Adhesive binding of rokitamycin to Staphylococcus aureus ribosomes

FEMS MicrobiologyLetters72 (19~} 93-96 Publishedby EIs¢,ier

93

FEMSLE04177

Adhesive binding of rokitamycin to Staphylococcus aureus ribosomes Kikutarou Endou, Mayumi Matsuoka and Yoshinori Nakajima Dictsion ol Allcroblolo~'. Hokkaido Institute of Pharmac~tlcal Scient es. Hokkatdo, Japan

Received12 Apn11990 Accepted I Junc 1990 Key words: Macrolide antibiotic; Bactericidal effect; Irreversible binding; TMS-19-Q

1, SUMMARY Rokitamycin (RKM), a Y'-O-propionyl derivative of leucomycin As, is bactericidal against staphylococci near the minimum inhibitory con. centrations. RKM bound to ribosomes beforehand is only slightly displaced by erythromycin or josamycin, or even by RKM itself. The adhesive binding of the RKM-ribosome complex might prove to be the lethal event for susceptible staphylococci,

2. INTRODUCTION Rokitamycin (RKM, formerly TMS-19-Q) is a semisynthetic derivative of leucomycin (LM) A 5, namely 3"-O-propionyl LM A~ [1,21. This drug is a bactericidal against susceptible staphylococci near the minimum inhibitory concentration (MIC), whereas both erythromycin (EM) and josamycin (JM; LM-A~) are bacteriostatic [3-6]. Inhibitory levels of RKM against bacteria, howe,.er, are

Correspondence m: YoshinofiNakajima, Divisionof Microbi-

ology, Hokkaido Inslitute of Pharmaceutical Sciences, 7-1 Katsuraoka-cho. Otaru. Hokkaido,047-02 Japan, 0378.1097/90/$03,50

somewhat greater than those of EM [3]. We confirmed this result through repeated tests: 50,% inhibitory doses of RKM, EM. and JM to the growth of Staphyloeoccus aureus NCTC8325 were 0.13, 0,07, and 0.19/xg/ml. respectively. The apparent dissociation constants (Kd) of RKM-, EM-, and JM-ribosome comple::es were determined by means of a Scatchard plot based on data from the macrolide-binding to 70S ribosomes obtained from strain NCTC8325. However. since we could not obtain a congruous curve to the plot. the apparent K~s of RKM were estimated from the concentcation of the antibiotic at half-saturation: their constants being 3.4 × 10 -8, 1.6 × 10 -5, and 2.7 × 10- s M, respectively. To give consideration only to the extent of affinity (apparent K d ) though, cannot by any means account for the bactericidal activity of RKM. Accordingly. therefore, the aim of this investigation was to elucidate the characteristics of the RKM-ribosome complex.

3. MATERIALS AND METHODS 3.1. Strains and ribosomes S, aureus NCTC8325 was used to provide ribosomes, and Micrococcus luteus ATCC9341

served as an indicator organism.

D 1990 Federation of European Microbiological Societies

94 Ammonium chlo~;~c-washed ?0S ribosomes from S. aureus NCTC8325 were prepared as previously described [7]." 3.2. Chemicals EM was provided from Japan Upjohn Co., Ltd. and JM from Yamanouchi Pharmaceutical Co., Ltd. RKM, N[14C]methyl-RKM (4.32 m C i / retool), N[t4C]methyl-JM (,*.55 mCi/mmol), and N-[14C]methyl-EM (3.68 mCi/mmol) were obtained from Research Laboratories, Toyojozo Co., Ltd., Japan, and the same t4C-labeled EM (8.3 mCi/mmol) also was kindly supplied by J.C.-H. Man, Abbott Laboratories. North Chicago, IL, Potassium-N-2-hydroxyethylpiperazJne.N "-2ethanesulfonate (K-Hepes), ethylene glycol bis(~-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and RNas¢-free sucrose were bought from Nacalai Tcsque Inc. All other chemicals were purchased from commercial sources. 3.3. Displacement of a prebound ['4C]macrolide antibiotic The reaction mixture of a 10 mM K-Hepes buffer, pH 7.6, (containing 16 mM magnesium acetate, 50 mM ammonium chloride, 1 mM EGTA. 0.1 mM dithiothreitol), 5 Az6o units of NCTC 8325's ribosomes (155 pmol [8]), and 150 pmol [t4C]macrolide (hot) antibiotic was incubated in a total volume of 0.45 ml at 37°C for 30 rain, Then the indicated amounts of non-radloactive (cold) antibiotic (150, 375, and 750 pmol/0.05 ml) were immediately added into this reaction mixture, and further incubated at 37°C for 30 rain. A portion (0.4 ml) of this new sample was filtered through a YMT membrane (Anficon MPS-3 ultrafiltration unit) by centrifugation (1500 Xg) for 10 min at 4"C. After 0.2. to 0,3 ml-portions had been filtered, each of these tubes was analyzed, Although test results show that the YMT membrane has the least ability to bind to hot RKM or JM, the amount ol the bound drug was not negligible (not exceeding 20%), ltence, the radioactivity remaining on the membrane in parallel experiments in the absence of the ribosomes was subtracted from the difference of the recorded radioactivity between the two samples. The result was taken as the amount of the drug bound to ribosomes,

3.4. Binding of a hot antibiotic when the hot and cold antibiotics were mixed together Binding of [14C]macrolide antibiotics to NCTC8325 ribosomes, being mixed together with the hot drag and the cold analog, was carried out in the same reaction mixture as described above except that the hot (150 pmol) and the cold antibiotics (150, 375, and 750 pmol) were mixed beforehand, and were then added to the antibioticfree reaction mixture. The reaction mixtures were incubated at 37°C for 30 rain. The amount of the drug bound to ribosomes was determined as described above. 4. RESULTS AND DISCUSSION Fig. 1 shows that the Ka is closely correlated with the extent of the ribosomes" affinity to the % of [==CIn~cni:ie ~ltibiotics t2ou¢~l to r 10os¢cn~ ( t up tow ) e~l

0o:' latt~ (bd (ed t. Bindingof [t4C]macrolidea n t i h i o l i c s added previously to ribosomes from S. aureux NCTC8325. Each 0,45 ml reaction mixturecontained 5 A2eoanil~of NCTC8325ribosorr.¢,~ and 150 pmol of tR) [~aCIRKM,rE) [I~C[EMor (,]) ['4C[JM, Aner a 3 0 - m l n iftcubation,the reaction m i x t u r e was supplied with the followingcold antibiollcs;]SO pmol(a t. a~, aj), 375 pmol (b~, b~, hjl and 750 pmol(c,, c~, cj) in a total volumeof 0,5 ml. The reaction mixturewas further incubated for 30 rain, filtered, and counted, Sub~fiptsr, ¢, and j were referred to as rokitamycln,el~thlxam~cin,and josamy¢in,respectively, ~ T h e relativebindingof [14Clmacrolidcantibtoucwas referred to as Fig.

ratio (%) o f the b o u n d a m o u n t o f the original or post-diluted

hot drug (At to thai of the ribosomes 1155 pmoll, namely a relativebinding= t00× A/155. When 150 pmoi of [=4C]RKM, [Z4CIEM,or [I~C],IM",curemixed with 155 pmol of the rib0some.s, the amount of the drugs bound to them was 63 (R, 0: 40.6%), 118 (E, 0: 76,1%), and 79 (J, 0: 51.05) pmol, respectively.

q5 antibiotics (E, 0 > J, 0 ~-R, 0), and that RKM bound previously to the ribosomes is hardly displaced even by cold RKM itself (R, 0: 40.6% ~ R, ar; R, bt; R, er), whereas bound EM and JM are relatively easily released from the ribosomes by the correspondin$ cold drugs (E, G: 76.1% ~ E, a~; E, b,; E, % ) 0 , 0: 51.0% ~ J , aj; J, bj; J, cj), In short, RKM is the most cohesive of those three antibiotics, followed by JM and then EM (in the case of a homogeneous combination such as hot and cold RKM, EM, and JM in Fig. 1). The degree of the mutual-exclusive binding of a hot drug appears to depend in general on how much lower the K,~ of the antibiotic is, and how much more of a cold drug than another hot drug is present in a binding-reaction mixture. The results of the mutually exclusive binding in a heterogeneous combination such as hot EM and either cold RKM or JM, and hot JM and either cold RKM or EM (Fig. I) agreed with the ones that several workers had previously obtained [9-11].

(top row) and 155 ~ ~iDc*soma und~Tdaced by non-rat~et~,a INC]fc~tan~/cln (F~ I~Cle~ryt hr~nycin rE) t~

RK~t:,,)

I~.ll~lLg~J

I

JM Ibll t e l ) ~

Fig, 2. Bindingof (l'~ClmacrolideantibiolicsIo Ncrcg325 ribosome~ when the hot and cold drugs were mixed together. Each 0.5 ml reaction mixturecontained 5 A~) unizsof ribosomes, and the antibioticsolutionwhichcontained 150pmol of either (R) [I4C]RKM or (E) 114CIEMwere previouslymixed with the followingcold antibiotic: 150 pmol (a,, a~, a jJ, 375 praol (bp b~, bib and 750 pmol(c,, %, cj). and was incubated at 37°C for 30 rain, After incubation,the mixturewas filtered, and counted. Symbolsare the sa~ as those in Fig. I a See the legendat ~ in Fis. I.

However, the exclusive binding of pre-bound RKM to the ribosomes greatly differs from that of the other macrolide antibiotics such as EM and JM (Fig. 15. We inquired why the curious undissociable binding of RKM had taken place. We examined whether there is some contaminant in the hot RKM, which is responsible for the unique RKMbinding to the ribosomes. Reciprocally, pre-bound cold RKM did not allow any hol RKM, EM, or JM to bind to the ribosomes (data not shown). Moreover. the purity of the radioactive RKM, EM, and JM was assessed to be more than 97.1, 97.5, and 98.1%, respectively, by means of bioautograph), and thin-layer chromatography using three ind~,pc,adent separate-solvent systems, i.e. chloroforn -acetone (1 : 1 v / v 5. chloroform-methanol-ammonia (9:1:0.1). and chloroform-ethanol (9:15. Hot RKM binds to only 50S ribosomal subunits. Pre-bound [14C]RKM to the large particles could not be displaced either by cold RKM (data not shown). Hence there seems to be no adhesive binding due to some contaminant present in hot RKM. When hot t150 pmol) and cold antibiotics (150. 375, or 750 pmol) were mixed together simultaneously, the binding of the hot antibiotic to the ribosomes was decreased in inverse proportion to the increase of the cold drug (Fig. 2). The decreasing profile of bound [~4CIRKM, which is brought about I)5' increasing the amour.t of cold EM (R. a~; R, b~; R, c~ in Fig. 2), greatly differs from that of [=aC]EM which is unable to be displaced by either cold RKM (E, at; E, bT; E, cr) or JM (E, aj; E. bj; E. c~). This result suggests that even when hot RKM and cold EM were mixed together, there seems to exist an RKM-speeific binding site (about 30%5 on the ribosome (R, a¢; R, be; R, % in Fig. 2). since hot RKM is competitively removed by cold RKM (R, at; R, br; R, cr), whereas the profile in the mixture of hot and cold RKM (R, a,; R, br; R, Cr) resembles that of hot and cold EM (E, ae; E, b~.; E, ce). The binding of [t4C]RKM in the case of a combination as shown at (R, art in Fig. 2 attained 40.2% even in the presence of an additional 150 pmol of cold RKM ((R, O: 40.6%) in the same figure). This can be accounted for by the

96 fact that original [t4C]RKM, when its 300 pmol were nlixed with 155 pmol of the ribosomes, can bind to about 80% of the ribosomes (124 pmol). Moreover, the finding is confirmed by the fact that antibiotic-ribosome complexes sedimented in a sucrose gradient [8] obtained similar results to those in the membrane filtration just stated above (data not shown). In conclusion the fashion of R K M - b i n d l n g to ribesomes appears to differ particularly from that of EM- and JM-binding. This undissoeiable binding of the RKM-ribosome complex might become lethal for susceptible staphylococci. The question of whether the antibiotic and the ribosomes form covalent binding remains to be resolved.

ACKNOWLEDGEMENT We wish to thank F. Yamazaki for his skilled assistance.

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