Mechanism of Action of Isoniazid on Mycobacterium bovis Strain BCG

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INFCTION AND IMMUNITY, Apr. 1971, p. 53-534 Copyright o 1971 Amerian Soiety for Mirobiology Vol. 3, No. 4 Printed in U.S.A. Mehanism of Ation of Isoniazid on Myobaterium bovis Strain BCG J.

1 INFCTION AND IMMUNITY, Apr. 1971, p Copyright o 1971 Amerian Soiety for Mirobiology Vol. 3, No. 4 Printed in U.S.A. Mehanism of Ation of Isoniazid on Myobaterium bovis Strain BCG J. KNNTH MCLATCHY Departmnenit of Cliniial Laboratories, Nationial Jewish Hospital and Researh Center, Denver, Colorado 86 Reeived for publiation 14 Deember 197 The mehanism of ation of isoniazid (INH) on Myobaterium bovis strain BCG was studied. The rates of synthesis of deoxyribonulei aid (DNA), ribonulei aid (RNA), and protein after the addition of INH to growing ultures were followed by measuring the inorporation of 3H-thymidine, 3H-uridine, and '4C-Lvaline, respetively. After the addition of INH, the rate of DNA synthesis began to derease and was abolished within 4 hr. RNA synthesis eased after 6 hr, and protein synthesis was inhibited after 7 hr. Thus, it appears that inhibition of the synthesis of DNA is one of the earliest events after INH addition. The inhibition of the synthesis of DNA was further found to orrespond to losses in viability of treated ultures. Degradation of preexisting DNA in INH-treated strain BCG was not deteted. Isoniazid (INH), the most ommonly used of antituberulosis drugs, has been reported by a number of investigators to affet a variety of metaboli proesses in suseptible myobateria. Youatt (9) has reently reviewed the literature onerning the mehanism of ation of INH and suggested that many of the ontraditions and disrepanies in the literature are the result of the variety of experimental onditions (aeration, temperature, growth phase of bateria, drug onentrations, et.) used in the investigations. However, a number of investigators found that INH affets only growing suseptible myobateria. These observations suggest that the drug, or one of its metabolites, has a primary effet on a vital metaboli proess or on the synthesis of an essential maromoleule. This paper reports the results of studies on the effets of INH on the inorporation of radioative preursors of deoxyribonulei aid (DNA), ribonulei aid (RNA), and protein by Myobaterium bovis strain BCG. MATRIALS AND MTHODS Bateria and onditions of growth. An INH-suseptible, nonpathogeni myobaterium, M. bovis BCG, was grown in liquid medium at 37 C with vigorous shaking for aeration. The mineral salts medium of Sauton (6) ontaining.5% Tween 8 (SMT; Hill Top Laboratories, Cininnati, Ohio) was employed. Transfers were made into fresh medium at frequent intervals to avoid baterial lumping. Baterial growth was measured by determining the optial density (OD) of ultures in a Coleman Junior II spetrophotometer (Coleman Instrument Corp., Maywood, Ill.) at 6 nm. An OD of.1 orresponds to approximately 7 X 16 olony-forming units per ml of SMT or 5,ug of baterial protein per ml. The numbers of viable bateria were determined by serial dilution plate ounting on 7H11 agar (1). The plates were inubated at 37 C in an atmosphere of 5% CO for 3 weeks prior to ounting. Baterial protein was measured by the method of Lowry et al. (3) Ṁeasurement of the synthesis of ellular maromoleules. The distribution of radioativity in maromoleular frations of strain BCG growing in the presene of various radioative preursors was determined by using a modifiation of the membrane-filter frationation tehnique of Roodyn and Mandel (5). Thymidine-methyl-3H (5,Ci/ml; 16. Ci/mM, Amersham-Searle, Des Plaines, Ill.), 3H-uridine (5,uCi/ml; Ci/mM, New ngland Nulear Corp., Boston, Mass.), and '4C-L-valine (uniformly labeled; 5M,Ci/ml; 9 mci/mm, New ngland Nulear Corp.) were added separately to growing ultures of strain BCG (OD =.), and samples were taken after 8 hr. Inorporation into DNA was determined by pipetting the samples into equal volumes of 5.5 N NaOH and inubating at 37 C for hr. After ooling in ie, the samples were aidified by the addition of.5 volume of 6 N HCI, and the DNA was preipitated with 1 volume of 1% trihloroaeti aid. The preipitates were olleted on membrane filters (.,m pore size, Millipore Corp., Bedford, Mass.) and washed with 1 ml of old 5% trihloroaeti aid and followed by 5 ml of old 1% trihloroaeti aid. After air drying the filters were plaed in vials ontaining 1 ml of sintillation solution [1 mg of 1,4-bis--(5-phenyloxazolyl)-benzene per liter and 4 g of,5-diphenyloxazole per liter in toluene], and the radioativity was determined in a Mark I liquid sintillation ounter (Nulear-Chiago Corp., Des Plaines, Ill.). 53

2 VOL. 3, 1971 ISONIAZID ACTION ON M. BOVIS BCG 531 In some experiments, the effets of.5 pug of INH/ ml of SMT on rates of synthesis of DNA, RNA, and protein were studied by adding the drug to growing ultures of strain BCG (O.D. -.3) at the same time as 3H-thymidine, 3H-uridine, and "4C-valine (,uci/ml eah), respetively. At intervals, samples were pipetted into equal volumes of ie-old 1% trihloroaeti aid. After min, the aid-insoluble preipitates were olleted on membrane filters, washed, dried, and ounted as desribed above. Measurement of uptake of labeled ompounds. To study the uptake of 14C-INH (1,ug/ml; 9.6 mci/mm, Amersham-Searle, Des Plaines, Ill.) and 3H-thymidine (5,uCi/ml), respetively, the ompounds were added to ultures of strain BCG growing in SMT. At intervals, samples were pipetted diretly onto membrane filters, washed with volumes of hilled SMT, and dried and, the radioativities were determined. Measurement of the effet of INH on the stability of preexisting DNA in strain BCG. Strain BCG (OD =.) was grown for 4 hr in SMT plus pci of 3H-thymidine per ml to label the DNA. The bateria were then washed free of exogenous 3H-thymidine by repeated entrifugations and resuspensions in fresh SMT. Half of the washed bateria were then resuspended in SMT and half in SMT plus 5 pg of INH per ml, inubated at 37 C, ana pipetted into old 1% trihloroaeti aid. The radioativities were determined as previously desribed. :6 M._ io7 N16 C U1- io5 FIG. 1. ffet of isoniazid on the viability of strain BCG. =._ o ( C.1._ I I I 4 6 X8 1 1 FIG.. ffet of isoniazid on the optial density of a ulture of strain BCG. t CY - x Ia.Q n _ a a Control -* * BCG B C G - I N HR FIG. 3. Uptake of 14C-isoniazid by strain BCG and an isoniazid-resistant mutant of strain BCG. RSULTS ffets of INH on growth and viability. The addition of.5,ug of INH per ml to growing ultures of strain BCG aused a derease in viability beginning after 1 hr of inubation. After 1 hr, a 97% loss in viability was evident (Fig. 1). ven though a redution in viable ount ourred, a slight inrease in turbidity for about 1 hr was deteted (Fig. ). The generation time of strain BCG growing under these onditions was about 3 hr. Uptake of 14C-INH. Strain BCG rapidly takes up radioative INH. In fat, the bateria appear to be saturated with the drug within or 3 hr after addition of 1 Mg of '4C-INH to a growing ulture (Fig. 3). The uptake of 14C-INH by an INH-resistant mutant of strain BCG (BCG- INHR) is shown for omparison. This result onfirms the observation of a number of other workers that resistant organisms are defiient in INH uptake. The isolation and haraterization of the INH-resistant mutant are desribed elsewhere (4). Distribution of radioativity in 3H-thymidine, 3H-uridine, and '4C-valine-labeled bateria. Prior

53 MCLATCHY INFC. IMMUN. TABL 1. Distribution of radioativity in hemial frations of strain BCG Fration Radioative traera 'H-thymi- 'H-uridine 14C-valine dine Lipid....4 1.7 3.4 Protein... <1. <1. 96.

3 53 MCLATCHY INFC. IMMUN. TABL 1. Distribution of radioativity in hemial frations of strain BCG Fration Radioative traera 'H-thymi- 'H-uridine 14C-valine dine Lipid Protein... <1. < Deoxyribonulei aid <1. Ribonulei aid <1. a Results expressed as a perentage of the radioativity in the old trihloroaeti aid residue. ) NM C 4) 4 mi ). N x L FIG. 5. ffet of isoniazid on the synthesis of RNA as measured by the inorporation of 3H-uridine. The baterial protein ontent of eah sample was normalized to I mg, and the data are expressed as ounts/minute of H-uridine inorporated into RNA per milligram ofbaterial protein FIG. 4. ffet of isoniazid on the synthesis of DNA as measured by the inorporation of 3H-thymidine. The baterial protein ontent of eah sample was normalized to 1 mg, and the data are expressed as ounts/minute of 3H-thymidine inorporated into DNA per milligram ofbaterial protein. to studying the effets of INH on the rates of synthesis of maromoleules by strain BCG, it was neessary to determine the distribution of possible maromoleular preursors into various hemial frations. 3H-thymidine, 3H-uridine, and 4C-valine (5,uCi eah) were added separately to growing ultures of strain BCG, and the radioativity of the various frations was determined after 8 hr. The results are shown in Table 1. It an be seen that the inorporation of radioative thymidine, uridine, and valine an be used as a measure of DNA, RNA, and protein synthesis, respetively. ffet of INH on synthesis of DNA, RNA, and protein. The synthesis of DNA, as measured by inorporation of,uci of 8H-thymidine per ml, began to derease about 3 hr after INH addition and was ompletely inhibited within 4 hr (Fig. 4). RNA synthesis, as measured by inorporation of MCi of 3H-uridine per ml, ontinued normally for about 4 hr after addition of INH and then began to derease (Fig. 5). C., a1) _ aiw.- a X CL CL _ ~~~Controlr I I I I ISI I I z I I.1 I FIG. 6. ffet of isoniazid on the synthesis of protein as measured by the inorporation of 14C-L-valine. The baterial protein ontent of eah sample was normalized to I mg. The data are expressed as ountsl minute of 4C-L-valine inorporated per milligram of protein. The synthesis of ellular proteins, as measured by the inorporation of,uci of '4C-L-valine/ml, was unaffeted until 6 to 7 hr after addition of INH (Fig. 6). Therefore, the earliest event, if not the primary event, seen after addition of INH to a growing ulture of strain BCG, was an inhibition of DNA synthesis. RNA and protein synthesis were affeted only later. The time of inhibition of

4 VOL. 3, 1971 ISONIAZID ACTION ON M. BOVIS BCG 533 C, >n % C Fi thymi TABL T'ime after Counts per sample min per onversion of thymidine to thymidine triphosresus;pension (hr) phate has not been eliminated. Degradation of Control 5 lag INH/ml preexisting DNA by INH was not deteted. 3,54,644 and omplete inhibition of DNA synthesis by 4,55,753 INH might also suggest that only initiation of 8,475,673 DNA synthesis is affeted and not the polym- 1,669,578 erization. However, it should be remembered 4,63,697 that 4 hr is a relatively short period in an organism with a generation time of 3 hr. The inhibition of DNA synthesis, whih is omplete 4 hr after DNAK synthesis and loss of viability oinide addition of the drug, might also be explained if appr oximately. this time period were required for the ells to Up )take of thymidine. Sine inorporation of take up the amount of INH required for ba- was used as an index of DNA terial killing. This does not seem to be the ase 'H-tiiymidine synti iesis, the effet of INH on the uptake of sine 14C-INH uptake is maximal within to 3 hr. 'H-tihymidine was measured. The transport of There is a possibility that the lag period preedhymidine was not signifiantly affeted by ing the omplete inhibition of DNA synthesis is 'H-ti INH until 3 to 4 hr and then began to derease required for onversion of isoniazid to a bio- (Fig. 7). The initial uptake represents logially ative metabolite. It has not been es- gradiually entry of the exogenous thymidine into the metatablshe *... boli pool. The later slowdown probably results from ellular into ffiar on DNA. that pre-e ativ an effet on general phosphate metabolism. Th e experiments reported here indiate that xperiments are now in progress to study the one of the early events after the addition of effets of INH on several enzymes whih atalyze isoniiazid to a growing ulture of strain BCG is steps in the synthesis of DNA. The metaboli )ition of DNA synthesis; this is followed fate of INH is also being investigated. inhit 3 later by inhibition of RNA synthesis and even Control later by inhibition of protein synthesis. The in- 5 hibition of DNA synthesis oinides with the loss of viability; DNA synthesis is inhibited by - 99% within 3.5 to 4 hr; a 9% loss of viability is evident within about 4 hr. The ontinued syn / / thesis of protein and the inrease in absorbany lo //o in treated ultures indiate that INH does not have a primary effet on energy metabolism. 5 _ < The mehanism of the inhibition of DNA synthesis is not yet lear. However, several possibilities have been eliminated. The drug does not seem to affet the uptake of nulei aid preursors; neither the uptake of thymidine nor the ;. 7. ffet of isoniazid on the uptake of 3H- inorporation of uridine was inhibited early. In idine by strain BCG. addition, sine RNA synthesis ontinued for a time after the essation of DNA synthesis, it,. ffet of isoniazid (INH) on the stability appears that INH does not affet the formation of, preexisting deoxyribonulei aid in strain of the purine and pyrimidine bases and their BCG onversion to the orresponding ribonuleotides. However, a speifi inhibition of a step in the The above observations lead to a onsideration,581,38 of the possibility that INH had a diret effet on 1,539,663 the synthesis of DNA. The lak of an immediate la ompound or whether an INH-metabolite is the INH inhibits effetor (9). Other workers (, 7) have also reported the in- synthesis hibition of nulei aid synthesis in sensitive xisting DNA was also onsidered. Table myobateria by INH. However, in both reports, indiates that no loss of aid-insoluble radio- nulei aid synthesis was determined by measurity after exposure to the drug was deteted. ing the inorporation of 3p into ellular frations. Youatt (8) has suggested that INH might have

534 MCLATCHY INFC. IMMUN. ACKNOWLDGMNTS j This investigation was supported by Publi Health Servie researh grant A1-819 from the National Institute of Allergy and Infetious Diseases.

5 534 MCLATCHY INFC. IMMUN. ACKNOWLDGMNTS j This investigation was supported by Publi Health Servie researh grant A1-819 from the National Institute of Allergy and Infetious Diseases. I thank Susan Stalling for her able tehnial assistane. LITRATURF, CITD 1. Cohn, M. L., R. W. Waggoner. and J. K. MClathy The 7H 1 medium for the.ulltivation of myobateria. Amer. Rev. Resp. Dis. 98: Gangadharam, P. R. J., F. M. Harold, and W. B. Shaefer Seletive inhibition of nulei aid synthesis in Myobaterium tuberulosis by isoniazid. Nature (London) 198: Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Minden, P., J. K. MClathy,. J. Bardana, Jr., and R. S. Farr Antigeni differenes between Myobaterium bovis strain BCG and an isoniazid-resistant mutant. Infe. Immun. 3: Roodyn, D. B., and H. G. Mandel A simple membrane frationation method for determining the distribution of radioativity in hemial frations of Baillus ereus. Biohim. Biophys. Ata 41: Willis, H. S., and M. M. Cummings Culture media, p. 16. In Diagnosti and experimental methods in tuberulosis, nd ed. Charles C Thomas, Publisher, Springfield, Ill. 7. Wimpenny, J. W. T ffet of isoniazid on biosynthesis in Myobaterium tuberulosis var. bovis BCG. J. Gen. Mirobiol. 47: Youatt, J Changes in the phosphate ontent of myobateria produed by isoniazid and ethambutol. Aust. J. xpt. Biol. Med. Si. 43: Youatt. J A review of the ation of isoniazid. Amer. Rev. Resp. Dis. 99:

Molecular Morphology of Ribosomes

Molecular Morphology of Ribosomes Eur. J. Biohem. 33,481485 (1973) Moleular Morphology of Ribosomes Loalization of Ribosomal Proteins in 54 Subunits Chester J. MCHALSX, Brue H. SELLS, and Martin MORRSON Laboratories of Biohemistry, St.