Molecular Characterization of Three Chloramphenicol ...

JOURNAL OF BACTERIOLOGY, Aug. 1982, p. 737-741 0021-9193/82/080737-05$02.00/0

Vol. 151, No. 2

Molecular Characterization of Three Chloramphenicol Acetyltransferases Isolated from Haemophilus influenzae MARILYN ROBERTS,'* ANGELA CORNEY,2 AND WILLIAM V. SHAW2 Department of Pediatrics, School of Medicine, University of Washington, Seattle, Washington 98195,1 and Department ofBiochemistry, University of Leicester, Leicester LE] 7RH, England2

Received 28 January 1982/Accepted 1 April 1982

Three plasmid-mediated chloramphenicol acetyltransferases isolated from different Haemophilus influenzae strains were purified and characterized. All three enzymes had properties in common with the gram-negative family of chloramphenicol acetyltransferases. The Haemophilus enzymes and the enteric type II enzyme were sensitive to 5,5'-dithiobis(2-nitrobenzoic acid), gave the same elution patterns from a highly substituted resin containing a bound chloramphenicol base, and had similar reactions to antisera. All four differed from each other in subunit molecular weight, enzyme activity, and partial protein digestion patterns. The data suggest that the three Haemophilus enzymes belong to the less common type II group and are related, but is not identical, to each other and to the enteric type II enzyme.

R factor-containing Haemophilus influenzae has only recently been detected. In a relatively short time, the Haemophilus population, previously regarded as uniformly susceptible, has become resistant to a number of antibiotics, including ampicillin (4), kanamycin (3), tetracycline, and chloramphenicol (11, 16). The mechanisms for resistance to ampicillin and tetracycline in H. influenzae appear to be very similar to those mediated by R factors in the Enterobacteriaceae (4, 5, 9). Chloramphenicol-resistant H. influenzae strains have steadily increased in the last few years. In most cases, chloramphenicol resistance is plasmid mediated and due to the production of an enzyme which catalyzes the acetylation of chloramphenicol in the presence of acetyl coenzyme A (1, 9). Bacterial resistance to chloramphenicol is mediated through the enzymatic action of chloramphenicol acetyltransferase (CAT). Two distinct groups of CAT variants comprising seven classes have been described: the constitutive group, found in Escherichia coli and related gramnegative genera, and the inducible group, associated with gram-positive species (6, 14, 17, 18). Each variant may be characterized by a number of physiochemical parameters. CAT variants specified from gram-negative organisms fall into one of three classes: type I, type II, or type III (18). Each enzyme comprises four identical subunits which catalyze the o-acetylation of chloramphenicol with acetyl coenzyme A as the donor. All three classes of enzymes share some properties with one another but can be differentiated by electrophoretic mobility, kinetic data, susceptibility to inhibitors, substrate affinity,

reactivity with antiserum, and affinity chromatography (6, 17). In this study, three H. influenzae enzymes from different strains, carrying distinct R plasmids, were characterized and compared with the known type I, type II, and type III CAT variants from the gram-negative family of enzymes. MATERIALS AND METHODS Bacterial strains. The bacterial strains and plasmids used are listed in Table 1. Media. The solid medium used for the growth of E. coli was 3.5% brain heart infusion (BHI) agar, and that

used for the growth of H. influenzae was BHI agar supplemented with 10%N sterile lysed horse blood. The

liquid medium was BHI broth for E. coli and BHI broth supplemented with 10 ,ug of hemin, 10 ,u8 of Lhistidine, and 2 Fg of NAD per ml for H. influenzae. Preparation and assay of CAT. Crude cell lysates were prepared by sonic disruption, as previously described, and stored at -10°C (13). Protein concentrations were determined by the method of Lowry et al. (8). The enzymatic acetylation of chloramphenicol was followed at 412 nm with a Pye-Unicam SP 1800 dual-beam recording spectrophotometer at 370C (14). Enzyme purificaton. All enzymes were purified from crude extracts by affinity chromatography employing a highly substituted Sepharose support, as previously described (6, 13, 18). The purified enzymes were run on polyacrylamide gels to determine purity. A single band was seen for each enzyme with Coomassie staining. Less than 5% impurities were found in the preparation when the more sensitive silver stain was used. Inhibition of CAT by DTNB. The crude lysates were tested for sensitivity to 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; a reagent which modifies reactive thiol groups). A 100-141 amount of each extract was incubat-

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ROBERTS, CORNEY, AND SHAW

J. BACTERIOL.

TABLE 1. Bacterial strains and their plasmids Strain

Date

Geographic area

Capsule Biotype Plasmid type type

Resistance'

MIC

(~~~~~~~g/ml)

CAT activity

(pmol/ ~~~inper mg)b

H. influenzae HC234 R375 R385 E. coli J53

1976 The Netherlands 1978 Colorado 1979 Pennsylvania

b b

II II I

pRI234 Cm, Tc pMR375 Cm, Tc, Ap pMR385 Cm, Tc

25 40 25

0.56 3.30 0.28

2.80 Cm (type I) 350 Km, Tc, Ap Cm (type II) J53 Sa 150 0.56 Sm, Km, Su Cm (type III), Sm 200 2.79 J53 R387 a Plasmid-mediated resistance: Cm, chloramphenicol; Ap, ampicillin; Tc, tetracycline; Sm, streptomycin; Su, sulfonamide; Km, kanamycin. b In crude extract.

ed for 10 min at 37°C. A 10-~Ll sample was removed, and its enzymatic activity was assayed. After the initial activity was determined, 10 ILI of a DTNB solution was added to the sample, making a final concentration of 1 mM. The extracts were incubated at 37°C, and samples were removed at 7, 15, and 30 min and assayed for enzyme activity. Immunological studies. Crude antisera raised against purified CAT variants were used. The type I reagent was made from a goat immunized with electrophoretically pure type I enzyme specified by plasmid JR66b (6). The CAT from JR66b is identical to the CAT encoded by plasmid R429 (used as the type I variant in this study) (6). Type II used purified enzyme specified from plasmid Sa, whereas type III was from plasmid R387. Both were raised in rabbits. Ouchterlony diffusion tests were performed in 0.75% agarose prepared in 0.8% NaCl-0.02% KCl-0.115% Na2HPO4-0.2% KH2PO4 in plates or on microscope slides. Gels were stained with 0.123% (wt/vol) amido black in 5% (wt/ vol) acetic acid and destained with 7% acetic acid. The type I and type III antisera gave strong reactions with the homologous CAT, whereas the type II antisera gave a very weak reaction. The animals were reimmunized, and the imnmunoglobulin G (IgG) fraction was purified (6). This preparation still gave a faint precipitin band with the homologous CAT. SDS-polyacrylamlde gel electrophoresis. Electrophoresis with 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels, using the system of Laemmli (7), was used to determine the apparent subunit molecular weights of CAT variants and for purification of enzymes for protein digests. Samples were heated in water to boiling and then boiled for 10 min before loading onto the gel. Gels were stained with Coomassie blue or silver stain. Protein dige ion in gel slices. Column-purified enzymes were run on 12% SDS-polyacrylamide gels, using 1.2-mm-thick spacers, and stained with Coomassie blue (2). The individual bands were cut out of the gel with a scalpel blade and placed in 10 ml of buffer A containing 0.125 M Tris-hydrochloride (pH 6.8), 1 mM EDTA, and 1% SDS, gently mixed, and soaked for 10 min at room temperature. The buffer was removed, replaced with fresh buffer, and soaked 10 min; a third

R429

change of buffer was then used. At the end of 30 min, the slices were removed and stored dry at -10°C until needed. A 17.5% SDS-polyacrylamide gel containing 1 mM EDTA with a long stacking gel, using 1.5-mm spacers, was used for the digestion gels. Before loading the samples, the wells were filled with buffer A. Then a gel slice was pushed to the bottom of a well with a spatula. Spaces around the gel piece were filled by overlaying 10 ,ul of buffer A containing 20% glycerol followed by 10 Il of buffer A containing 10%o glycerol, 0.1% bromphenol blue, and 1 pLg of papain or 2 pLg of Staphylococcus aureus V8 protease. The wells were filled to the top with buffer A. The top reservoir was filled with running buffer containing 1 mM EDTA. The samples were electrophoresed into the gel until the dye was 4 mm from the bottom of the stacking gel. The current was turned offfor 30 min and then turned on and run S to 6 h. The gel was stained with Coomassie blue or silver stain to visualize the bands (10). Silver stain. The protein-digested gels were soaked 1 h in 50%o methanol-10%o acetic acid and then overnight in 5% methanol-109o acetic acid. The next day the gels were soaked 30 min in 10%o unbuffered glutaraldehyde, rinsed, and soaked in distilled water for 2.5 h with gentle agitation. The gels were then stained in 200 ml of fresh 0.075% NaOH-7.76% AgNO3-2% NH40H for 10 min, rinsed, placed in 200 ml of fresh 0.005% sodium citrate-0.0199o formaldehyde to develop, and then rinsed extensively with water to stop development (10). Determination of MICs. The minimal inhibitory concentrations (MICs) were determined by agar dilutions, using Steers replicators. An inoculum of 105 cells was used, as described by Syriopoulou et al. (15).

RESULTS H. influenzae strains HC234, R375, and R385 were selected for study because they differ in

measurable characteristics (biotype and capsule product), were isolated at different times from different geographical areas, and carried different R plasmids (Table 1). Crude extracts were prepared from each Haemophilus strain and from E. coli strains carrying R429, Sa, or R387,

CATs FROM H. INFLUENZAE

VOL. 151, 1982

plasmids which encode for the type I, type II, or type III gram-negative CAT variants. All extracts were capable of acetylating chloramphenicol in the presence of acetyl coenzyme A (Table 1), but not when acetyl coenzyme A was missing. The type I, II, and III enzymes were stable (less than 20% loss of activity) to heating at 60°C (11). The Haemophilus extracts were heated to 60°C for 10 min and reassayed. All three enzymes retained greater then 80% of their measurable activity (data not shown). CAT variants from gram-negative species are produced constitutively, whereas CAT variants from gram-positive organisms are inducible (17). The three H. influenzae strains were grown in the presence of 0.5, 1.0, 5.0, and 10.0 ,g of chloramphenicol per ml. The amounts of enzyme per milligram of protein in the crude extracts were identical for the cells grown with and without chloramphenicol. Also, there was no difference in the MICs of cells exposed to chloramphenicol and those that were not (data not shown), indicating that the Haemophilus enzymes are not produced inducibly. Sensitivity to DTNB. The majority of the type II enzymes are sensitive to DTNB (6, 13, 17), a thiol reagent used in the CAT assay. When a type II enzyme is preincubated with DTNB, the activity is reduced by 80%o in 30 min, whereas less than 30% of the activity is lost by type I or type III enzymes. The DTNB sensitivities of the three H. influenzae enzymes were examined; all three enzymes were rapidly inactivated by DTNB (Fig. 1). The type II enzyme and the three Haemophilus CATs showed pseudo-firstorder inactivation in the presence of DTNB (Table 2). Affinity chromatography of crude extracts. Affinity chromatography is a rapid and reproducible means of classifying CAT variants (18). Crude extracts from each strain were dripped through a resin column. Greater than 99%o of the measurable activity was bound to the resin. All three Haemophilus enzymes were eluted in 0.6 M NaCl (Table 2). A total of 80 to 100%o of measurable activity was recovered for the three types of CAT variants and from crude extracts prepared from strains R385 and HC234, whereas only 45 to 50%o of the activity was recovered in extracts from strain R375. The reduced recovery from strain R375 could be due to inactivation of the enzyme or tight binding of the enzyme to the resin. However, the use of higher concentration salt washes (1, 2, or 5 M) did not elute additional activity from the column, suggesting that inactivation may occur with this enzyme. The lower recovery rate was consistently found with strain R375, but not with the other two Haemophilus enzymes. These differences suggest that the

739

100

90

.Type III

80

70 ..

6050

4,

-

40 30

rype II

20 20

HC,234

10

R

QI

0

5

10

15 20 Time (min)

25

30

FIG. 1. DTNB sensitivities of crude extracts.

Crude extracts of H. influenzae strains R385, HC234, and R375 and E. coli J53 (Sa type II) and R387 (type III) were prepared as described in the text. Extracts were incubated at 37°C in the presence of 1 mM DTNB. The CAT activity was assayed and compared with the activity at time zero.

enzyme from strain R375 has some unique physiochemical properties not found in the other two Haemophilus enzymes. Reaction with anti-CAT sera. Antiserum raised against the CAT encoded by JR66b has been shown to form precipitin bands with type I variants, but not with type II or type III enzymes in Ouchterlony gels. Similarly, antiserum raised against the type III enzyme encoded by R387 forms bands with type III variants, but not with type I or type 11 (6). The three'H. influenzae variants were tested against type I and type III antisera. No precipitin bands were formed with either antiserum (Table 2). The type II antiserum showed a very weak reaction with homologous CAT and no reaction with the Haemophilus type I and type III enzymes. It is unclear whether this poor reagent would detect nonhomologous but related CAT enzymes. Partial protein digestion of CAT variants. The apparent molecular weights for the three Haemophilus enzymes were determined on 12% SDS-polyacrylamide gels. The enzyme isolated

740

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ROBERTS, CORNEY, AND SHAW

J. BACTERIOL.

TABLE 2. Characteristics of CATs

Enzyme

Rate of" inhibition

Mol

R plasmid

by

Ma

DTNB (K)

Molar NaCI required for elution of affinity resin

1.0 24,000 0.001 R429 Type I 0.6 24,150 0.065 Sa Type II 0.3 25,000 0.007 R387 Type III 0.6 24,150 0.115 pRI234 H. influenzae 0.6 24,400 0.074 pMR375 H. influenzae 0.6 24,400 0.066 H. influenzae pMR385 a Apparent molecular weight of protein monomer. b The pseudo-first-order rate (constant [K]) for the inactivation of enzymes by DTNB graphically from the equation In E/Eo = -Kt, where E/Eo is the fraction of initial activity treatment for time t.

A

C

B

D

F

E

3:

..

:

....

......

S x

..

..

.,

.:

Ai

Y y

::: _

....

..

:. ' :B::: :. .'::.

.::

:3::

t.

::

,:.:

B

W

'i .....

.

.1

_

Reaction with antiserum I

III

+ -

+

-

-

was calculated remaining after

from strain HC234 ran with type II enzyme (molecular weight, 24, 150), whereas the other two CATs appeared to have identical molecular weights (24,400), which were slightly larger than that of the type II, subunit, but smaller than that of the type III subunit (Table 2). Cleveland et al. (2) have shown that totally unrelated proteins having similar molecular weights, when digested with papain, S. aureus V8 protease, or chymotrypsin, give patterns which are strikingly distinct, with no bands in common. The three Haemophilus variants and the three enteric-type enzymes were digested with papain or V8 protease, and the resulting patterns were examined in 17.5% SDS-polyacrylamide gels (Fig. 2). The type I, II, and III CATs gave distinct patterns, with no common band shared by all three enzymes. Two of the Haemophilus enzymes encoded by plasmids pMR385 and pMR375 gave very similar, if not identical, patterns, although the third Haemophilus enzyme gave different patterns with both papain and V8 protease. A few bands common to the E. coli and the Haemophilus enzymes were seen; however, the Haemophilus patterns could easily be distinguished from the type I, II, and III enzyme patterns.

DISCUSSION In this paper, we have compared the enzymes I. for chloramphenicol resistance in H. responsibleand influenzae E. coli. The resistant Haemophilus strains produce enzymes which have properB C D) F A E in common with the gram-negative family of ties CAT instead of having However, enzymes.similar to the most widespread FIG.:2. Digestion protein bands. (A) V8 protease characteristics digestiorn patterns stained with Coomassie blue. (B) v Papain s .ilver-staned digestion patters. Arrows idi- variant (type I), the Haemophilus enzymes apcate V8 Iprotease or papain band. Lane A, E. coli type pear to belong to the less common type II group. The three Haemophilus CATs differed from I. Lane B, E. coli type II. Lane C, E. coli type III. Lane D, H. influenzae HC234. Lane E, H. influenzae one another and the type II variant in subunit R375. LEane F, H. influenzae R385. molecular weight, partial protein digestion pat4WAIW Oftw4ft

VOL. 151, 1982

terns, CAT activity, and MIC levels. However, each was sensitive to DTNB, eluted from a highly substituted resin in 0.6 M NaCl, and was nonreactive with type I and type III antisera. The three Haemophilus enzymes did not react with the type II antiserum, but the poor precipitin reaction with the homologous enzyme suggests that a partial reaction would not be detected. The poor antibody response to the type II CAT was unexpected. Work is continuing to produce a more reactive antiserum. The Haemophilus enzymes encoded by pMR375 and pMR385 have very similar protein digestion patterns, yet these plasmids conferred very different CAT activity and MIC on their host. The higher level of resistance and activity was transferred with pMR375 to sensitive celis, suggesting that the high activity is plasmid mediated. The correlation between high CAT activity and high MIC levels has previously been described (12). The reason that some strains have significantly higher CAT levels is unclear. However, the specific activity of the enzyme from pMR375 is twofold higher than that of the other two Haemophilus CATs (data not shown). From the data, there is no indication that any of the three Haemophilus plasmids studied encode for more than a single CAT enzyme. Previously, Gaffney et al. (6) have shown that the type I CAT variants from different sources are not absolutely identical. They also noted that the type II group of enzymes represents a heterogeneous population which differs in a number of properties. Similarly, we found differences between the three Haemophilus CATs and the enteric type II enzyme. The physiochemical data indicate that the three Haemophilus CATs are unique, but belong to the type II CAT group. It is of interest to note that chloramphenicolresistant H. parainfluenzae also carries type II CAT enzymes (13; our unpublished data). Additionally, 45 chloramphenicol-resistant H. influenzae and H. parainfluenzae strains that we have examined are resistant to both chloramphenicol and tetracycline. The reason that Haemophilus species carry the uncommon type II variant, rather than the ubiquitous type I CAT, is not clear. Perhaps, only a single event occurred, with a donor plasmid encoding the type II CAT and tetracycline resistance which introduced these determinants into Haemophilus, and from this original type II gene, mutations occurred which made each enzyme slightly different. ACKNOWLEDGMENTS This work was supported by a special travel grant from the Burroughs Welcome Fund. M.R. was also supported by New Investigator Award Al 17761-01 from the National

CATs FROM H. INFLUENZAE

741

Institute of Allergy and Infectious Diseases. A.C. and W.S. have received a research studentship and project grant support from the Medical Research Council. W.S. is the recipient of a research leave feHlowship from the Wellcome Trust. LITERATURE CITED 1. Azemun, P., T. StuBl, M. Roberts, and A. L. Smith. 1981. Rapid detection of chloramphenicol resistance in Haemophilus influenzae. Antimicrob. Agents Chemother. 20:168-170. 2. Cleveland, D. W., S. G. Fischer, M. W. Klrschner, and U. K. Laemmll. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. 3. Dang Van, A., F. Goldstein, J. F. Acar, and D. H. Donanchaud. 1975. A transferable kanamycin resistance plasmid isolated from Haemophilus influenzae. Ann. Microbiol. Inst. Pasteur Ser. A. 126:397-399. 4. DeGnraff, J., L. P. Elweil, and S. Falkow. 1976. Molecular nature of two beta-lactamse-specifying plasmids isolated from Haemophilus influenzae type b. J. Bacteriol. 126:439-446. 5. Elwell, L. P., J. R. Saunders, M. H. Richmond, and S. Falkow. 1977. Relationships among some R plasmids found in Haemophilus irfluenzae. J. Bacteriol. 131:356362. 6. Gaffiey, D. F., T. J. Foster, and W. V. Shaw. 1978. Chloramphenicol acetyltransferases determined by R plasmids from gram-negative bacteria. J. Gen. Microbiol. 109:351-358. 7. Laemmll, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680485. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measured with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 9. Mendez, B., C. Tachibana, and S. B. Levy. 1980. Heterogeneity of tetracycline resistance determinants. Plasmid 3:99-108. 10. Oaldey, B. R., D. R. KirAch, and W. R. Morris. 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105:361-363. 11. Pakman, L. C., and W. V. Shaw. 1981. The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts. Biochem. J. 193:541-552. 12. Roberts, M. C., C. D. Swenson, L. M. Owens, and A. L. Smith. 1980. Characterization of chloramphenicol-resistant Haemophilus in.fluenzae. Antimicrob. Agents Chemother. 18:610-615. 13. Shaw, W. V., D. H. Bouanchaud, and F. W. Goldstdn. 1978. Mechanism of transferable resistance to chloramphenicol in Haemophilus parainfluenzae. Antimicrob. Agents Chemother. 13:326-330. 14. Shaw, W. V., and R. F. Brodsky. 1968. Characterization of chloramphenicol acetyltransferase from chloramphenicol resistant Staphylococcus aureus. J. Bacteriol. 95:2836. 15. Syriopoulou, V. P., D. W. Schefele, C. M. Sack, and A. L. Smith. 1980. Effect of inoculum size on the susceptibility of Haemophilus influenzae type b to beta-lactam antibiotics. Antimicrob. Agents Chemother. 16:510-513. 16. van Klingeren, B., J. D. A. van Embden, and M. DessensKroon. 1977. Plasmid-mediated chloramphenicol resistance in Haemophilus irfluenzae. Antimicrob. Agents Chemother. 11:383-387. 17. adeag, Y., J. E. Fltton, L. C. Packman, and W. V. Shaw. 1979. Characterization and comparison of chloramphenicol acetyltransferase variants. Eur. J. Biochem. 100:609-618. 18. Zaldenzaig, Y., and W. V. Shaw. 1976. Affinity and hydrophobic chromatography of three variants of chloramphenicol acetyltransferases specified by R factors in Escherichia coli. FEBS Lett. 62:266-271.