucleic cultures were streaked onto 5% sheep blood agar plates acid. Brown et al. (8) reported the isolation of and, after overnight incubation at 37°C, were stored the protein CAMP factor of the group B strep- at 4°C for up to 3 weeks. Liquid media and growth conditions. A variety tococci, which caused the rapid lysis of erythroof liquid culture media were used during the course with the of treated cytes beta-hemolysin Staph- of these studies. Todd-Hewitt broth (Difco) was preylococcus aureus. Finally, Hayano et al. (13, 14) pared to the manufacturer's directions, was according of a were able to demonstrate the production sterilized by autoclaving, and, after cooling, was adgroup B streptococcal sialidase (neuraminidase) justed to pH 7.0 and modified by addition of filterthat was active on a sialomucoid preparation sterilized glucose (0.25 to 2.00%, final concentration). from bovine submaxillary glands. Although The latter medium is referred to in the text as THGB. these workers described the production of neur- THGD refers to the dialyzable fraction of Todd-Hew189

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INFECT. IMMUN.

itt broth, which was routinely made by preparing a the enzyme preparation. Each assay mixture contained lOx solution of Todd-Hewitt broth that was dialyzed the following components in a volume of 0.5 ml: 1.0 at 40C for 24 h against a volume of distilled water mg of BSM, 10 mM CaCl2, 100 mM sodium acetate that at equilibrium would result in a lx concentration (pH 6.5), and group B streptococcal neuraminidase of diffusible components in the dialysate. FMC refers (generally 0.01 to 0.05 ml of a 20-fold-concentrated to a chemically defined medium that was prepared filtrate). Each set of assays included a substrate blank, according to the procedure of Terleckyj et al. (25), and if the filtrate had not been tested before, an except that the sodium phosphate concentration was additional tube was included in which the enzyme adjusted to 0.06 M. In addition, the sodium carbonate preparation was heat-inactivated for 2 min at 100°C. concentration was 0.019 M, and the pH of FMC was Heat treatment was always found to destroy 95 to routinely adjusted to 7.0. All broth cultures were 100% of the activity. Enzyme reactions were performed grown statically at 37°C in 50- to 100-ml volumes in in duplicate and initiated by addition of the enzyme 300-ml optically calibrated side-arm flasks. Growth (except where heat-inactivated enzyme was assayed, was monitored by measuring the absorbance in a in which case substrate was the initiator) to the reColeman Junior II spectrophotometer at 675 nm. The maining components, and the amount of released sialic observed optical density was multiplied by 1,000 and acid was quantitated by the colorimetric thiobarbituric converted to adjusted optical density units so that acid (TBA) assay of Aminoff (1), using N-acetylneurareadings would agree with Beer's Law and be propor- minic acid (Sigma) as a standard. Protein concentrational to bacterial mass (27). Turbidities were quanti- tions were determined in duplicate by the Folin phenol tated by dry-weight determinations of 10-mi samples reagent method of Lowry et al. (18), using bovine of washed, stationary-phase cell suspensions. Samples serum albumin (BSA) as the standard, and specific were dried at 84°C for 48 h and desiccated over Ca2SO4 activities were expressed as nanomoles of sialic acid before weighing. It was ascertained that 1 adjusted released per minute per milligram of extracellular optical density unit corresponded to 0.43,ug of cellular protein. To more accurately compare the absolute dry weight per ml. The various broth media were levels of neuraminidase present in cell filtrates, total inoculated with organisms that had grown for at least enzyme activity was also expressed on a cellular dryfour to five generations in the identical medium to weight basis. The following formula was used to degive an initial adjusted optical density reading of 5 to termine the total amount of activity present in con40 units (2.2 to 17.2 ,tg of dry weight per ml). centrated filtrates: Preparation of culture filtrates. Broth cultures at various stages of the growth cycle were quickly Total activity chilled in ice and harvested by centrifugation at 10,000 [volume of concentrated filtrate (milliliters) x g for 30 min at 40C and filtered through a 0.20-,um x nanomoles of sialic acid released sterile Nalgene filter unit. Filtrates were then adjusted per minute per milliliter] to pH 6.5 and concentrated by either of two methods. In some experiments, filtrates were precipitated by 0 [total volume of culture medium (milliliters) x milligrams of cell dry weight to 75% ammonium sulfate saturation, followed by susper milliliter at time of harvest] pension of the precipitate in a small volume of 0.01 M sodium acetate buffer (pH 6.5) and dialysis against 100 to 200 volumes of the same buffer at 4°C for 24 Neuraminic acid aldolase activity was determined by h. The second concentration method involved the use incubating enzyme samples with the sialic acid reacof immersible molecular separators (Millipore Corp., tion product of the group B neuraminidase or a comPTGC 001 K1) in which the entire filtrate was concen- mercial preparation of N-acetylneuraminic acid and trated to 5 to 10 ml and subsequently washed by 50 measuring the loss of TBA reactive material. To prevolumes of 0.01 M sodium acetate buffer. pare the group B sialic acid substrate, neuraminidase Substrate preparation. Bovine submaxillary mu- from culture filtrates of strain H36B or 110 was incucin (BSM; Sigma Chemical Co.) was dissolved in 0.01 bated with 5.0 mg of BSM, and the reaction was M sodium acetate (pH 6.5) at a concentration of 10 allowed to proceed to completion under standard conmg/ml. All solutions of human alpha-1 acid glycopro- ditions. Subsequently, the reaction mixture was ditein (Calbiochem) were prepared in the same manner alyzed against 10 ml of distilled water, and the resultas BSM. Alkali treatment of BSM was performed ing dialysate was concentrated by lyophilization. Apaccording to the procedure of Gibbons (11). Briefly, proximately 0.01 Amol of the sialic acid was then 50 mg of BSM was dissolved in 50 mi of 0.05 M incubated with autologous (H36B or 110) enzyme samsodium carbonate and heated for 20 min at 1000C. ples under the conditions of the neuraminidase assay. After cooling, the material was dialyzed against 1 liter The amount of TBA reactive material remaining in of distilled water at 0 to 4°C with daily changes for 5 the reaction mixture was determined and compared days. The nondiffusible components were subse- with a control containing no enzyme. quently concentrated by lyophilization and stored at Preparation of [3HJvaline-labeled crude extra-20°C until used. cellular neuraminidase. To obtain a crude radiolaEnzyme assays. The amount of neuraminidase beled neuraminidase preparation, strain 110 (type III) present in concentrated filtrates of group B strepto- cells were grown for five to six generations in 5 ml of cocci was quantitated by measuring the amount of THGD supplemented with 5,uCi of [3H]valine per ml. sialic acid released from saturating amounts of BSM. A portion of these uniformly labeled cells was inocuVarious preparations were incubated at 370C for var- lated into 50 ml of an identical medium and grown to ious times ranging from 2 to 10 min, depending on the early stationary phase of growth (0.14 mg of cell

VOL. 18, 1977

NEURAMINIDASE OF GROUP B STREPTOCOCCI

dry weight per ml). A 0 to 75% ammonium sulfate preparation of enzyme was then prepared as described above. To quantitatively determine the amount of extracellular radiolabeled macromolecular protein in the enzyme preparation, duplicate 50-Al samples of the 10-fold-concentrated radiolabeled supernatant material were precipitated with 5 ml of ice-cold 10% trichloroacetic acid. All trichloroacetic acid precipitates were collected on glass-fiber filters (Whatman GF/C), and the filters were washed three times with cold trichloroacetic acid and two times with 95% ethanol. Filters were transferred to scintillation vials, and precipitates were solubilized with 0.5 ml of 90% NCS (Amersham/Searle) for 2 at 500C. After cooling to 100C, 5 ml of a toluene-based scintillation cocktail (24) was added, and the samples were counted in a Searle Mark III scintillation counter equipped with computer conversion of counts per minute to disintegrations per minute.

RESULTS Effect of different culture media on production of extracellular group B streptococcal neuraminidase. The initial studies were designed to formulate a medium that would support the production of group B streptococcal neuraminidase in relatively high specific activity as compared with classical Todd-Hewitt broth. Therefore, strain H36B (serotype Ib) was grown to early exponential phase (0.07 to 0.11 mg of cell dry weight per ml), and concentrated cellfree filtrates were prepared by ammonium sulfate precipitation (or membrane filter concentration in the case of unsupplemented FMC) and assayed for neuraminidase. Similar levels of enzyme were present in filtrates from THGB and THGD when total activities from cells grown with the same standard glucose concentration (0.25%) were compared (Table 1). THGD, however, displayed a higher specific activity due to the relatively low protein content of THGD compared with that seen in THGB. The addition of a nonlimiting concentration of glucose (2.0%), however, appeared to decrease the amount of activity in filtrates prepared from THGD. Significantly, filtrates from unsupplemented FMC or FMC plus BSA (added after cell harvest) did not have detectable activity. In contrast, group B streptococci grown in FMC supplemented with 1.0 mg of BSA per ml throughout the growth cycle produced levels of enzyme comparable to those seen in whole Todd-Hewitt broth. The absence of neuraminidase in these former two filtrates did not appear to be related to the final pH at the time of harvest, since in all three cultures of FMC shown in Table 1, the final pH was nearly identical (pH range = 6.82 to 6.99). Relationship between the group B streptococcal growth cycle and neuraminidase

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production. Because of the apparent instability of neuraminidase activity during the growth cycle (see Table 1), experiments were performed to determine the optimal phase of the growth cycle for maximum yield of extracellular neuraminidase. H36B (serotype Ib) was selected as a laboratory strain, and strain 110 (serotype III) was chosen as a representative fresh clinical isolate. Cells were grown in THGD (0.25% glucose); at various times, 100-ml samples were withdrawn, and the cell-free supematant material was concentrated by 0 to 75% ammonium sulfate fractionation. Figure 1 shows the specific TABLE 1. Levels of extracellular neuraminidase produced by group B streptococcus strain H36B in various media Sp aacta Sp

Medium Medium

Total ac-

~~~~~tjvjty6

252 45 THGB (0.25% glucose) 177 59 THGD (0.25% glucose) 208 67 THGD (1.00% glucose) 117 33 THGD (2.00% glucose) ND NDc FMC (0.25% glucose) ND ND FMC (0.25% glucose); 1.0 mg of BSA per ml added after harvesting 187 43 FMC (0.25% glucose); 1.0 mg of BSA per ml present during growth cycle a Expressed as nanomoles of sialic acid released per minute per milligram of protein at 37°C; average of duplicate determinations. b Expressed as nanomoles per minute per milligram of cell dry weight. c ND, None detected; less than 5 nmol of sialic acid was released in a 10-min assay at 37°C. H36B

110 1000-

:5 120

~

00

e

80

-

,7oo

800600 -

-800 -600

400-

-400

-

250 200-

Yzx9

20

45

15

>=

E 100 -

60

80-:

60-

-200

i

i

40-

I

20

10

'I

20-

0 40 80 120 160 200240 280 320 360

1- 80 _1- 120 60 200 240 280 0 40 110

320 360

MINUTES OF GROWTH

FIG. 1. Effect of growth phase (x) on specific activity ((D) and specific activity per milligrams of dry cell weight (8) of extracellular neuraminidase from group B streptococcal strains H36B and 110. Cells were grown in THGD (0.25% glucose), and concentrated filtrates were obtained at various times during the growth cycle and assayed for neuraminidase as described in Materials and Methods.

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activities of neuraminidase present in these filtrates at various stages of the growth cycle. The consistent finding in both strains was an increase in the specific activity of enzyme during the exponential phase. After this time, the specific activity was found to decrease dramatically if cells were allowed to enter the stationary phase of growth. For example, the specific activity of neuraminidase elaborated by strain 110 decreased from 120 to 7.3 when cells were allowed to incubate for just 60 min after entry into the stationary phase. Similar decreases in activity were seen when specific activity was based on cell-dry weight (Fig. 1) or when the total amount of activity from these filtrates was compared (data not shown). The decrease in apparent activity during the stationary phase could have been due to the production of an extracellular neuraminic acid aldolase (7), which has been demonstrated in other streptococci (15). However, no neuraminic acid aldolase activity could be demonstrated, even when the actual sialic acid product liberated by the group B neuraminidase (strain H36B or 110) or a commercial preparation of N-acetylneuraminic acid was used as a substrate for the assay (data not shown). These results, together with the observation of the "stabilizing" effect of BSA on enzyme expression in FMC, suggested that group B extracellular protease(s) could be degrading the neuraminidase released into the culture filtrates during the growth cycle. Decay of cell-free neuraminidase activity from type II strain 110. To determine whether protease(s) was indeed responsible for inactivation of group B streptococcal neuraminidase, radiolabeled crude extracellular enzyme was prepared by growing the cells in THGD containing [3H]valine and concentrating the crude enzyme by ammonium sulfate precipitation. The enzyme preparation was harvested during the late exponential phase of growth so that maximum activity (Fig. 1) would be present in the preparation. The crude extracellular material was filtered just before initiation of the experiment to insure that no contaminating bacteria were present. The sample was then shifted from 4 to 37°C, and thereafter, at various time periods, portions were removed to determine (i) the amount of neuraminidase activity and (ii) the total disintegrations per minute present in 3H-labeled protein. Neuraminidase, in the absence of group B streptococcal cells, lost activity and trichloroacetic acid-precipitable counts during. incubation at 37°C (Fig. 2). The loss in neuraminidase activity occurred more quickly and to a greater extent (76%) than loss of trichloroacetic acid-precipitable counts (39%) at

z

60-\ fi 50

6

o\

40 30 -\

oH 0

3,0

560

120 so Ix0 TIME (minues)

180

2*

FIG. 2. Loss in enzyme activity (0) and trichloroacetic acid-precipitable counts (a) of group B (strain 110, type III) [H]valine-labeled extracellular protein.

180 min. Thus the group B streptococci appear to produce an extracellular protease that rapidly inactivates neuraminidase. This probably explains the lack of demonstrable neuraminidase activity in chemically defined medium and also the "stabilizing" effect of added BSA on enzyme production in FMC.

Effectiveness of various protein preparations in protecting group B streptococcal strain H36B neuraminidase from proteolytic digestion. In view of the above data on the presence of group B extracellular protease and its activity on extracellular neuraminidase, we chose to look at the ability of different protein preparations to protect neuraminidase activity when added to FMC at various concentrations. Exhaustively dialyzed solutions of ovalbumin (OVA), BSA, and human serum albumin (HSA) were added on a dry-weight basis to give the same theoretical number of susceptible peptide bonds (Table 2). The results indicate that the glycoprotein OVA was unable to protect the group B neuraminidase unless a relatively high concentration (1.0 mg/ml) was added. In marked contrast, BSA and HSA, both structurally pure proteins, were able to protect the enzyme at much lower levels. Interestingly, HSA, when added at a concentration of 250 ,ig/ml, provided protection of 84% of maximal neuraminidase activity. This concentration is quite similar to the total concentration of protein present in normal human cerebral spinal fluid (CSF) (21). The actual concentration of total protein present in normal human CSF varies from 150 to 450 jig/ml (15 to 45 mg/100 ml) (21). In addition, the major proteins present in normal human CSF consist of HSA and globulin in a ratio of

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5:1 (21). These results suggest the possible production of group B streptococcal neuraminidase during suppurative meningitis, since the enzyme was elaborated and protected from proteolytic digestion by the presence of HSA at-levels consistent with those found in CSF. Substrate specificity of the group B streptococcal neuraminidase. To determine whether the group B streptococcal neuraminidase showed specificity in its ability to cleave sialic acid, we compared the ability of an enzyme preparation from type III strain 110 to cleave sialic acid from a variety of glycoprotein preparations (Table 3). In each case, 1.0 mg of glycoprotein preparation was treated with either a commercial preparation of Vibrio cholerae neuraminidase (Behringwerke) or a group B (type III, strain 110) neuraminidase, and specific activity was calculated for each substrate (Table 3). The group B streptococcal neuraminidase was not able to hydrolyze human alpha-i acid glycoprotein (specific activity not detected) compared with BSM (specific activity = 137). The ability of these enzymes to act on alkali-treated BSM was also examined, since Aminoff (1) reported that bovine sialic acid, acetylated at C-7 or C-8, does not react in the TBA procedure. It was therefore felt that O-deacetylation of BSM with mild alkali might increase the apparent specific activity of our preparations, as has been shown for V. cholerae neuraminidase (11). As expected, the specific activity of the V. cholerae neuraminidase was increased by 90% over that measured with standard BSM. No increase was seen in specific activity when group B neuraminidase was assayed with BSM compared with alkali-treated BSM. The above results suggest, TABLE 2. Effectiveness of OVA, BSA, and HSA in protecting group B streptococcal strain H36B neuraminidase from proteolytic digestion Total activity' Exogenous Approx no. in protein

pOeaBSA, (OVA," or HSA)

(jLg/ml) 0 100 250 500 1,000

.

peptide bonds/mil

of (x 1017)b

-

-

but do not prove, that 0-acetylated derivatives of sialic acid may not be subject to cleavage by the group B streptococcal enzyme. Although the actual identity of the reaction product of the group B neuraminidase acting on BSM has not yet been ascertained, it is probable that it is a member of the sialic acid famnily of compounds, since the absorption spectrum of a concentrated dialysate of the reaction mixture was found to be nearly identical to that of N-acetylneuraminic acid (Fig. 3). DISCUSSION The most important factors involved in the maximal expression of neuraminidase activity in culture filtrates of the group B streptococci appear to be (i) the concentration of protective protein present in the media (Tables 1 and 2) and (ii) the time of harvest of the cells for preparation of the culture filtrates (Fig. 1). The finding that specific activity of the group B TABLE 3. Ability ofgroup B streptococcus type III strain 110 neuraminidase and V. cholerae neuraminidase to cleave sialic acid from various

glycoprotein preparationsa Glycoprotein prepn

Glycoprotepn

S

OVA

BSA

HSA

NDd ND ND ND

ND 135 224 277 292

ND 99 279

V. cholerae neuraminidase (nmol of sialic acid released/ mnin per ml)

0.6-

0V50)

0.0 5.0 12.5 25.0 50.0

17

Group B type III (110) neuraminidase (Inmolreleased/ of sialic acid min per mg of protein)

137 262 BSM . Alkali-treated 498 123 BSM . Human alpha-1 acid NDb 684 glycoprotein a Average of duplicate determinations. bND, None detected; less than 5 nmol of sialic acid released in a 10-min assay at 37°C.

S

V

193

N-

ACETYLNEU)RAMINC AMD

0.30 c',

304 333

a Corrected for 1,200-molecular-weight carbohydrate moiety in OVA. 'Calculation based on a molecular weight of 120 for an average amino acid. c Expressed as nanomoles of sialic acid released per minute per milligram of cell dry weight. d ND, None detected; less than 5 nmol of sialic acid released per 10-min assay at 37°C.

'

( nm)

FIG. 3. Absorption spectra of complexes obtained from the group B neuraminidase (H36B) reaction product (0) and N-acetylneuraminic acid (-) with the TBA reagent.

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neuraminidase decreased by 75 to 94% if cells were incubated for 60 min after deviation from the exponential rate of growth emphasizes the importance of harvesting cells during the late exponential phase of growth to obtain maximal levels of enzyme. The demonstration of extracellular group B streptococcal protease activity (Fig. 2) was an unexpected finding in the present study, since Deibel (9) could not demonstrate proteolytic activity against gelatin with an agar plate assay. However, an extracellular protease is produced in cultures of group A streptococci that degrades an autologous group A streptococcal product, the M protein (10). This proteolytic enzyme is excreted in an inactive form and achieves maximal activity only under reducing conditions that can be generated during the later stages of the growth cycle (10). An analogous situation in the group B streptococci may exist, since dramatic decreases in neuraminidase activity were seen when the bacterial mass had reached its highest density (Fig. 1). The role of neuraminidase in the pathophysiology of group B streptococcal disease is only speculative at the present time. However, several workers have sought to assign a role for neuraminidase in relationship to clinical bacterial meningitis associated with Streptococcus pneumoniae. O'Toole et al. (22) examined levels of free and total N-acetylneuraminic acid concentrations in patients with various types of bacterial meningitis. In their series of patients, abnormal levels of free N-acetylneuraminic acid were found in 17 of 35 patients with pneumococcal meningitis, whereas 28 patients with Haemophilus influenzae or Neisseria meningitidis had normal levels of free N-acetylneuraminic acid. These results correlated well with the ability of these organisms to elaborate neuraminidase in vitro (i.e., only pneumococci were able to produce the enzyme). The neuraminidase of the group B streptococci, however, appears to be fundamentally different in substrate specificity from the S. pneumoniae enzyme. S. pneumoniae neuraminidase is active on N-acetylneuraminyllactose (16) and human alpha-i acid glycoprotein (15), whereas the group B streptococcal neuraminidase exhibited no activity on human alpha-i acid glycoprotein. Additionally, although pneumococci produce an extracellular N-acetylneuraminic acid aldolase (15), such activity has not been found in our concentrated culture filtrates. Studies on the ability of various media to support enzyme production by the group B streptococci have revealed some insight into the regulation of enzyme synthesis. The fact that similar levels of enzyme were present in a chemically defined

INFECT. IMMUN.

medium supplemented only with BSA (Table 1) and the complex medium Todd-Hewitt broth suggests a constitutive mode of neuraminidase production by the group B streptococci. An intriguing but unproven possibility is that "endogenous" sialic acid present in the capsular carbohydrate (6) is acting as an "inducer" of the

neuraminidase. Type III group B streptococci have been shown to be particularly common among bacterial isolates from neonates with meningitis, comprising 75 to 94% of the group B streptococci isolated (3, 4, 28). In this regard, Baker et al. (5, 6) have shown that women who give birth to infants who subsequently suffer from invasive type III streptococcal disease are deficient in antibody to purified type III polysaccharide determinants. Although these studies and the work of others (2, 17, 19) provide compelling evidence for the role of the maternal immune response in determining whether group B streptococci establish infection in neonates, they do not explain the pathogenic mechanisms operative in the disease once the group B streptococci are well established in the infant's meninges and cerebral spinal fluid. Studies are currently in progress to determine whether type III group B streptococci from neonatal suppurative meningitis are more prone to elaborate extracellular neuraminidase than the other serotypes and the role of the enzyme, if any, in group B streptococcal disease. ACKNOWLEDGMENTS We thank Jack C. Homer for his skilled technical assistance in the laboratory and Evelyn Oginsky for her advice and helpful criticism during the design of the experiments presented herein. This work was supported in part by Public Health Service research grant DE-04444 from the National Institute of Dental Research and institutional grant 5 SO0 RR 05654-06 from the Division of Research Resources.

LITERATURE CITED 1. Aminoff, D. 1961. Methods for quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem. J. 81:384-392. 2. Anthony, B. F. 1976. Immunity to the group B streptococci: interaction of serum and macrophages with types Ia, Ib, and Ic. J. Exp. Med. 143:1186-1198. 3. Baker, C. J., and F. F. Barrett. 1973. Transmission of group B streptococci among parturient women and their neonates. J. Pediatr. 83:919-925. 4. Baker, C. J., F. F. Barrett, R. C. Gordon, and M. D. Yow. 1973. Suppurative meningitis due to streptococci of Lancefield group B: a study of 33 infants. J. Pediatr. 82:724-729. 5. Baker, C. J., and D. L. Kasper. 1976. Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med.

294:753-756.

6. Baker, C. J., D. L. Kasper, and C. E. Davis. 1976. Immunochemical characterization of the "native" type

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7.

8.

9.

10. 11.

12.

13.

14.

15.

16. 17.

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