the action of sulphanilamide on bacteriophages t1-t7

369

THE ACTION OF SULPHANILAMIDE ON BACTERIOPHAGES T1-T7.

F. J. RUTTEN, K. C. WINKLER AND P. G. DE HAAN.From the Laboratory of Hygiene, Univer8ity, Utrecht, Holland.

Received for publication April 25, 1950.

THE influence of sulphanilamide on bacteriophage multiplication has beenstudied on several occasions without success, presumably because multiplicationof the host-bacteria was inhibited (Delbruck and Luria, 1944; Wahl, Nitti andFaquet, 1946).

In our studies on the action of sulphonamides we have been able to obtaingood growth of Escherichia coli in media containing 2000 mg./l. of sulphanilamideby the addition to the medium of the five non-competitive sulphanilamide antag-onists (Winkler and de Haan, 1948). This opened the possibility of studyingthe influence of high concentrations of sulphanilamide on bacteriophage multi-plication, without interfering with bacterial growth. The results of these experi-ments are reported here.

MATERIALS AND METHODS.

Through the courtesy of Prof. Delbruck we were able to use E8cherichiacoli B and the bacteriophages T1-T7 of Demerec and Fano (1945).

Eacherichia coti was cultured in a synthetic medium (medium A) containingper litre: Glucose, 2 g.; NH4C1, 0 2 g.; KH2PO4, 0-6 g.; K2HPO4, 0-6 g., andMgSO4, 0*05 g.; pH, 6-6. The medium was completely free of p-aminobenzoicacid. The non-competitive sulphanilamide antagonists, methionine, xanthine,thymine and valine were each added at a concentration of 30 mg./l. (medium B);in some instances serine was added at a concentration of 20 mg./l.

Bacterial counts were made by the technique of Julius (1938), which is amodified plating method and has given reliable results in our previous work(Julius, 1941, 1942; Havinga, Julius, Veldstra and Winkler, 1946). In pre-liminary experiments bacterial growth and lysis were measured by turbiditywith a Moll extinctiometer, as is usual in our laboratory.

Bacteriophages were prepared by inoculating the basal medium with 107viable bacteria and 105 bacteriophage particles per ml., and filtering after lysishad occurred through G5 on 3 sintered glass filters. The filtrate generallycontained 108 to 109 bacteriophage particles per ml. Plaque counts were madewith Gratia's (1936) double layer plate method according to the modification ofHershey, Kalmanson and Bronfenbrenner (1943). In experimentsr with bacterio-phage T4, 20 Lg. tryptophan per ml. was added.

One-step growth curves were obtained by the technique of Ellis and Delbruick(1939). Adsorption mixtures, unless stated otherwise, contained between 0 5and 2 x 108 bacteria and about 1 to 5 x 107 bacteriophage particles. Adsorption

F. J. RUTTEN, K. C. WINKLER AND P. G. DE HAAN

and dilution tubes were mixed by continuous aeration. Eight minutes afteradsorption decimal dilution tubes down to 1 in 100,000 were prepared. Thenumber of free bacteriophage particles was estimated after centrifugation of the1 in 100 dilution at 10,000 r.p.m. About 90 per cent adsorption was generallyobtained (Table II). The dilution tubes 1 in 1000 and 1 in 100,000 were usedas growth tubes, and the number of plaque-forming particles estimated at fre-quent intervals with the double layer plates. Burst sizes varied from 60 to 160.

Time in hoursFIG. 1.-Influence of varying concentrations of sulphanilamide on bacteriolysis of Esc8 erichia

coli B by bacteriophage T1.

Without bacteriophage.------ With bacteriophage T1 (all cultures are lysed).

Resistant growth in lysed culture.

RESULTS.

Preliminary experiments.Non-competitive sulphonamides antagonists for E. coli B.-E. coli B behaved in

general like our E. coli (Strain 2) studied previously (Winkler and de Haan, 1948).Methionine, xanthine, thymine and valine seem to be the end-products of thereactions blocked by sulphanilamide. By the addition of these four non-com-petitive antagonists normal growth was obtained with E. coli B in the presenceof as much as 2000 mg. sulphanilamide/l. The growth rate was decreased to agreater extent, however, than with E. coli (Strain 2), especially with small inocula.A further difference between the two strains was- that serine did not act as anon-competitive antagonist for E. coli B. These features did not affect the prin-ciple, however, and it remained possible to study the action of high sulphanilamide

370

ACTION OF SULPHANILAMIDE ON BACTERIOPHAGES

concentrations on bacteriophage multiplication without unduly reducing bacterialgrowth.

Inhibition of bacteriotysis by sulphanilamide.Tubes containing varying concentrations of sulphanilamide (0, 125, 250, 500,

1000 and 2000 mg./l.) in a medium with non-competitive sulphanilamideantagonists (medium B) were inoculated with 3 x 106 bacteria or with bothbacteria and 3 x 104 bacteriophage particles. In the case of phage T4 and T620 ,ig./l. tryptophan was added. Turbidities were estimated at intervals.

Time in hours

FIG. 2.-Influence of varying concentrations of sulphanilamide on bacteriolysis of Escherichiacoli B by bacteriophage T2.

Without bacteriophage. With bacteriophage.

Results with bacteriophage T1 are given in Fig. 1. In the controls withoutbacteriophage sulphanilamide reduced the growth-rate in spite of the presenceof the five antagonists. It should be realized, however, 'that without non-com-petitive antagonists about 60 mg./l. sulphanilamide suppressed visible growthcompletely. All cultures to which bacteriophage T1 was added were lysed. Inthe control tube without sulphanilamide a delayed growth was observed due toa resistant E. coli. It was omitted in the figure.

The influence of sulphanilamide on lysis by bacteriophage T2 is shown inFig. 2. Cultures without bacteriophage showed growth as before. With bacterio-phage the result was entirely different. The culture with bacteriophage, butwithout sulphanilamide, showed complete lysis. In the presence of 125 or 250mg./l. sulphanilamide, lysis was less complete. Above 500 mg./l. sulphanilamide

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no lysis occurred and bacterial growth equalled that of cultures without bacterio-phage. This growth is not due to bacteriophage-resistant bacteria.

Analogous results were obtained with the bacteriophages T4 and T6' whereasno such lysis-inhibition by sulphanilamide was observed with the bacteriophagesT1, T3 and T7.

The effect described was obtained with varying inocula of both bacteriophageand E. coli B. Although the inhibition of bacteriolysis was more pronouncedwith small inocula (presumably by delayed bacteriophage adsorption) it wasalways present.

The effect was not due to an increase in generation time induced by sulphanila-mide. By culturing E. coli at lower temperatures it was shown that an increase ingeneration time in itself did not affect bacteriolysis. Again, a decrease of thegeneration time in the presence of sulphanilamide by the addition of casein-hydrolysate even seemed to enhance the inhibition of bacteriolysis by sulphanila-mide.

All causes for an unspecific effect thus being eliminated, we concluded thathigh concentrations of sulphanilamide inhibit bacteriolysis of E. coli B by bacterio-phages T2, T4 and T6, whereas the lysis by the bacteriophages T1, T3 and T7 isunaffected.

Inhibition of bacteriophage multiplication by sulphanilamide.It was soon established by plaque counts that in the cultures in which sul-

phanilamide inhibits bacteriolysis no bacteriophage multiplication takes place,whereas in the control tubes without sulphanilamide the number of bacteriophageparticles increased from 103 to about 108 or 109.

Mechanism of action of virus inhibition by sulphanilamsde.In the chemotherapy of viruses such as the bacteriophages, there are at least

three possible points of attack: (a) extracellular inactivation of virus particles,(b) inhibition of adsorption of virus to host cells, and (c) intracellular inhibition*of virus multiplication.

To ascertain with which stage of the life-cycle of the bacteriophage sulphanila-mide interferes, we first studied the effect of 2000 mg./l. sulphanilamide on freeT4 bacteriophage particles (Table I) and on adsorption (Table II). With bacterio-_phage T2 analogous results were obtained. It is evident from the results shown in

TABLE I.-Survival of Free Bacteriophage T4 in 2000 mg./l. Sulphanilamide.

Number of bacteriophage particles x 108 atTime in hours.

0. 24. 48.Medium A . . 081 . 0 22 . 0 26Medium B . . 0 81 . 0-36 . 0 33Medium B + 2000 mg./l.

S.A. . . . 0-81 . 0*26 . 0-26

S.A. = Sulphanilamide.

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'F'ABLE II.-Influence of Sulphanilamide and of Bacteriophage T4 on Escherichiacoli B.

Growth in Adsorption in Viable bacteria Bacteriophage Per cent

medwthinm medium.nMl. (X 107). particles/ml. adsorptionmedium. medium. ml. ( x 107). (x 107). in 8 min.A . A . 6*2 . 1-2 . 84A . A . 3*2 . 2*0 . 84A .A+2000mg./I.S.A.. 23 . O-32 . 94

A . 23 . 0 32 . 96A . B+2000mg./l.S.A.. 8 . 2-1 . 95

A . 8 . 21 . 95A . B . 6-2 . 1*75 . 95

A . 6 2 . 175 . 95B+2000 mg./l. . B+2000mg./l.S.A. . 10 . 49 . 93

S.A.. B+2000mg./l.S.A. . 6 6 . 4 6 . 91

S.A. = Sulphanilamide.

the tables that sulphanilamide does not inactivate free bacteriophage particlesand that sulphanilamide does not inhibit the adsorption of bacteriophage particlesto E. coli B.

The effect of sulphanilamide on intracellular bacteriophage multiplication.

The only reliable method of showing the influence of a drug on intracellularbacteriophage multiplication is the one-step growth technique in which one lifecycle of the virus is singled out. Any inhibition of intracellular multiplicationwill result in an extension of the latent period or in a reduction of the meannumber of bacteriophage particles liberated per bacterium.

One-step growth curves were obtained with bacteriophage T4 in medium Aor B, or without sulphanilamide, using E. coli B grown in medium A as an inoculum.With sulphanilamide a very slight increase in the latent period was often observed,but the burst size was never affected. Using bacteria grown in medium A,sulphanilamide had no direct effect on the multiplication of bacteriophage T4 inone-step growth.

The delayed effect of sulphanilamide.The above effect was not unexpected. Bacteriophage multiplication might

be inhibited by interference with a supposed phage metabolism, although it isgenerally agreed that bacteriophage multiplication is largely dependent onbacterial metabolism (anabolism). Bacteriophage multiplication might thus beinhibited by interference with aspects of bacterial metabolism not affectingbacterial growth. For instance, inhibition of a surplus production by certainenzyme systems might inhibit virus multiplication without affecting bacterialgrowth. Again some enzyme systems might be essential for phage multiplication,but not essential for bacterial growth.

The above results exclude any direct action on bacteriophage metabolism.Yet the action of sulphanilamide on bacterial growth is generally delayed, i.e.

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374 F.!J. RUTTEN, K. C. WINKLER AND P. G. DE HAAN

only becomes evident after one or two normal divisions. Presumably the end-products of the inhibited enzymes are still present in sufficient amount to permitone or two divisions. Now in " one-step growth " experiments bacteriophage isadsorbed onto the bacteria within a few minutes, and the host cell is infectedbefore the sulphanilamide effect can develop. Inhibition of bacterial metabolismmight well be without effect on bacteriophage reproduction unless sulphanilamidewas added before the addition of bacteriophage.

" One-step growth " experiments were set up as before, using as inoculumE. coli B grown in medium B with 2000 mg./l. sulphanilamide. In this case acomplete suppression of bacteriophage multiplication (T4) was observed when2000 mg./l. sulphanila.mide was added in the one-step experiment (Fig. 3).

80Time in minutes

FIG. 3.-Influence of sulphanilamide on growth of bacteriophage T4. Abscissa, time in minutes.Ordinate, number of bacteriophage particles per bacterium.

Curve 1 ------ multiplication of bacteriophage T4 in bacteria grown in medium B plus2000 mg./l. sulphanilamide.

Curve 2 Control, bacteria grown either without sulphanilamide or non-competitiveantagonists (= medium A), or grown in medium A plus 12 mg./l. sulphanilamide to obtainthe same growth rate as in the culture producing the host bacteria for Curve 1.

This effect was not due to a reduced growth rate in the culture from whichthe host cells were derived. By heavy inoculation the growth rate in sulphanila-mide plus non-competitive antagonists was made equal to the control. Variousother experiments showed beyond doubt that even in experiments in which thegrowth rate of the culture from which the host cells were obtained had beenreduced, this reduction did not affect bacteriophage multiplication. The bac-teriophage multiplication is shown in Fig. 3. It is evident that in bacteriacultured in 2000 mg./I. sulphanilamide (with non-competitive antagonists),bacteriophage T4 cannot multiply. That this effect is indeed specific is shownby the fact that reproduction of bacteriophage T1 was not inhibited under thesame conditions. We thus conclude that in bacteria grown in the presence ofsulphanilamide and non-competitive antagonists, intracellular bacteriophagereproduction (T4) is inhibited.


Bacterial enzymes inhibited in the range of sulpahnitamide concentration whichinhibits bacteriophage.

The experiments reported in the previous section show that sulphanilamideacts by interfering with bacterial metabolism, as it is the growth of the host cellsin media containing sulphanilamide which is essential for the effect. No non-specific sulphanilamide effect is involved as bacteriophage reproduction is re-estab-lished by the addition of p-aminobenzoic acid. Some p-aminobenzoic acid-dependent reaction is thus involved.

Cone. SA in mg./L.FIG. 4.-Influence of non-competitive antagonists on sulphanilamide inhibition of growth

of E. coli B, with and without bacteriophage. Abscissa, concentration sulphanilamidein mg. /1. Ordinate, turbidity.

Controls without bacteriophage.c = Medium A.m = Medium A + methionine.x = Medium A + methionine + xanthine.th. = Medium A + methionine + xanthine + thymine.v = Medium A + methionine + xanthine + thymine + valine.---- - With bacteriophage.

With bacteriophage the cultures c, m and x are completely lysed (not shown in fig.) orshow resistant growth (R). Only on the addition of thymine and valine does growthoccur. It was assured that this growth was not bacteriophage-resistant.

The activity of sulphanilamide against virus reproduction might then be dueto the inhibition of one of the four p-aminobenzoic acid-dependent enzymesystems leading to methionine, xanthine, thymine and valine, or to an interferencewith some as yet unknown function of p-aminobenzoic acid. Which of the fourenzyme systems of the former possibility is involved might be studied by com-paring the sulphanilamide concentration at which each system is inhibited withthe concentration at which bacteriophage inhibition occurs.

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376 F. J. RUTTEN, K. C. WINKLER AND P. G. DE HAAN

To this end E. coli B was cultured in medium A with increasing concentrationsof sulphanilamide in five series of tubes. The first series acted as control.Methionine was added to the second, methionine and xanthine to the third.The fourth series contained methionine, xanthine and thymine. The fifth alsovaline, thus containing medium B. To a duplicate set of tubes bacteriophagewas added. At regular intervals turbidity readings were taken. Fig. 4 showsthe results at 48 hours. In the series without bacteriophage a step-wise increasein the tolerated sulphanilamide concentration was observed, as in previous work

7 PABA/L.FIG. 5.-Influence of non-competitive sulphanilamide antagonists on the growth of E. coli

in the presence of sulphanilamide and p-aminobenzoic acid, with and without bacteriophage.Abscissa, concentration p-aminobenzoic acid in mg. /1. Ordinate, turbidity. Constant sul.phanilamide concentration 2000 mg./l. Bacterial growth at 48 hours.

Control without bacteriophage. With bacteriophage (p).I, Ip medium A.II, iIp ,, A with methionine.III, IIIp ,, A ,, and xanthine.IV, IVp ,, A ,, ,, and serine.V, Vp ,, A ,, ,, ,, and thymine.VI, VIp ,, A ,, ,, ,, ,, and valine.

With bacteriophage all cultures show lysis, except Vp and VIp. Resistant growth was omittedfrom the figure and only indicated as R.

(Winkler and de Haan, 1948). With bacteriophage all cultures showed lysis,until thymine was added. On the addition of thymine high sulphanilamideconcentrations inhibited bacteriophage multiplication (lysis).

The experiment was varied by culturing E. coli in five series of tubes allcontaining 2000 mg./I. sulphanilamide. Varying concentrations of p-amino-benzoic acid were added to each series and the same concentrations of non-competitive antagonists as before were used. The results are represented in


Fig. 5. Without bacteriophage the addition of each additional non-competitiveantagonist decreased the concentration of p-aminobenzoic acid which supportedbacterial growth, until with all four non-competitive antagonists growth withoutp-aminobenzoic acid was obtained.

On the addition of bacteriophage all cultures were lysed until thymine wasadded. Again, as soon as the p-aminobenzoic acid concentration fell below thelevel which antagonizes inhibition by sulphanilamide of the thymine-synthesizingsystem (10 ,ug./l.)-that is as soon as this system is inhibited, and the bacteriacan only grow by the addition of thymine-bacteriophage inhibition began.

In both experiments bacteriophage inhibition began as soon as the thymine-synthesizing system was inhibited. This might be a coincidence, bacteriophagemultiplication being inhibited at the same concentration of sulphanilamide asthe thymine-synthesizing enzyme, or might indicate a narrower relationship;the thymine supply might be sufficient for bacterial growth but insufficient forbacteriophage synthesis or the (still inhibited) thymine-synthesizing enzyme itselfmight be involved in bacteriophage synthesis. The,same reasoning might beapplied to valine.

We conclude that the action of sulphanilamide on bacteriophage reproductionis not due to inhibition of the methionine and xanthine synthesizing system.Only the synthesis of thymine and valine or further unknown systems areinvolved.

Search for sub8tances which antagonize the sulphanilamide effect on bacteriophage.

Whether some unknown function of p-aminobenzoic acid was involved in theinhibition of bacteriophage multiplication was studied by trying to find substanceswhich antagonize the sulphanilamide effect on bacteriophage non-competitively.The following substances were tested: Various inorganic salts, ascorbic acid,eight vitamins of the B group, yeast autolysate, adenosinetriphosphoric acid, allavailable keto acids, twenty-two amino-acids, the peptides valyl-glycine, alanyl-glycine, leucylglycine, glycineanhydrid, glycylglycine, alanylglycylglycine, leucyl-glycylglycine and casein hydrolysate. Furthermore, creatine, sarcosine, glyco-cyamine, hippuric acid, thymine, thymidine, guanosine, uracil, ribonucleic acidand desoxyribonucleic acid were added.

The bacteriophage inhibition by sulphanilamide was only antagonized byglycine and the glycine-containing peptides. Even with these substances theeffect was very slight and not sufficient to warrant any conclusion.

DISCUSSION.

The above experiments show that the intracellular multiplication of thebacteriophages T2, T4 and T6 on E. coti B can be inhibited by high concentrationsof sulphanilamide, when the interference with bacterial growth is antagonizedby the addition of four non-competitive antagonists. The specificify of thiseffect is stressed by the fact that multiplication of the bacteriophages T1, T3 andT7 is unaffected by su]phanilamide. It was further shown that sulphanilamidedoes not interfere with an eventual bacteriophage metabolism, but that theeffect is due to some interference with bacterial metabolism. The effect must bedue to some p-aminobenzoic acid-dependent system.

377


Some digression into our present views (Winkler and de Haan, 1948) on sul-phanilamide action may be allowed here. We suppose that sulphanilamideinterferes with the synthesis from p-aminobenzoic acid of several substanceswhich act as, or form part of, prosthetic groups for the enzymes synthesizingmethionine, xanthine, serine, thymine and valine from their precursors. In someinstances pteroylglutamic acid is one of these prosthetic groups (probably syn-.thesizing thymine), but in E. coli B there is no evidence for this.

In our present case the bacteria are able to grow in the presence of highconcentrations of sulphanilamide after the addition of the four non-competitiveantagonists. In these bacteria the enzymes synthesizing methionine, xanthine,thymine and valine do not function (possibly because the prosthetic group ismissing), but growth is permitted by the supply of the end-products.

The activity of sulphanilamide against virus reproduction might then be dueto at least four causes:

1. One or more of the four enzymes leading to methionine, xanthine, thymineand valine are involved:

(a) The supplied concentration of the four end-products is sufficientfor bacterial reproduction, but insufficient for the more exacting bacterio-phage multiplication.

(b) The presence of the intermediate products derived from p-amino-benzoic acid (prosthetic groups ?) is essential for bacteriophage repro-duction as the bacteriophage uses them for its own purposes, for instanceon apo-enzymes of its own.

(c) The presence of one of the four supposed bacterial apo-enzymes isessential for the bacteriophage. In bacteria grown in sulphanilamidethese apo-enzymes might not be available but blocked by compoundscontaining sulphanilamide (e.g. folic acid analogous with .sulphanilamideinstead of p-aminobenzoic acid).

2. Other enzymes, dependent on intermediate products derived from p-amino-benzoic acid and the end-products of which are not yet known, are involved.

It was established that a fourfold increase in the concentration of the four non-competitive antagonists does not re-establish bacteriophage multiplication;possibility l(a) is thereby excluded.

It was shown that the methionine and xanthine-synthesizing systems arenot involved. There is a probability that the thymine and valine synthesizingsystems are involved, but other unknown systems could not be completelyexcluded.

Definite evidence on this subject might be obtained if it could be shownthat a p-aminobenzoic acid-less E. coli B mutant strain, grown without p-amino-benzoic acid on a medium containing methionine, xanthine, thymine and valinewas bacteriophage-resistant. Such an experiment would at the same time showthat the- bacteriophage requires the p-aminobenzoic acid-containing inter-mediates which probably catalyse the synthesis of methionine, xanthine, serineand valine.

It is thought that the submitted facts are a possible approach to virus chemo-therapy as it is shown that interference with host-cell metabolism can inhibitvirus propagation without unduly affecting the host cell.

378

ACTION OF SULPHANILAMIDE ON BACTERIOPHAGES 379

SUMMARY.

It is shown that high concentrations of sulphanilamide completely inhibitthe intracellular multiplication of the bacteriophages T2, T4 and T6 on Escherichiacoli B, if the interference of sulphanilamide with bacterial growth is antagonizedwith four non-competitive sulphanilamide antagonists. This effect is due toan interference with bacterial metabolism and might be a possible approach tovirus chemotherapy.

REFERENCES.DELBRtYCK, M., AND LURIA, S. E.-(1944) Proc. Ind. Acad. Sci., 53, 28.DEMEREC, M., AND FANO, U.-(1945) Genetics, 30, 119.ELLIS, E. L., AND DELBRUCK, M.-(1939) J. gen. Physiol., 22, 365.GRATIA, A.-(1936) Ann. Inst. Pasteur, 57, 652.HAVINGA, E., JuLIus, H. W., VELDSTRA, H., AND WINKLER, K. C.-(1946) 'Modern

Development of Chemotherapy.' Amsterdam (Elsevier Publishing Company,Inc., New York).

HERSHEY, A: D., KALMANSON, G., AND BRONFENBRENNER, J.-(1943) J. Immunol.,46, 267.

JULIUS, H. W.-(1938) Ant. van Leeuwenhoek, 5, 28.-(1941) Ibid., 7, 153.-(1942)Ibid., 8, 86.

WAHL, R., NITTI, F., AND FAQUET, M.-(1946) Ann. Inst. Pasteur, 72, 290.WINKLER, K. C., AND DE HAAN, P. G.-(1948) Arch. Biochem., 18, 97.

the action of sulphanilamide on bacteriophages t1-t7

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