ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1977, p. 735-742 Copyright © 1977 American Society for Microbiology
Vol. 11, No. 4 Printed in U.S.A.
Bactericidal Factor Produced by Haemophilus influenzae b: Partial Purification of the Factor and Transfer of Its Genetic Determinant R. A. VENEZIA,* P. M. MATUSIAK, AND R. G. ROBERTSON Department of Microbiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642
Received for publication 7 October 1976
When aerobically grown on complex media, Haemophilus influenzae b and unencapsulated variants, Rb strains, produced a bactericidal factor that was active against other Haemophilus species and certain genera of the Enterobacteriaceae. A total of 341 clinical isolates of Haemophilus were tested for susceptibility to the factor. Ninety-three percent of H. influenzae (nontypable), 75% of H. haemolyticus, 71% of H. parainfluenzae, and 22% of H. parahaemolyticus were susceptible. H. influenaze b strains were resistant producers of the bactericidal factor and H. influenzae f strains were susceptible nonproducers. Only one strain each of H. aegyptius and H. aphrophilus was isolated and each was susceptible and resistant, respectively. 143 clinical isolates of the Enterobacteriaceae were tested and of those 82% of Escherichia coli, 85% of Salmonella sp., and all Citrobacter sp., Shigella sp., and Yersinia sp. were sensitive to the bactericidal factor produced by H. influenzae b. Attempts to isolate the bactericidal activity from mechanically disrupted, solubilized, or osmotically shocked cells failed to release active bactericidal factor. However, we partially purified the bactericidal factor from the spent culture medium of aerobically grown H. influenzae b by a series of extractions. The ability to produce the bactericidal factor was transferable to nonproducer strains without also genetically transforming for type b encapsulation. The converse was also true in that type b capsules were produced by transformed H. influenzae Rd strains but no bactericidal factor was detected from these strains. Additionally, nitrosoguanidineinduced mutants of H. influenzae b lost the ability to produce bactericidal factor without loss of their type-specific capsule, demonstrating that production of the bactericidal factor was genetically separable from production of the type capsule of H. influenzae b. We have reported that Haemophilus influenzae b produces a bactericidal factor that is active against Haemophilus species and certain members of the Enterobacteriaceae but not against gram-positive bacteria (13). Preliminary characterization of the bactericidal factor showed that it was a heat-stable protein of between 50,000 and 100,000 daltons in size, as determined by membrane filter retention. It was produced throughout the growth cycle of H. influenzae when cultured aerobically. The bactericidal factor appeared to be different from "colicin-like" bacteriocins in that its production was not inducible by mitomycin C, it did not kill by "single-hit" kinetics and, as opposed to the strain specificity of bacteriocin production, we have observed that all H. influenzae b strains produced the factor. None of the clinical nontypable H. influenzae strains tested pro735
duced the bactericidal factor. It is interesting that H. influenzae Rb, i.e., type b strains that have lost the ability to produce a capsule, retained the capacity to produce the bactericidal factor. We have hypothesized that capsule production occurs independently of bactericidal factor production. In this report, we present data to support that hypothesis and look more extensively into the spectrum of activity among the Haemophilus species and the Enterobacteriaceae. Additionally, our attempts at the isolation of the bactericidal factor from bacterial cells and the spent culture medium suggest that it may be produced as an extracellular protein. MATERIALS AND METHODS Bacterial strains. The clinical isolates of the Haemophilus species and the Enterobacteriaceae were
736
VENEZIA, MATUSIAK, AND ROBERTSON
obtained from the Bacteriology Laboratory, Strong Memorial Hospital, Rochester, N.Y. H. influenzae b (105) and H. influenzae Rd (C25) were studied as the bactericidal factor producer and susceptible strains, respectively. Media. Brain heart infusion (BHI) agar and 3.7% BHI broth (Difco) were prepared and supplemented with hemin (Kodak) and nicotinamide adenine dinucleotide (NAD; Sigma) as previously described (sBHI) (13). Other media tested for supporting the production of the bactericidal factor included chocolate agar (Baktron), Trypticase soy broth (Difco), and 1.5% proteose peptone no. 3 (Difco), supplemented with 1.2% agar (Difco), 2 ug of NAD per ml, and 10 jug of hemin per ml. Additionally, IsoVitaleX (BBL) or GC supplement without antibiotics (Oxoid) was added as indicated according to the manufacturer's instructions. The defined medium, utilizing the base medium described by Michalka and Goodgal (9), was modified by the addition of 0.2% milk casein hydrolysate (Difco), 0.01% glutamine (Sigma), 0.005% tryptophan (Sigma), 0.02% cystine (Sigma), 0.002% Tween 80 (Sigma), and 0.5% soluble starch (Difco) (final concentrations). In all cases, percent compositions were by weight/volume. We increased the glucose concentration to 0. 5% and decreased the supplement of hemin to 10 ,ug/ml and that of NAD to 2 ,ug/ml (final concentrations). Strains of 105 and C25 were selected for growth on this medium. Preparation of the bactericidal factor. Isolation of the partially purified bactericidal factor was accomplished by extraction of the spent culture of strain 105 grown in sBHI. The crude extract was obtained by the addition of (NH4)2SO4, to 55% saturation, to the cell-free, spent culture medium at 00C. After the mixture was stirred for 2 h, the precipitate was collected by centrifugation at 12,000 x g for 30 min at 40C, and the supernatant fluid was dialyzed extensively against 50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7, until no free sulfate ion was detected by addition of 2% BaCl, pH 6. The retained fraction was extracted with an equal volume of chloroform-methanol (2:1, vol/vol). The interface that formed between the aqueous methanol and chloroform phases was collected by centrifugation at 3,000 x g for 5 min and washed twice with an upper phase (chloroformmethanol-Tris-hydrochloride buffer, 3:48:47) (11). The interface was resuspended in Tris-hydrochloride buffer to the original volume and dissolved by heating in a 1000C water bath for 15 min. The solution was centrifuged, and then the supernatant fluid was titrated with concentrated HCl until a precipitate formed at pH 3.2. It was left overnight at 4°C, and the precipitate was removed by centrifugation at 5,000 x g for 15 min at 40C. The activity remained in the supernatant fluid and was stable at 4°C after filter sterilization. For long-term storage the bactericidal factor was maintained at - 70°C. Protein estimates were performed according to Lowry (7). In some preparations the chloroform-methanol extraction was repeated. The assays for bactericidal activity have been previously described (13).
ANTIMICROB. AGENTS CHEMOTHER.
Gel electrophoresis. Polyacrylamide gel electrophoresis was performed using either of two sodium dodecyl sulfate (SDS) systems: Biophore precast gels and SDS buffer (Bio-Rad) or SDS-polyacrylamide gels according to Laemmli (8). All samples were concentrated by precipitation with nine parts of cold acetone, redissolved with an SDS-denaturing buffer (Bio-Rad), and heated for 4 min in a 1000C water bath. The gels were fixed and stained with Coomassie blue (3). To isolate the bactericidal factor from unfixed gels, gel slices were homogenized in 2 ml of water and refrigerated overnight, and the acrylamide was pelleted by centrifugation at 1,500 x g for 10 min. The supernatant fluid was lyophilized and redissolved with water to 50% of the sample volume. The factor was precipitated with nine parts of cold acetone (14), and the precipitate, after pelleting at 1,500 x g for 10 min, was dissolved in Tris-hydrochloride buffer for assaying its activity. Disruption of washed cells. H. influenzae b was grown in 1 liter of sBHI at 37°C with shaking at 200 rpm until early stationary phase (260 to 280 Klett units, filter 54). The cells were pelleted by centrifugation at 17,000 x g for 10 min, washed once in deionized water, and resuspended to give a heavy slurry with 0.01 M Tris-hydrochloride buffer, pH 8. All resuspensions were with that buffer unless otherwise indicated. The cell suspension was divided into four equal parts and kept at 00C or frozen at - 20°C until treatment. The spent culture from those cells was used for extraction of the bactericidal factor. Four types of cellular disruption were used on the cell suspension. (i) Sonication. An aliquot of the cell suspension was sonically treated with a Bronwill Biosonic III (Bronwill Scientific, Rochester, N.Y.) in an ice bath until approximately greater than 80% cellular breakage was estimated by observation with a light microscope (3 min). The cellular debris was pelleted at 17,000 x g for 10 min at 40C. Both the supernatant fluid and the resuspended debris were assayed for activity. One milliliter of a crude extract of the bactericidal factor was exposed to sonication for 1 min at 00C to determine if activity was lost by this treatment. (ii) Na2CO3 solubilization. The cells of a second aliquot were pelleted and resuspended in 25 ml of 1% Na2CO3, pH 11, according to Tunevall (12). The resulting highly viscous solution was dialyzed overnight at 40C against Tris-hydrochloride buffer and treated with deoxyribonuclease I (Sigma) until viscosity decreased. The treated solution was heated to 800C for 10 min and clarified by centrifugation at 9,000 x g for 30 min. Both the resuspended cellular debris and the deoxyribonuclease-treated supernatant fluid were tested for activity. (iii) Heat disruption of cells. The cells from a third aliquot were resuspended in 5 ml of Tris-hydrochloride buffer and heated in a water bath at 650C for 15 min. The lysed cellular suspension was assayed for activity. (iv) Cell homogenization. Cells of the fourth aliquot were resuspended in 0.01 M Tris-hydrochloride buffer, pH 7.5 with 1 mM ethylenediaminetetraace-
H. INFLUENZAE b BACTERICIDAL FACTOR
VOL. 11, 1977
tate, and disrupted by homogenization with acidcleaned glass beads (0.10 to 0.11 mM) in a liquid
CO2-cooled Bronwill homogenizer. The resulting liquid was decanted and centrifuged at 5,000 x g for 10 min at 4°C. The supernatant fluid was heated to 65°C for 15 min. A precipitate was formed by addition of (NH4)2SO4 to 55% saturation at 40C, pelleted by centrifugation at 12,000 x g for 30 min at 4°C, dissolved to 10% of the original volume in Trishydrochloride buffer, and assayed for activity. (v) Osmotic shock. Freshly grown cells from both midexponential and early stationary phases of growth were osmotically shocked according to Heppel (5). The released cellular fluid was concentrated by lyophilization and tested for activity. Transformation of C25. A streptomycin-resistant mutant of strain 105 was selected as previously described (13). Transforming deoxyribonucleic acid (DNA) from 105 was isolated by the phenol extraction technique of Yasbin et al. (15). C25 cells were made competent, and the transformation reaction was according to the methods of Barnhart and Herriott (2). Competent cells and transformed cells were stored at -70°C in sBHI with 15% glycerol. The streptomycin resistance marker was selected by plating transformants on sBHI agar containing 200 jg of streptomycin sulfate, grade B (Calbiochem), per ml. Controls of transformation experiments either involved the use of deoxyribonuclease-treated DNA from strain 105 or no DNA was added to the transformation reaction. NTG treatment of strain 105. Nitrosoguanidine (NTG; Aldrich) treatment of strain 105 was accomplished according to Adelberg et al. (1). Briefly, 0.33 mg of NTG per ml (final concentration), pH 6, was added to one part of 0.1 M sodium acetate, pH 5, and five parts of saline containing approximately 1010 cells/ml. The mixture was incubated for 10 min at 37°C, and the reaction was terminated with an equal volume of ice-cold BHI, resuspended in 100 ml of sBHI, and incubated at 31°C for 2.5 h (four to five generations). The cells were stored at -70°C in 15% glycerol. This gave a final cell concentration of 2 x 106 cells/ml. The NTG-treated 105 cells were diluted to give 200 colonies per sBHI agar plate after 18 h of incubation at 37°C in a 10% CO2 atmosphere. Single colonies were streaked onto an indicator lawn (C25). All colonies that failed to give zones of inhibition for three transfers were scored as H. influenzae b nonproducers of the bacteridical factor. Crude extracts from broth cultures of these mutants were assayed for bactericidal activity. Control cells were not treated with NTG. RESULTS
H. influenzae grew on each of the media tested; however, bactericidal activity by strain 105 was observed only when grown on the complex media. Although all media allowed good to excellent growth of H. influenzae with capsule formation by type b strains, the defined medium would not support the production of bacte-
737
ricidal activity by strain 105 unless Oxoid GC supplement was added. In a similar experiment, GC supplement or IsoVitaleX had to be added to GC base medium, Trypticase soy medium, or the proteose peptone no. 3 medium. Only highly complex and enriched media supported the release or production of the bactericidal activity by strain 105. The crude extract of the bactericidal factor inhibited the indicator lawn grown on each of the media, indicating that no substances inhibitory to the bactericidal activity were present in those media tested. The bactericidal activity produced by strain 105 was typical of that produced by other H. influenzae b strains. The bactericidal factor killed strains of H. influenzae (nontypable), H. parainfluenzae, and H. haemolyticus (Table 1). The least susceptible strains were H. parahaemolyticus; only one H. aegyptius and one H. aphrophilus was isolated and they were susceptible and resistant, respectively. The members of the Enterobacteriaceae, which were susceptible to the bactericidal factor, were strains of Escherichia coli and closely related genera (Table 2). Lawns of 50 of 61 E. coli strains were susceptible to streaked overlays of strain 105 and crude extracts of the bactericidal factor. However, Proteus sp., Enterobacter sp., Klebsiella sp., and the other Enterobacteriaceae tested were uniformly resistant to the bactericidal activity. The bactericidal factor was isolated from the spent culture of strain 105 grown in sBHI (Fig. 1). The extraction procedures resulted in a preparation containing active bactericidal factor that resolved as a single diffuse band on 12% polyacrylamide SDS gels (Fig. 2). When the band was cut from unstained gels and extraneous SDS was removed by precipitation in TABLE 1. Strains of Haemophilus species susceptible to the bactericidal factor % tiblea Suscep-
Haemophilus sp.
No. of strains
susNo. ceptible
aegyptius aphrophilus haemolyticus influenzae (nontypable) H. influenzae type b H. influenzae type f H. parahaemolyti-
1 1 12 121
1 0 9 113
75 93
11 3 68
0 3 15
0 100 22
124
88
71
H. H. H. H.
cus
H. parainfluenzae
a An insignificant number of strains was isolated to give a meaningful calculation for percent suscep-
tible.
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VENEZIA, MATUSIAK, AND ROBERTSON
TABLE 2. Strains of Enterobacteriaceae susceptible to the H. influenzae b bactericidal factor No. of
No.
sus-
%
Suscep-
tiblea strains ceptible 100 5 5 Citrobacter sp. 82 50 61 E. coli 85 11 13 Salmonella sp. 100 7 7 Shigella sp. 1 1 Yersinia sp. 0 0 29 Enterobacter sp. 0 0 33 Klebsiella sp. 0 1 Pectobacterium sp. 0 0 33 Proteus sp. 0 0 5 Providencia sp. 0 0 7 Serratia sp. a An insignificant number of strains was isolated to give a meaningful calculation for percent susceptible.
ANTIMICROB. AGENTS CHEMOTHER.
tor. All transformants were both producers of the bactericidal factor and resistant to it, but none was also transformed for a type b capsule. By use of anti-H. influenzae Rd rabbit serum, we were able to select for C25 strains transformed to type b capsule production, because anti-Rd rabbit serum would agglutinate Rd type strains and leave type b transformants in the supernatant fluid. These encapsulated transformants were not producers of the bactericidal factor and were sensitive to its activity. Control cells treated with deoxyribonucleasedegraded DNA, or no DNA, did not show transformation to streptomycin resistance, to production of the bactericidal factor, or to encapsulation. In addition, strains resistant to the bactericidal factor, but not producers of it, arose among both the transformed cells and control cells grown in the presence of the bactericidal factor. When strain 105 was treated with NTG under the conditions described, 2 of 200 colonies resulted in mutants that were encapsulated but had lost the ability to produce the bactericidal factor both on sBHI agar and in broth. They remained resistant to the bactericidal activity. Control cells failed to produce spontaneous mutants.
acetone, no activity was lost as compared with control samples treated in a similar manner with SDS and acetone. Although further determinations are in progress, we feel that we have obtained the bactericidal factor in a partially purified form after the three-step extraction procedure. The specific activity has increased approximately 16 times that of the crude extract (Table 3). All four means of disruption and osmotic DISCUSSION shock of the washed cells of strain 105 failed to The range of activity ofthe bactericidal factor release active bactericidal factor. None of the by H. influenzae b varied among the produced when bactericidal activity inactivated methods crude extracts were tested under similar condi- Haemophilus species isolated. Certain species tions. The disruption of washed cells by use of a were observed to be very susceptible and these liquid C02-cooled Bronwill homogenizer re- included H. influenzae (nontypable), H. haesulted in the release of material that inhibited molyticus, and H. parainfluenzae. H. parahaelawns of both strains C25 and 105. The activity molyticus was least susceptible, and an insuffiof the inhibitory material was destroyed upon cient number of H. aegyptius and H. aphrophiheating to 65°C for 15 min in a water bath. Both lus were isolated to made a determination. The the lack of specificity of the bactericidal effect number of encapsulated strains of H. influand its heat lability were inconsistent with enzae was also low. However, all the type b those properties of the bactericidal factor. The strains were producers of the bactericidal factor technique of disruption was applied to midlog- and maintained the same spectrum of activity phase cells and the results were the same. The as test strain 105. In a previous study in which washes from the cells did not contain any bacte- all six serotypes were tested, none other than type b produced or was resistant to the bacteriricidal activity. The competent cells of strain C25 were trans- cidal factor (13). It was interesting to find that a formed with DNA from a streptomycin-resist- large percentage of E. coli and related genera ant 105 strain. The transformants were selected were susceptible to this bactericidal factor. The for streptomycin resistance and then tested for less closely related genera, i.e. Klebsiella, Proability to produce bactericidal activity. Of 200 teus, and Enterobacter, were uniformnly resiststreptomycin-resistant transformants screened, ant. According to a recent report by Johnson none was as able to produce the bactericidal (6), the Enterobacteriaceae can be divided into activity. However, when competent cells were three groups or classifications based on biotreated with DNA isolated from strain 105 chemical, physiological, and morphological and subsequently grown in the presence of the characters. From the results of their taxonomibactericidal factor, transformants were ob- cal study, Escherichia, Shigella, Salmonella, tained that could produce the bactericidal fac- and Yersinia fell into one closely related group
VOL. 11, 1977
H. INFLUENZAE b BACTERICIDAL FACTOR
739
CULTURE FLUID 55% saturated (NH4)2SO4
Precipitate
Supernatant fluid (discard)
Tris buffer to 4% original volume
Boiling-water bath (30 min)
Pellet (discard)
Supernatant fluid Dialyze
C:M (2:1, vol/vol)
Chloroform (discard)
Aqueous Methanol (discard)
Interface Dissolve in Tris buffer Boiling-water bath (30 min)
Supernatant fluid
Pellet (discard)
pH 3.2 Concn HCl
Supernatant fluid
Precipitate (discard)
pH 7 Partially purified bactericidal factor (stored -70°C) Fig. 1 Venezia et al. FIG. 1. Summary scheme of the purification of the bactericidal factor from culture fluid of H. influenzae b. C:M, Chloroform-methanol.
differing from both the Proteus-Providencia group and a Klebsiella, Serratia, Enterobacter group. It is not surprising that genera of the group closely related to E. coli would all be susceptible to the same bactericidal factor. The exact reason for the spectrum of activity of the factor extending from the genus Haemophilus
to those particular members of the Enterobacteriaceae is not readily apparent. However, we can suggest that similar receptor sites for the factor may exist on both the Haemophilus species and theE. coli group and quite possibly the metabolic response to the effect of the bacteri-
cidal factor may be similar. This is currently being investigated. The bactericidal factor is readily extracted from the spent culture medium of aerobically grown H. influenzae b. However, we have observed that only when complex media were used for growth did production of the bactericidal factor occur. The H. influenzae strains grew on simple media when supplied with hemin and NAD. We observed good growth on both supplemented Trypticase soy broth and the defined medium, but H. influenzae b did not exhibit bactericidal activity unless each
740
...S.....
~
~
~
~
AL
_
FIG. 2. (A) The partially purified bactericidal factor resolved as a very diffuse band near the migratory front (arrow) of 12% polyacrylamide SDS gels (Bio-Rad) (42 mglml). (B) The crude preparation of the bactericidal factor resolved as multiple bands on the same type of gels (250 p.Ag/ml).
was
ANTIMICROB. AGENTS CHEMOTHER.
VENEZIA, MATUSIAK, AND ROBERTSOI
enriched with
a
commercial
The need for enriched
or
complex
supplement. media sug-
trated and tested for bactericidal activity, none was observed. The failure to isolate active bactericidal factor from disrupted, solubilized, or osmotically shocked bacterial cells can be interpreted either as a lack of significant intracellular pools of the factor or as evidence that it exists in an inactive or characteristically different form within the cell. It seems unlikely that inactivation of the bactericidal factor occurred by the action of bacterial proteases. Enzymatic activity was kept to a minimum by maintaining the cells at less than 00C prior to treatment and heat treating the lysates after treatment. In any case, solubilization of the cellular envelope by heat or alkali treatment would argue against enzymatic inactivation of the bactericidal factor. In addition, the bactericidal factor was not inactivated by any of the means of cell fractionation and remained active in control preparations when treated under similar conditions. Previously, we showed that the bactericidal factor is released into the growth medium throughout the growth cycle ofH. influenzae b (13). No net increase in bactericidal activity was noted during the stationary phases of growth when autolysis was increased. The presence of the bactericidal factor in the medium throughout the entire growth curve, the requirement for complex growth medium, the sensitivity to proteolytic activity, and the inability to isolate active factor from washed cells suggest that the bactericidal factor may be an "exoprotein." Final determination as to the bactericidal factor's exoprotein nature will rest in demonstrating that during growth each cell is capable of producing and releasing it without damage to the cellular integrity. We were unable to use either molecular-sieve or ion-exchange column chromatography for the purification of the bactericidal factor from crude extracts. This was attributed to inactivaTABLE 3. Partial purification of the bactericidal factor from the culture supernatant fluid of H. influenzae b Fraction
growth conditions for either its production by
Crude extract, (NH4),S04 100°C, 30 min Dialysis
the cell
Chloroform-methanol
that the bactericidal factor may be a metabolic "luxuriant" product requiring
gests to
us
or
its release into the culture medium.
We have
attempted several methods for the
isolation of the bactericidal factor from washed
cells. The bactericidal factor in its extracellular form is resistant to heat short
or
alkali conditions for
periods of time. When either the released
cellular fluid
or
the cellular debris
was concen-
Bactericidal ac- Protein (mg/ Sp actb tivitya (%) ml) 38.5 47 14.1 3.26
Vol (ml)
33 34.5 19
50 54 47
14.8 8.0 0.96
3.39 6.75 48.96
interface 18.5 43 pH 3.2 0.84 51.19 a Percentage of C25 killed after 10 min of incubation at room temperature with 100 jul of each fraction when added to 5 x 104 colony-forming units of C25 per ml. ° Bactericidal activity per milligram of total protein per milliliter.
VOL. 11, 1977
H. INFLUENZAE b BACTERICIDAL FACTOR
tion of the factor or adsorption to the column supports. The bactericidal factor also displayed the unusual property of nonsTecific binding to cellulose or cellulose-like derivatives. However, we have developed a series of extractions that lead to a partial purification of the factor. The partially purified factor forms a single diffuse band on SDS-polyacrylamide gels after electrophoresis. The band migrates near the front on 12% gels and maintains all the bactericidal activity. This suggests that the molecular size of the factor is smaller than 14,000 daltons. However, we previously stated the factor was larger than 50,000 daltons based on retention by membrane filters. When the electrophoresis is performed without SDS, the bactericidal factor remains near the top of the 12% gels, and this indicates that aggregates of the molecule form in the absence of SDS. This also could explain the larger size estimated by the membrane filters. What clouds the issue is that the factor appears as a diffuse band atypical of SDS-denatured proteins on electrophoresis with SDS buffers. We suspect that the bactericidal factor may represent a class of molecules of approximately the same size. This heterogeneity would account for the diffuseness of the factor on SDS-containing gels. However, we cannot rule out the presence of acyl groups or other molecules bound to the protein of the bactericidal factor that contribute to this heterogeneity. For these reasons we prefer to state that the bactericidal factor is partially purified. It is interesting that the bactericidal factor remains active after partitioning between organic and aqueous phases. This condition suggests that the bactericidal factor is an amphipathic molecule. This configuration would allow the molecule to accommodate its interface location without undergoing an irreversible unfolding of its molecular structure. In addition, Reynolds (10) has observed that SDS interacts with some proteins in a way suggestive of the hydrophobic interactions of proteins within a biological membrane. This may explain why the active site of the bactericidal factor remains intact after denaturation in the presence of SDS. In any case, these characteristics indicate that the bactericidal factor is an interesting molecule that warrants further investigation to test this hypothesis. Since we have found that in addition to H. influenzae b strains H. influenzae Rb strains produce this bactericidal activity, it seemed unlikely that the activity was a consequence of encapsulation. We tested this by genetic transformation and mutagenesis. The selection for the streptomycin resistance marker was our
741
monitor as to whether genetic transformation was occurring. This marker appeared to transform with the same frequency as reported by others (4). We were able to transfer by genetic transformation the ability to produce the bactericidal factor to a nonproducing strain without also transferring the capacity to produce a capsule. The converse was also true. In addition, H. influenzae b, when exposed to a mutagen, lost the bactericidal activity but maintained its characteristic capsule. This confirms the hypothesis, suggested by the observation that H. influenzae Rb strains produce the bactericidal activity, that production of the bactericidal factor is independent of encapsulation. Whether this proves to be useful as an epidemological marker for H. influenzae Rb strains will be determined by clinical laboratories that screen for bactericidal factor production in association with disease-causing H. influenzae strains. ACKNOWLEDGMENTS We thank the Diagnostic Laboratory, Strong Memorial Hospital, Rochester, N.Y., and in particular Barbara Doerflein, for their cooperation in obtaining strains used in this
study. This investigation was supported by U.S. Public Health Service training grant no. GM 00592 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. C. C. Cheir. 1965. Optimal conditions for mutagenesis by N-methyl-Nnitro-N-Nitroso-Guanidine in Escherichia coli K-12. Biochem. Biophys. Res. Commun. 18:788-795. 2. Barnhart, B. J., and R. M. Herriott. 1963. Penetration of deoxyribonucleic acid into Haemophilus influenzae. Biochim. Biophys. Acta 76:25-39. 3. Fairbanks, G., T. L. Steck, and D. F. H. Wallach. 1971. Electrophoretic analysis of the major polypeptides of the human erthrocyte membrane. Biochemistry 10:2606-2617. 4. Goodgal, S. H., and R. W. Herriott. 1961. Studies on transformation ofHaemophilus influenzae. I. Competence. J. Gen. Physiol. 44:1201-1227. 5. Heppel, A. 1971. The concept of periplasmic enzymes, p. 223-247. In L. I. Rothfield (ed.), Structure and function of biological membranes. Academic Press Inc., New York. 6. Johnson, R., R. R. Colwell, R. Sakazaki, and K. Tamura. 1975. Numerical taxonomy study oftheEnterobacteriaceae. Int. J. Syst. Bacteriol. 25:12-37. 7. Kabat, E. A., and M. M. Mayes. 1961. Experimental immunochemistry, p. 556-558. Charles C Thomas Publishers, Springfield, Ill. 8. Laemmli, U. K. 1970. Cleavage of structural progeins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 9. Michalka, J., and S. H. Goodgal. 1969. Genetic and physical maps of the chromosome of Haemophilus influenzae. J. Mol. Biol. 45:407-421. 10. Reynolds, J. A., and C. Tanford. 1970. The gross conformation of protein-sodium dodecyl sulfate complexes. J. Biol. Chem. 245:5161-5165. 11. Soto, E. F., J. M. Pasquini, R. Placido, and J. L. Latorre. 1969. Fractionation of lipids and proteolipids from cat grey and white matter by chromatography
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on an organaphilic dextran gel. J. Chromatogr. 41:400409. 12. Tunevall, G. 1953. Studies on Haemophilus influenzae. Haemophilus influenzae antigens studied by the gel precipitation method. Acta. Pathol. Microbiol. Scand. 32:193-197. 13. Venezia, R. A., and R. G. Robertson. 1975. Bactericidal substance produced by Haemophilus influenzae b. Can. J. Microbiol. 21:1587-1594.
ANTIMICROB. AGENTS CHEMOTHER. 14. Weber, K., and M. Osborne. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 211:4406-4412. 15. Yasbin, R. E., G. A. Wilson, and F. E. Young. 1975. Transformation and transfection in lysogenic strains ofBacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol. 121:269304.
Vol. 11, No. 4 Printed in U.S.A.
Bactericidal Factor Produced by Haemophilus influenzae b: Partial Purification of the Factor and Transfer of Its Genetic Determinant R. A. VENEZIA,* P. M. MATUSIAK, AND R. G. ROBERTSON Department of Microbiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642
Received for publication 7 October 1976
When aerobically grown on complex media, Haemophilus influenzae b and unencapsulated variants, Rb strains, produced a bactericidal factor that was active against other Haemophilus species and certain genera of the Enterobacteriaceae. A total of 341 clinical isolates of Haemophilus were tested for susceptibility to the factor. Ninety-three percent of H. influenzae (nontypable), 75% of H. haemolyticus, 71% of H. parainfluenzae, and 22% of H. parahaemolyticus were susceptible. H. influenaze b strains were resistant producers of the bactericidal factor and H. influenzae f strains were susceptible nonproducers. Only one strain each of H. aegyptius and H. aphrophilus was isolated and each was susceptible and resistant, respectively. 143 clinical isolates of the Enterobacteriaceae were tested and of those 82% of Escherichia coli, 85% of Salmonella sp., and all Citrobacter sp., Shigella sp., and Yersinia sp. were sensitive to the bactericidal factor produced by H. influenzae b. Attempts to isolate the bactericidal activity from mechanically disrupted, solubilized, or osmotically shocked cells failed to release active bactericidal factor. However, we partially purified the bactericidal factor from the spent culture medium of aerobically grown H. influenzae b by a series of extractions. The ability to produce the bactericidal factor was transferable to nonproducer strains without also genetically transforming for type b encapsulation. The converse was also true in that type b capsules were produced by transformed H. influenzae Rd strains but no bactericidal factor was detected from these strains. Additionally, nitrosoguanidineinduced mutants of H. influenzae b lost the ability to produce bactericidal factor without loss of their type-specific capsule, demonstrating that production of the bactericidal factor was genetically separable from production of the type capsule of H. influenzae b. We have reported that Haemophilus influenzae b produces a bactericidal factor that is active against Haemophilus species and certain members of the Enterobacteriaceae but not against gram-positive bacteria (13). Preliminary characterization of the bactericidal factor showed that it was a heat-stable protein of between 50,000 and 100,000 daltons in size, as determined by membrane filter retention. It was produced throughout the growth cycle of H. influenzae when cultured aerobically. The bactericidal factor appeared to be different from "colicin-like" bacteriocins in that its production was not inducible by mitomycin C, it did not kill by "single-hit" kinetics and, as opposed to the strain specificity of bacteriocin production, we have observed that all H. influenzae b strains produced the factor. None of the clinical nontypable H. influenzae strains tested pro735
duced the bactericidal factor. It is interesting that H. influenzae Rb, i.e., type b strains that have lost the ability to produce a capsule, retained the capacity to produce the bactericidal factor. We have hypothesized that capsule production occurs independently of bactericidal factor production. In this report, we present data to support that hypothesis and look more extensively into the spectrum of activity among the Haemophilus species and the Enterobacteriaceae. Additionally, our attempts at the isolation of the bactericidal factor from bacterial cells and the spent culture medium suggest that it may be produced as an extracellular protein. MATERIALS AND METHODS Bacterial strains. The clinical isolates of the Haemophilus species and the Enterobacteriaceae were
736
VENEZIA, MATUSIAK, AND ROBERTSON
obtained from the Bacteriology Laboratory, Strong Memorial Hospital, Rochester, N.Y. H. influenzae b (105) and H. influenzae Rd (C25) were studied as the bactericidal factor producer and susceptible strains, respectively. Media. Brain heart infusion (BHI) agar and 3.7% BHI broth (Difco) were prepared and supplemented with hemin (Kodak) and nicotinamide adenine dinucleotide (NAD; Sigma) as previously described (sBHI) (13). Other media tested for supporting the production of the bactericidal factor included chocolate agar (Baktron), Trypticase soy broth (Difco), and 1.5% proteose peptone no. 3 (Difco), supplemented with 1.2% agar (Difco), 2 ug of NAD per ml, and 10 jug of hemin per ml. Additionally, IsoVitaleX (BBL) or GC supplement without antibiotics (Oxoid) was added as indicated according to the manufacturer's instructions. The defined medium, utilizing the base medium described by Michalka and Goodgal (9), was modified by the addition of 0.2% milk casein hydrolysate (Difco), 0.01% glutamine (Sigma), 0.005% tryptophan (Sigma), 0.02% cystine (Sigma), 0.002% Tween 80 (Sigma), and 0.5% soluble starch (Difco) (final concentrations). In all cases, percent compositions were by weight/volume. We increased the glucose concentration to 0. 5% and decreased the supplement of hemin to 10 ,ug/ml and that of NAD to 2 ,ug/ml (final concentrations). Strains of 105 and C25 were selected for growth on this medium. Preparation of the bactericidal factor. Isolation of the partially purified bactericidal factor was accomplished by extraction of the spent culture of strain 105 grown in sBHI. The crude extract was obtained by the addition of (NH4)2SO4, to 55% saturation, to the cell-free, spent culture medium at 00C. After the mixture was stirred for 2 h, the precipitate was collected by centrifugation at 12,000 x g for 30 min at 40C, and the supernatant fluid was dialyzed extensively against 50 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7, until no free sulfate ion was detected by addition of 2% BaCl, pH 6. The retained fraction was extracted with an equal volume of chloroform-methanol (2:1, vol/vol). The interface that formed between the aqueous methanol and chloroform phases was collected by centrifugation at 3,000 x g for 5 min and washed twice with an upper phase (chloroformmethanol-Tris-hydrochloride buffer, 3:48:47) (11). The interface was resuspended in Tris-hydrochloride buffer to the original volume and dissolved by heating in a 1000C water bath for 15 min. The solution was centrifuged, and then the supernatant fluid was titrated with concentrated HCl until a precipitate formed at pH 3.2. It was left overnight at 4°C, and the precipitate was removed by centrifugation at 5,000 x g for 15 min at 40C. The activity remained in the supernatant fluid and was stable at 4°C after filter sterilization. For long-term storage the bactericidal factor was maintained at - 70°C. Protein estimates were performed according to Lowry (7). In some preparations the chloroform-methanol extraction was repeated. The assays for bactericidal activity have been previously described (13).
ANTIMICROB. AGENTS CHEMOTHER.
Gel electrophoresis. Polyacrylamide gel electrophoresis was performed using either of two sodium dodecyl sulfate (SDS) systems: Biophore precast gels and SDS buffer (Bio-Rad) or SDS-polyacrylamide gels according to Laemmli (8). All samples were concentrated by precipitation with nine parts of cold acetone, redissolved with an SDS-denaturing buffer (Bio-Rad), and heated for 4 min in a 1000C water bath. The gels were fixed and stained with Coomassie blue (3). To isolate the bactericidal factor from unfixed gels, gel slices were homogenized in 2 ml of water and refrigerated overnight, and the acrylamide was pelleted by centrifugation at 1,500 x g for 10 min. The supernatant fluid was lyophilized and redissolved with water to 50% of the sample volume. The factor was precipitated with nine parts of cold acetone (14), and the precipitate, after pelleting at 1,500 x g for 10 min, was dissolved in Tris-hydrochloride buffer for assaying its activity. Disruption of washed cells. H. influenzae b was grown in 1 liter of sBHI at 37°C with shaking at 200 rpm until early stationary phase (260 to 280 Klett units, filter 54). The cells were pelleted by centrifugation at 17,000 x g for 10 min, washed once in deionized water, and resuspended to give a heavy slurry with 0.01 M Tris-hydrochloride buffer, pH 8. All resuspensions were with that buffer unless otherwise indicated. The cell suspension was divided into four equal parts and kept at 00C or frozen at - 20°C until treatment. The spent culture from those cells was used for extraction of the bactericidal factor. Four types of cellular disruption were used on the cell suspension. (i) Sonication. An aliquot of the cell suspension was sonically treated with a Bronwill Biosonic III (Bronwill Scientific, Rochester, N.Y.) in an ice bath until approximately greater than 80% cellular breakage was estimated by observation with a light microscope (3 min). The cellular debris was pelleted at 17,000 x g for 10 min at 40C. Both the supernatant fluid and the resuspended debris were assayed for activity. One milliliter of a crude extract of the bactericidal factor was exposed to sonication for 1 min at 00C to determine if activity was lost by this treatment. (ii) Na2CO3 solubilization. The cells of a second aliquot were pelleted and resuspended in 25 ml of 1% Na2CO3, pH 11, according to Tunevall (12). The resulting highly viscous solution was dialyzed overnight at 40C against Tris-hydrochloride buffer and treated with deoxyribonuclease I (Sigma) until viscosity decreased. The treated solution was heated to 800C for 10 min and clarified by centrifugation at 9,000 x g for 30 min. Both the resuspended cellular debris and the deoxyribonuclease-treated supernatant fluid were tested for activity. (iii) Heat disruption of cells. The cells from a third aliquot were resuspended in 5 ml of Tris-hydrochloride buffer and heated in a water bath at 650C for 15 min. The lysed cellular suspension was assayed for activity. (iv) Cell homogenization. Cells of the fourth aliquot were resuspended in 0.01 M Tris-hydrochloride buffer, pH 7.5 with 1 mM ethylenediaminetetraace-
H. INFLUENZAE b BACTERICIDAL FACTOR
VOL. 11, 1977
tate, and disrupted by homogenization with acidcleaned glass beads (0.10 to 0.11 mM) in a liquid
CO2-cooled Bronwill homogenizer. The resulting liquid was decanted and centrifuged at 5,000 x g for 10 min at 4°C. The supernatant fluid was heated to 65°C for 15 min. A precipitate was formed by addition of (NH4)2SO4 to 55% saturation at 40C, pelleted by centrifugation at 12,000 x g for 30 min at 4°C, dissolved to 10% of the original volume in Trishydrochloride buffer, and assayed for activity. (v) Osmotic shock. Freshly grown cells from both midexponential and early stationary phases of growth were osmotically shocked according to Heppel (5). The released cellular fluid was concentrated by lyophilization and tested for activity. Transformation of C25. A streptomycin-resistant mutant of strain 105 was selected as previously described (13). Transforming deoxyribonucleic acid (DNA) from 105 was isolated by the phenol extraction technique of Yasbin et al. (15). C25 cells were made competent, and the transformation reaction was according to the methods of Barnhart and Herriott (2). Competent cells and transformed cells were stored at -70°C in sBHI with 15% glycerol. The streptomycin resistance marker was selected by plating transformants on sBHI agar containing 200 jg of streptomycin sulfate, grade B (Calbiochem), per ml. Controls of transformation experiments either involved the use of deoxyribonuclease-treated DNA from strain 105 or no DNA was added to the transformation reaction. NTG treatment of strain 105. Nitrosoguanidine (NTG; Aldrich) treatment of strain 105 was accomplished according to Adelberg et al. (1). Briefly, 0.33 mg of NTG per ml (final concentration), pH 6, was added to one part of 0.1 M sodium acetate, pH 5, and five parts of saline containing approximately 1010 cells/ml. The mixture was incubated for 10 min at 37°C, and the reaction was terminated with an equal volume of ice-cold BHI, resuspended in 100 ml of sBHI, and incubated at 31°C for 2.5 h (four to five generations). The cells were stored at -70°C in 15% glycerol. This gave a final cell concentration of 2 x 106 cells/ml. The NTG-treated 105 cells were diluted to give 200 colonies per sBHI agar plate after 18 h of incubation at 37°C in a 10% CO2 atmosphere. Single colonies were streaked onto an indicator lawn (C25). All colonies that failed to give zones of inhibition for three transfers were scored as H. influenzae b nonproducers of the bacteridical factor. Crude extracts from broth cultures of these mutants were assayed for bactericidal activity. Control cells were not treated with NTG. RESULTS
H. influenzae grew on each of the media tested; however, bactericidal activity by strain 105 was observed only when grown on the complex media. Although all media allowed good to excellent growth of H. influenzae with capsule formation by type b strains, the defined medium would not support the production of bacte-
737
ricidal activity by strain 105 unless Oxoid GC supplement was added. In a similar experiment, GC supplement or IsoVitaleX had to be added to GC base medium, Trypticase soy medium, or the proteose peptone no. 3 medium. Only highly complex and enriched media supported the release or production of the bactericidal activity by strain 105. The crude extract of the bactericidal factor inhibited the indicator lawn grown on each of the media, indicating that no substances inhibitory to the bactericidal activity were present in those media tested. The bactericidal activity produced by strain 105 was typical of that produced by other H. influenzae b strains. The bactericidal factor killed strains of H. influenzae (nontypable), H. parainfluenzae, and H. haemolyticus (Table 1). The least susceptible strains were H. parahaemolyticus; only one H. aegyptius and one H. aphrophilus was isolated and they were susceptible and resistant, respectively. The members of the Enterobacteriaceae, which were susceptible to the bactericidal factor, were strains of Escherichia coli and closely related genera (Table 2). Lawns of 50 of 61 E. coli strains were susceptible to streaked overlays of strain 105 and crude extracts of the bactericidal factor. However, Proteus sp., Enterobacter sp., Klebsiella sp., and the other Enterobacteriaceae tested were uniformly resistant to the bactericidal activity. The bactericidal factor was isolated from the spent culture of strain 105 grown in sBHI (Fig. 1). The extraction procedures resulted in a preparation containing active bactericidal factor that resolved as a single diffuse band on 12% polyacrylamide SDS gels (Fig. 2). When the band was cut from unstained gels and extraneous SDS was removed by precipitation in TABLE 1. Strains of Haemophilus species susceptible to the bactericidal factor % tiblea Suscep-
Haemophilus sp.
No. of strains
susNo. ceptible
aegyptius aphrophilus haemolyticus influenzae (nontypable) H. influenzae type b H. influenzae type f H. parahaemolyti-
1 1 12 121
1 0 9 113
75 93
11 3 68
0 3 15
0 100 22
124
88
71
H. H. H. H.
cus
H. parainfluenzae
a An insignificant number of strains was isolated to give a meaningful calculation for percent suscep-
tible.
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VENEZIA, MATUSIAK, AND ROBERTSON
TABLE 2. Strains of Enterobacteriaceae susceptible to the H. influenzae b bactericidal factor No. of
No.
sus-
%
Suscep-
tiblea strains ceptible 100 5 5 Citrobacter sp. 82 50 61 E. coli 85 11 13 Salmonella sp. 100 7 7 Shigella sp. 1 1 Yersinia sp. 0 0 29 Enterobacter sp. 0 0 33 Klebsiella sp. 0 1 Pectobacterium sp. 0 0 33 Proteus sp. 0 0 5 Providencia sp. 0 0 7 Serratia sp. a An insignificant number of strains was isolated to give a meaningful calculation for percent susceptible.
ANTIMICROB. AGENTS CHEMOTHER.
tor. All transformants were both producers of the bactericidal factor and resistant to it, but none was also transformed for a type b capsule. By use of anti-H. influenzae Rd rabbit serum, we were able to select for C25 strains transformed to type b capsule production, because anti-Rd rabbit serum would agglutinate Rd type strains and leave type b transformants in the supernatant fluid. These encapsulated transformants were not producers of the bactericidal factor and were sensitive to its activity. Control cells treated with deoxyribonucleasedegraded DNA, or no DNA, did not show transformation to streptomycin resistance, to production of the bactericidal factor, or to encapsulation. In addition, strains resistant to the bactericidal factor, but not producers of it, arose among both the transformed cells and control cells grown in the presence of the bactericidal factor. When strain 105 was treated with NTG under the conditions described, 2 of 200 colonies resulted in mutants that were encapsulated but had lost the ability to produce the bactericidal factor both on sBHI agar and in broth. They remained resistant to the bactericidal activity. Control cells failed to produce spontaneous mutants.
acetone, no activity was lost as compared with control samples treated in a similar manner with SDS and acetone. Although further determinations are in progress, we feel that we have obtained the bactericidal factor in a partially purified form after the three-step extraction procedure. The specific activity has increased approximately 16 times that of the crude extract (Table 3). All four means of disruption and osmotic DISCUSSION shock of the washed cells of strain 105 failed to The range of activity ofthe bactericidal factor release active bactericidal factor. None of the by H. influenzae b varied among the produced when bactericidal activity inactivated methods crude extracts were tested under similar condi- Haemophilus species isolated. Certain species tions. The disruption of washed cells by use of a were observed to be very susceptible and these liquid C02-cooled Bronwill homogenizer re- included H. influenzae (nontypable), H. haesulted in the release of material that inhibited molyticus, and H. parainfluenzae. H. parahaelawns of both strains C25 and 105. The activity molyticus was least susceptible, and an insuffiof the inhibitory material was destroyed upon cient number of H. aegyptius and H. aphrophiheating to 65°C for 15 min in a water bath. Both lus were isolated to made a determination. The the lack of specificity of the bactericidal effect number of encapsulated strains of H. influand its heat lability were inconsistent with enzae was also low. However, all the type b those properties of the bactericidal factor. The strains were producers of the bactericidal factor technique of disruption was applied to midlog- and maintained the same spectrum of activity phase cells and the results were the same. The as test strain 105. In a previous study in which washes from the cells did not contain any bacte- all six serotypes were tested, none other than type b produced or was resistant to the bacteriricidal activity. The competent cells of strain C25 were trans- cidal factor (13). It was interesting to find that a formed with DNA from a streptomycin-resist- large percentage of E. coli and related genera ant 105 strain. The transformants were selected were susceptible to this bactericidal factor. The for streptomycin resistance and then tested for less closely related genera, i.e. Klebsiella, Proability to produce bactericidal activity. Of 200 teus, and Enterobacter, were uniformnly resiststreptomycin-resistant transformants screened, ant. According to a recent report by Johnson none was as able to produce the bactericidal (6), the Enterobacteriaceae can be divided into activity. However, when competent cells were three groups or classifications based on biotreated with DNA isolated from strain 105 chemical, physiological, and morphological and subsequently grown in the presence of the characters. From the results of their taxonomibactericidal factor, transformants were ob- cal study, Escherichia, Shigella, Salmonella, tained that could produce the bactericidal fac- and Yersinia fell into one closely related group
VOL. 11, 1977
H. INFLUENZAE b BACTERICIDAL FACTOR
739
CULTURE FLUID 55% saturated (NH4)2SO4
Precipitate
Supernatant fluid (discard)
Tris buffer to 4% original volume
Boiling-water bath (30 min)
Pellet (discard)
Supernatant fluid Dialyze
C:M (2:1, vol/vol)
Chloroform (discard)
Aqueous Methanol (discard)
Interface Dissolve in Tris buffer Boiling-water bath (30 min)
Supernatant fluid
Pellet (discard)
pH 3.2 Concn HCl
Supernatant fluid
Precipitate (discard)
pH 7 Partially purified bactericidal factor (stored -70°C) Fig. 1 Venezia et al. FIG. 1. Summary scheme of the purification of the bactericidal factor from culture fluid of H. influenzae b. C:M, Chloroform-methanol.
differing from both the Proteus-Providencia group and a Klebsiella, Serratia, Enterobacter group. It is not surprising that genera of the group closely related to E. coli would all be susceptible to the same bactericidal factor. The exact reason for the spectrum of activity of the factor extending from the genus Haemophilus
to those particular members of the Enterobacteriaceae is not readily apparent. However, we can suggest that similar receptor sites for the factor may exist on both the Haemophilus species and theE. coli group and quite possibly the metabolic response to the effect of the bacteri-
cidal factor may be similar. This is currently being investigated. The bactericidal factor is readily extracted from the spent culture medium of aerobically grown H. influenzae b. However, we have observed that only when complex media were used for growth did production of the bactericidal factor occur. The H. influenzae strains grew on simple media when supplied with hemin and NAD. We observed good growth on both supplemented Trypticase soy broth and the defined medium, but H. influenzae b did not exhibit bactericidal activity unless each
740
...S.....
~
~
~
~
AL
_
FIG. 2. (A) The partially purified bactericidal factor resolved as a very diffuse band near the migratory front (arrow) of 12% polyacrylamide SDS gels (Bio-Rad) (42 mglml). (B) The crude preparation of the bactericidal factor resolved as multiple bands on the same type of gels (250 p.Ag/ml).
was
ANTIMICROB. AGENTS CHEMOTHER.
VENEZIA, MATUSIAK, AND ROBERTSOI
enriched with
a
commercial
The need for enriched
or
complex
supplement. media sug-
trated and tested for bactericidal activity, none was observed. The failure to isolate active bactericidal factor from disrupted, solubilized, or osmotically shocked bacterial cells can be interpreted either as a lack of significant intracellular pools of the factor or as evidence that it exists in an inactive or characteristically different form within the cell. It seems unlikely that inactivation of the bactericidal factor occurred by the action of bacterial proteases. Enzymatic activity was kept to a minimum by maintaining the cells at less than 00C prior to treatment and heat treating the lysates after treatment. In any case, solubilization of the cellular envelope by heat or alkali treatment would argue against enzymatic inactivation of the bactericidal factor. In addition, the bactericidal factor was not inactivated by any of the means of cell fractionation and remained active in control preparations when treated under similar conditions. Previously, we showed that the bactericidal factor is released into the growth medium throughout the growth cycle ofH. influenzae b (13). No net increase in bactericidal activity was noted during the stationary phases of growth when autolysis was increased. The presence of the bactericidal factor in the medium throughout the entire growth curve, the requirement for complex growth medium, the sensitivity to proteolytic activity, and the inability to isolate active factor from washed cells suggest that the bactericidal factor may be an "exoprotein." Final determination as to the bactericidal factor's exoprotein nature will rest in demonstrating that during growth each cell is capable of producing and releasing it without damage to the cellular integrity. We were unable to use either molecular-sieve or ion-exchange column chromatography for the purification of the bactericidal factor from crude extracts. This was attributed to inactivaTABLE 3. Partial purification of the bactericidal factor from the culture supernatant fluid of H. influenzae b Fraction
growth conditions for either its production by
Crude extract, (NH4),S04 100°C, 30 min Dialysis
the cell
Chloroform-methanol
that the bactericidal factor may be a metabolic "luxuriant" product requiring
gests to
us
or
its release into the culture medium.
We have
attempted several methods for the
isolation of the bactericidal factor from washed
cells. The bactericidal factor in its extracellular form is resistant to heat short
or
alkali conditions for
periods of time. When either the released
cellular fluid
or
the cellular debris
was concen-
Bactericidal ac- Protein (mg/ Sp actb tivitya (%) ml) 38.5 47 14.1 3.26
Vol (ml)
33 34.5 19
50 54 47
14.8 8.0 0.96
3.39 6.75 48.96
interface 18.5 43 pH 3.2 0.84 51.19 a Percentage of C25 killed after 10 min of incubation at room temperature with 100 jul of each fraction when added to 5 x 104 colony-forming units of C25 per ml. ° Bactericidal activity per milligram of total protein per milliliter.
VOL. 11, 1977
H. INFLUENZAE b BACTERICIDAL FACTOR
tion of the factor or adsorption to the column supports. The bactericidal factor also displayed the unusual property of nonsTecific binding to cellulose or cellulose-like derivatives. However, we have developed a series of extractions that lead to a partial purification of the factor. The partially purified factor forms a single diffuse band on SDS-polyacrylamide gels after electrophoresis. The band migrates near the front on 12% gels and maintains all the bactericidal activity. This suggests that the molecular size of the factor is smaller than 14,000 daltons. However, we previously stated the factor was larger than 50,000 daltons based on retention by membrane filters. When the electrophoresis is performed without SDS, the bactericidal factor remains near the top of the 12% gels, and this indicates that aggregates of the molecule form in the absence of SDS. This also could explain the larger size estimated by the membrane filters. What clouds the issue is that the factor appears as a diffuse band atypical of SDS-denatured proteins on electrophoresis with SDS buffers. We suspect that the bactericidal factor may represent a class of molecules of approximately the same size. This heterogeneity would account for the diffuseness of the factor on SDS-containing gels. However, we cannot rule out the presence of acyl groups or other molecules bound to the protein of the bactericidal factor that contribute to this heterogeneity. For these reasons we prefer to state that the bactericidal factor is partially purified. It is interesting that the bactericidal factor remains active after partitioning between organic and aqueous phases. This condition suggests that the bactericidal factor is an amphipathic molecule. This configuration would allow the molecule to accommodate its interface location without undergoing an irreversible unfolding of its molecular structure. In addition, Reynolds (10) has observed that SDS interacts with some proteins in a way suggestive of the hydrophobic interactions of proteins within a biological membrane. This may explain why the active site of the bactericidal factor remains intact after denaturation in the presence of SDS. In any case, these characteristics indicate that the bactericidal factor is an interesting molecule that warrants further investigation to test this hypothesis. Since we have found that in addition to H. influenzae b strains H. influenzae Rb strains produce this bactericidal activity, it seemed unlikely that the activity was a consequence of encapsulation. We tested this by genetic transformation and mutagenesis. The selection for the streptomycin resistance marker was our
741
monitor as to whether genetic transformation was occurring. This marker appeared to transform with the same frequency as reported by others (4). We were able to transfer by genetic transformation the ability to produce the bactericidal factor to a nonproducing strain without also transferring the capacity to produce a capsule. The converse was also true. In addition, H. influenzae b, when exposed to a mutagen, lost the bactericidal activity but maintained its characteristic capsule. This confirms the hypothesis, suggested by the observation that H. influenzae Rb strains produce the bactericidal activity, that production of the bactericidal factor is independent of encapsulation. Whether this proves to be useful as an epidemological marker for H. influenzae Rb strains will be determined by clinical laboratories that screen for bactericidal factor production in association with disease-causing H. influenzae strains. ACKNOWLEDGMENTS We thank the Diagnostic Laboratory, Strong Memorial Hospital, Rochester, N.Y., and in particular Barbara Doerflein, for their cooperation in obtaining strains used in this
study. This investigation was supported by U.S. Public Health Service training grant no. GM 00592 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. C. C. Cheir. 1965. Optimal conditions for mutagenesis by N-methyl-Nnitro-N-Nitroso-Guanidine in Escherichia coli K-12. Biochem. Biophys. Res. Commun. 18:788-795. 2. Barnhart, B. J., and R. M. Herriott. 1963. Penetration of deoxyribonucleic acid into Haemophilus influenzae. Biochim. Biophys. Acta 76:25-39. 3. Fairbanks, G., T. L. Steck, and D. F. H. Wallach. 1971. Electrophoretic analysis of the major polypeptides of the human erthrocyte membrane. Biochemistry 10:2606-2617. 4. Goodgal, S. H., and R. W. Herriott. 1961. Studies on transformation ofHaemophilus influenzae. I. Competence. J. Gen. Physiol. 44:1201-1227. 5. Heppel, A. 1971. The concept of periplasmic enzymes, p. 223-247. In L. I. Rothfield (ed.), Structure and function of biological membranes. Academic Press Inc., New York. 6. Johnson, R., R. R. Colwell, R. Sakazaki, and K. Tamura. 1975. Numerical taxonomy study oftheEnterobacteriaceae. Int. J. Syst. Bacteriol. 25:12-37. 7. Kabat, E. A., and M. M. Mayes. 1961. Experimental immunochemistry, p. 556-558. Charles C Thomas Publishers, Springfield, Ill. 8. Laemmli, U. K. 1970. Cleavage of structural progeins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 9. Michalka, J., and S. H. Goodgal. 1969. Genetic and physical maps of the chromosome of Haemophilus influenzae. J. Mol. Biol. 45:407-421. 10. Reynolds, J. A., and C. Tanford. 1970. The gross conformation of protein-sodium dodecyl sulfate complexes. J. Biol. Chem. 245:5161-5165. 11. Soto, E. F., J. M. Pasquini, R. Placido, and J. L. Latorre. 1969. Fractionation of lipids and proteolipids from cat grey and white matter by chromatography
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on an organaphilic dextran gel. J. Chromatogr. 41:400409. 12. Tunevall, G. 1953. Studies on Haemophilus influenzae. Haemophilus influenzae antigens studied by the gel precipitation method. Acta. Pathol. Microbiol. Scand. 32:193-197. 13. Venezia, R. A., and R. G. Robertson. 1975. Bactericidal substance produced by Haemophilus influenzae b. Can. J. Microbiol. 21:1587-1594.
ANTIMICROB. AGENTS CHEMOTHER. 14. Weber, K., and M. Osborne. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 211:4406-4412. 15. Yasbin, R. E., G. A. Wilson, and F. E. Young. 1975. Transformation and transfection in lysogenic strains ofBacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol. 121:269304.
