GR.OWTI-I INHIBITION OF BACTERIA BY SYNTHETIC PTERINS

Transcription

1 GR.OWTII INHIBITION OF BACTERIA BY SYNTHETIC PTERINS I. STUDIES WITH STREPTOCOCCUS FAECALIS, LACTOBACILLUS CASEI, AND LACTOBACILLUS ARABINOSUS* BY LOUISE J. DANIEL, L. C. NORRIS, M. L. SCOTT, API D G. F. HEUSER WITH THB TECHNICAL ASSI~TAKCE OF LENA STRUGLIA (Prom the Agricultural Experiment Stutiwn and the School of Nutrition., Cornell University, Ithaca) (Received for publication, May 9, 947) Woods () has shown that paminobenzoic acid (PABA) antagonizes the sulfanilamide inhibition of bacterial growth. Lampen and Jones (2) found that pteroylglutamic acid (folic acid) and related compounds are also active antagonists of sulfonamide inhibition of certain microorganisms. They concluded from their results that t,he inhibition of bacterial growt,h by sulfonamides was caused by interference in the synthesis of folic acid at the PABA site in the folic acid molecule. The studies reported in t,his paper demonstrate that growth inhibition of bacteria is likewise obtained with pterinsl related structurally to the pteridine portion of the folic acid molecule. The studies include work with three organisms, Streptococcus faecalis, Lactobacillus casei, and Lactobacillus arabinosus. Xtreptococcus faecalis and Lactobacillus casei require folic acid in the medium and Lactobacillus arabinosus synt,hesizes its own requirements of this vitamin. Studies with other microorganisms and biological results obt,ained with chicks will be reported in later publications. EXPERIMENTAL Cultures and Me&aCultures of Streptococcus faecalis (S. lactis R), La.ctobacillus cusei, a.nd La,cfobacillus arabinosus were obtained from the American Type Culture Collection. They were maintained as agar stabs in liver, glucose, yeast, and tryptone agar, the composition of which is described by Nymon, Gunsalus, and Gortner (3). Weekly transfers were Downloaded from by guest on August 6, 28 * This work was undertaken in cooperation with the Office of Naval Research, Navy Department, Washington, D. C., and was aided by a grant to Cornell University by the Nutrition Foundation, Inc., New York. The work was conducted in the Nutrition Laboratories of the Department of Poultry Husbandry. The synthesis of these compounds is being reported elsewhere by M. F. Mallette, E. C. Taylor, Jr., and C. K. Cain, of the Department of Chemistry, Cornell University, Ithaca, New York. G89

2 69 INHIBITION OF BACTERIA BY PTERINS. I made through a broth of the same composition, except for the omission of agar. The medium used for Streptococcus faecalis was that of Luckey, Briggs, and Elvehjem (4), modified to include 2 millimicrograms of pyridoxamine per tube. For Lactobacillus casei the medium of Teply and Elvehjem () was employed, except that tryptone was substituted for peptone at a level of 2 mg. per tube. The tryptone was adsorbed at ph 6.6 to 7. with Darco G6 for three 3 minute periods. For Lactobacillus arabinosus the medium of Skeggs and Wright (6) was used with the inclusion of adequate calcium pantothenate. In some of the studies with Lactobacillus arabinosus PABA was omitted from the medium. Compounds UsedThe synthetic pterins used in these studies have the following structure,2 where R and R are hydrogen, methyl, phenyl, or carboxyl, as well as compounds having phenanthro and acenaphtho groups fused to the pyrimido(4,b)pyrazine nucleus. N This structure differs from that of the pteridine portion of the folic acid molecule by having an amino group in the 4 position instead of a hydroxyl group. The formulae of these compounds are presented in Table I. per cent glycerol was found to be a somewhat better solvent for these compounds than was water alone. Synthetic folic acid3 was used in all the experiments. MethodsIn studies involving Streptococcus faecalis and Lactobacillus casei varying amounts of folic acid were added to the medium, as well as increasing amounts of the pterins. In one study with Lactobacillus arabimsus increasing levels of the pterins were.added to the medium without the addition of folic acid. In a later study with this organism the an N Downloaded from by guest on August 6, 28 2 The pteridine portion of folic acid has the following structure: 3 The authors are indebted to the Lederle LeboreLtories Division, American Cyansmid Company, Pearl River, New York, for synthetic folic acid.

3 DANIEL, NORRIS, SCOTT, AND HEUSER 69 tagonistic effect of folk acid was demonstrated by showing that folk acid is capable of overcoming the inhibitory effect of the pterin. In the studies with Streptococcus jaecalis and Lactobacillus casei the assay tubes were inoculated from a liquid culture grown in basal medium containing millimicrograms of VVilliams folic acid concentrate.4 The CompoundNo. TABLE I Synthetic Pterins Studied structure Name R = R = CHz 2 R=H 2,4Diamino7methylpyrimido(4,b) R = CHz pyrazine 3 R=R COOH 4 R=H 2,4Diamino7carboxypyrimido(4,b) R = COOH pyrazine R = R = CsHz R R==R =H \ / R I I? / \ a3 / / \ 2,4Diamino6,7dimethylpyrimido (4,b)pyrazine 2,4Diamino6,7dicarboxypyrimido (4,b)pyrazine 2,4Diamino6,7diphenylpyrimido (4,b)pyrazine 2,4Diaminopyrimido(4,b)pyrazine 2,4Diaminophenanthro(9,e) pyrimido(4,b)pyrazine 2,4Diaminoacenaphtho (4,b)pyraeine (,2e)pyrimido Downloaded from by guest on August 6, 28 inoculum for Lactobacillus arabinosus contained the basal medium without added folic acid. Streptococcus jaecalis and Lactobacillus arab,inosus were inoculated by loop from 24 hour cultures, whereas Lactobacillus casei was centrifuged, washed once with ph 7. phosphate buffer, and resuspended in ml. of sterile saline, to which had been added 2 y 4 The per cent folic acid concentrate was kindly supplied by Dr. R. J. Williams, of the University of Texas, Austin, Texas.

4 692 INHIBITION OF BACTERIA BY PTERINS. I of sterile pyridoxal. A sterile ml. serological pipette was used to inoculate Lactobacillus casei with a drop of this suspension. Streptococcus faecalis and Lactobacillus arabinosus were incubated at 3 for 6 to 8 TABLE II Inhibition of Growth of Streptococcus faecalis by S 3% thetic Pterins Gal r I F;yd,id ai?om~ Antieter bacterial Compound read indext added ing* y per ml, loo loo 2 _ mrf per JO??& loo ,OC 3 2, 2,,6,, : my per JOml. Galvanmete,ead c ink? i loo loo ooo ooo , * A reading of represents no growth. t Ratio of concentration of inhibitor to the concentration of metabolite at which complete inhibition of growth of the organism occurs. $Millimicrograms. Downloaded from by guest on August 6, 28 hours, and Lactobacillus casei at 37 for 2 hours. After the incubation period the growth was measured turbidimetrically with a Coleman spectrophotometer.

5 DANIEL, NORRIS, SCOTT, AND HEUSER 693 Results Since the inhibitory effect of the various pterins differed with the organism studied, the results with each organism are presented separately. comp;yd _ 2 6 TABLE III Inhibition of Growth of Lactobacillus casei by Synthetic Pterins Compound added y per ml. 2 2 looo _ Folk acid added my per ml. 2 c i * A reading of represents no growth. 7 C ialvanometer reading* Antibacterial index ,,,,OOO 8, 8, 4,,, 8, Downloaded from by guest on August 6, 28 Streptococcus faecalisthe results of the studies on Streptococcus faecalis are presented in Table II. They show that several of the pterins have an extremely marked antibacterial action for this organism. Competitive antagonism between these pterins and folic acid has been demonstrated over a lofold range in concentration for most of the compounds. For the pterin possessing the phenyl group in the 6 and 7 positions, this com

6 694 INHIBITION OF BACTERIA BY PTERINS. I petitive antagonism has been shown over a looofold range in concentration. The antibacterial index for this compound was found to be the same at the two widely separated concentrations of folic acid. TABLE Inhibition of Growth of Lactobacillus arabinosus by Synthetic Pterins IV Compound No. Compound added Galvanometer reading Plus PABAt Minus PABAt 2 6 y per ml loo * A reading of represents no growth. t y of PABA added per tube. $ Medium contains no added PABA F i Downloaded from by guest on August 6, 28 Studies with 2amino4hydroxy, 2,4dihydroxy, and 2mercapto4 hydroxy analogues of these compounds showed that they had no antibacterial effect on Streptococcus faecalis. Some of them, chiefly the carboxy derivatives, exhibited slight folk acid activity. The substitution of a hydroxyl group for an amino group in the 4 position completttly nullified the antifolic acid activity of the pterins. Replacing the amino group in the 2 position by a hydroxyl or mercapto group also produced compounds having no inhibitory effect.

7 DANIEL, NORRIS, SCOW, AND HEUSER 69 Lactobacillus case&the results of studies conducted with Lactobacillus casei are given in Table III. The pterins had much less effect on this organism than on Streptococcus faecalis, although both organisms require approximately the same amount of preformed folk acid in the basal medium. The Z,&diamino6,7diphenylpteridine had the greatest inhibitory effect of all the compounds studied. The phenanthro and acenaphtho compounds are so insoluble that it was impossible to obtain TABLE V Antagonism of Pterin Inhibition of Growth of Lactobacillus arabinosus by Folic Acid Compound added* Folic acid added Galvanometer resdingt Antibacterial index. 7 Qw ml. y Qn IO ml * 2,4Diamino6,7diphenylpyrimido(4,b)pyrazine. t A reading of represents no growth. The medium contained no added PABA Downloaded from by guest on August 6, 28 sufficiently concentrated solutions to show an effect with Lactobacillus casei. The carboxy compounds stimulated growth to a slight extent. Lactobacillus arabinosusthe results of studies with Lactobacillus arabinosus are presented in Tables IV and V. Except for the 2,4diamino 6,7diphenylpteridine, relatively large amounts of pterins were found to be required to inhibit the growth of this organism. The addition of paminobenzoic acid caused an antagonism of the pterin inhibition, since 2 to times the amount of compound was needed to prevent the growth of Lactobacillus arabinosus when I?AR,4 was present. It is evident from the results in Table V that folic acid completely overcame the inhibition

8 696 INHIBITION OF BACTERIA BY PTERINS. I by the pterin at low concentrations of the compound. However, above a certain concentration of the pterin the inhibitory effect was only partially reversible by folic acid. The carboxy derivatives showed little or no inhibition of this organism. DISCUSSION The results present,ed in this report demonstrate that certain synthetic pterins possess strong antibacterial activity which is antagonized competitively by folic acid. In contrast to sulfonamide inhibition which appears to affect only those bacteria which synthesize folic acid, and in opposition to pyrithiamine, thiopanic acid, 3pyridinesulfonic acid, and many other antimetabolites which are active in inhibiting growth only in those bacterial species which. require the metabolite as an essential nutrient, the pterins reported here have the unique capacity of inhibiting the bacteria which synthesize folic acid as well as those requiring the preformed vitamin. In view of the ineffect,iveness of the 4hydroxy pterins in inhibiting bacterial growth it is apparent that the antibacterial activity of the compounds described in this report hinges upon the presence of an amino group in the 4 position of the pteridine nucleus. Under these conditions the substituent groups present at the 6 and 7 positions have a marked influence upon the antibacterial potency. From the results thus far obtained those pterins possessing large, stable groups at the Ci and 7 positions shorn the greatest antifolic acid activity. R oolley (7) has proposed that a compound has the greatest antimetabolite activity when its structural arrangement most nearly coincides with that of the metabolite, except for one essential detail. However, t his hypothesis does not hold in explaining the action of the pterins, since the structures such as the phenanthro compound possessing the greater antimetabolite activity are farther removed from the structure of the pterin of folic acid than is that of the 6,7dimethylpteridine, which showed considerably less antibacterial activity. A possible explanation of the effects of these compounds is that they act by combining with a specific protein (apoenzyme), thus preventing the normal attachment of folic acid in the format.ion of an enzyme required for bacterial growth. Since the only functional group common to both the inhibitory pterins and the pterin of folic acid is the amino group in the 2 posit.ion, it is possible that union with the apoenzyme occurs at t,his position. On the other hand, since the antifolic acid pterins also have an amino group in the 4 position, it may be that union in this instance occurs at t,he 4 position, and is thus responsible for the inhibitory effect. In any case, the effect upon antibacterial activity produced by the groups Downloaded from by guest on August 6, 28

9 DANIEL, NORRIS, SCOTT, AND HEUSER 697 on the 6 and 7 positions may be due to their influence upon chemical reactivity of the amino group in the 2 and 4 positions. The striking dissimilarity in the response of Lactobacillus casei and Streptococcus faecalis to the pterins suggests that more than one system is involved in the metabolism of folic acid. Lactobacillus casei may possess several separate mechanisms, of which one is affected by the pterins, and the others are not. The fact that some pterin inhibition of Lactobacillus casei occurs indicates that one of its metabolic pathways is similar to the system functioning in Streptococcus faecalis. SUMMARY Several synthetic pt,erins have been shown to possess marked antibacterial activity which is antagonized competitively by folic acid. These $erins have the unique capacity of inhibiting bacteria which synthesize folic acid, as well as those requiring the preformed vitamin. A possible explanation for the effects of these compounds is that they act by competing with folic acid for a place in a metabolic reaction essential for t,he growth of the bacteria. BIBLIOGRAPHY. Woods, I). D., Brit. J. h xp. Path., 2, 74 (94). 2. Lsmpen, J. O., and Jones, M. J., J. Biol. Chem., 66, 43 (946). 3. Nymon, M. C., Gunsalus, I. C., and Gortner, W. 4., Science, 2, 2 (94). 4. Luckey, T. D., Briggs, G. M., Jr., and Elvehjem, C. A., J. Biol. Chem., 62, 7 (944).. Teply, L. J., and Elvehjem, C. A., J. Biol. Chem., 67, 33 (94). 6. Skeggs, H. It., and Wright, I,. D., J. Biol. Chem., 66, 2 (944). 7. Woolley, D. W., in Green, D. E., Currents in biochemical research, ru ew York, 37 (946). Downloaded from by guest on August 6, 28

10 GROWTH INHIBITION OF BACTERIA BY SYNTHETIC PTERINS: I. STUDIES WITH STREPTOCOCCUS FAECALIS, LACTOBACILLUS CASEI, AND LACTOBACILLUS ARABINOSUS Louise J. Daniel, L. C. Norris, M. L. Scott, G. F. Heuser and With the technical assistance of Lena Struglia J. Biol. Chem. 947, 69: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites references, of which can be accessed free at tml#reflist Downloaded from by guest on August 6, 28