inhibition acetylsalicylic strains p-aminobenzoic · p-aminobenzoic acid marker. reversal of...

INHIBITION BY ACETYLSALICYLIC ACID OF RICKETTSIAL STRAINSRESISTANT TO p-AMINOBENZOIC ACID

EMILIO WEISS,'HARRY R. DRESSLER, AND EARL C. SUITOR, JR.WITH THE TECHNICAL ASSISTANCE OF I. E. FISHBURNE

Virology Division, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland

Received for publication March 11, 1959

Two p-aminobenzoic acid-resistant mutants ofthe Madrid E strain of Rickettsia prowazekii,pr and prr have been described in previouspublications (Weiss et al., 1957; 1959). It hasbeen shown that certain properties, such asantigenic specificity, infectivity, and virulencefor the chick embryo and cotton rat, have notchanged in these strains. However, when thestudy was extended to susceptibility to com-pounds chemically related to p-aminobenzoicacid, new characteristics were detected. Thesecharacteristics are described in this paper.'

MATERIALS AND METHODS

Rickettsiae. The Madrid E strain (Perez Ga-Ilardo and Fox, 1948) of R. prowazekii and itsfollowing substrains were used: psr, pr, and prr,which can be considered as first, second, andthird-step mutants, respectively, to p-amino-benzoic acid resistance; Ar, resistant to acetyl-salicylic acid; Qr, resistant to 2, 3-dimethyl-quinoxaline-1 ,4-dioxide; and pQr resistant toboth p-aminobenzoic acid and the quinoxalinecompound. The selection of these strains wasdescribed in previous publications (Weiss et al.,1957; 1959), except for psr and Ar which aredescribed in later sections of this paper.2 Rickett-sial pools were prepared from the infected yolksacs of chick embryos and diluted in Bovarnick'sisotonic solution (Bovarnick et al., 1950).

Chemical compounds. The chemical compound(lswere obtained from several commercial sources.Solutions were prepared immediately before use,except for leucovorin and sulfadiazine which werereceived as solutions. The compounds were dis-solved in distilled water with sufficient sodium

1 Some of this work was presented at the SeventhInternational Congress for Microbiology in Stock-holm, Sweden, August 4 to 9, 1958.

2 The p-aminobenzoic acid-resistant strains weredesignated PABr, PABrr, and PABrrQr in a pre-vious publication (Weiss et al., 1959).

bicarbonate to bring the pH to between 7.0 and7.4, sterilized by filtration, and further diluted,as needed, in Hanks' balanced salt solution(Hanks and Wallace, 1949). When two or morecompounds were to be used, their solutions weremixed just before injection. The solutions wereinjected into eggs via the yolk sac, immediatelyprior to rickettsial inoculation, in a volume of0.1 ml per egg. Untreated embryos were giventhe diluent.

Chick embryos. The eggs used were WhiteLeghorn from a flock maintained on an antibiotic-free diet. The age of the embryos was 7 or 8 days,the temperature of incubation 35 C, and theperiod of observation 13 days. Rickettsial suspen-sions were injected into the yolk sac in volumesof 0.4 ml per egg.

Determinations of the effect of chemicals weremade by comparing mean survival times of con-trol and treated groups of embryos. Most groupswere composed of approximately 20 embryos,exclusive of those which died because of injec-tion trauma. Yolk sacs from approximately onefourth of the embryos were smeared and checkedfor the presence of rickettsiae. The few embryossurviving at 13 days were arbitrarily given 14 asthe day of death. Some of the compounds weretoxic in high concentration and tended to reduceembryo survival time. However, interferencewith rickettsial growth could be demonstrated,even though in some instances it was not possibleto separate death due to infection from deathdue to toxicity.A statistical analysis of the data obtained in

the course of this work indicated that the reli-ability of the tests was of the same order as thatreported by other investigators who have studiedchemotherapeutic effects on rickettsial infectionin eggs (Ormsbee et al., 1955; Robbins et al.,1951). On the basis of these analyses, the follow-ing guiding criteria can be formulated for thesignificance of differences in single experiments.

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RICKETTSIAE INHIBITION BY ACETYLSALICYLIC ACID

With concentrations of rickettsiae sufficient tokill untreated embryos in approximately 6 days,the least increase in survival time of a group ofdrug-treated embryos that can be considered asindicating significant protection is 1.0 day. Theleast difference between two protected groupsthat can be considered significant is 1.5 days.However, differences are not significant whenboth protected groups survive at least 4.0 dayslonger than the control group.Most of the data presented in this paper are

based on experiments repeated two or moretimes.

EXPERIMENTAL PROCEDURES AND RESULTS

Selection of the p8r strain. The series of eggpassages of the parent strain in the presence ofp-aminobenzoic acid which had led to the isola-tion of pr (Weiss et al., 1957) was subsequentlyutilized for the selection of psr. A pool preparedfrom the 15th passage, which had been stored at-50 C for over 3 years, was passed once more inthe presence of 22,umoles of p-aminobenzoic acidper egg. From this 16th passage PIr was obtainedby limit-dilution isolation (see Weiss et al., 1957,for procedure). As shown in table 1, the suscepti-bility of this strain to p-aminobenzoic acid ap-peared to be intermediate between the parentstrain and pr.

Reversal of p-aminobenzoic acid inhibition byp-hydroxybenzoic acid. Snyder and Davis (1951)and Takemori and Kitaoka (1952) have shownthat the rickettsiostatic effect of p-aminobenzoicacid can be competitively reversed by p-hydroxy-benzoic acid. Ratios of at least 1 :10 of p-hydroxy-to p-aminobenzoic acid were required for com-plete reversal.As shown in table 2, the above described re-

sults were reproducible with the present parentstrain. Complete or almost complete reversal was

TABLE 1Effect of p-aminobenzoic acid on rickettsial strains

Chick Embryos

Strain Untreated Treated (22 ,Amoles/Survival (mean egg)Increased survivaldays) (mean days)

Parent .......... 5.8 4.6per........... 6.1 1.4pr .............. 6.2 0.3

TABLE 2Reversal of p-aminobenzoic acid inhibition by

p-hydroxybenzoic acid

Strain

Parent

Micromoles/Egg Chick Embryos

p-Amino- _enHyo Untreatedbenzoic benz~oix Survivalacid nezs cd(mean days)

088888880

000.250.512488

6.0

TreatedIncreasedsurvival

(mean days)

5.52.41.50.80.50.50.10.5

per 0 0 6.332 0 1.532 8 0.20 8 -0.1

pr 0 0 6.722 0 0.622 7 -0.30 7 -0.2

prr 0 0 6.30 36 0.00 72 0.5

obtained with a ratio of 1:8, but only partialreversal with 1:16. Furthermore, table 2 showsthat the slight inhibitory effect of p-aminobenzoicacid on p8r was also reversed by p-hydroxybenzoicacid. The same is possibly true of pr, althoughdifferences in chick embryo survival times weretoo small to be significant. The same type of testcould not be extended to prr, because of its ap-parently complete resistance to p-aminobenzoicacid. But it is obvious from the results shown intable 2, that even large amounts of p-hydroxy-benzoic acid, such as 36 or 72 umoles per egg,did not inhibit this strain. The results presentedin table 2 thus indicate that p-hydroxybenzoicacid has similar effects on the p-aminobenzoicacid-resistant as on the susceptible strains.The specificity of the reversing effect of p-hy-

droxybenzoic acid on the parent strain is illus-trated in table 3, which lists a number of com-pounds that did not inhibit the parent strain orreverse the inhibition by p-aminobenzoic acid.

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WEISS, DRESSLER, AND SUITOR

This list includes compounds either chemicallyrelated to p-aminobenzoic acid or involved in itsmetabolism, as demonstrated in other micro-organisms.

Effect of salicylic acid and acetylsalicylic acid.Robbins et al. (1951) have shown that salicylicacid inhibits the Wilmington strain of Rickettsiatyphi to a moderate degree. In the present studies,inhibition of the parent Madrid E strain with thiscompound could be demonstrated only with greatdifficulty, but pr and prr were inhibited to anappreciable extent by concentrations which didnot affect the parent strain. Typical results areillustrated in table 4. Some of the experimentssuggested that the susceptibility of prr to sali-cylic acid was slightly greater than that of pr.As shown in table 5, the three p-aminobenzoic

acid-resistant strains were also inhibited by con-centrations of acetylsalicylic acid which did notaffect the parent strain. This effect appeared tobe more marked than that obtained with salicylicacid and the evidence that the susceptibility toacetylsalicylic acid increases with the level ofp-aminobenzoic acid resistance of the strain is

TABLE 3Effects of various compounds on rickettsial strains

Effect

p-Amino- p-Aminoben-benzoic acid- zoic acid-resis-

sensitive strains tant strains

Compound Rever- Rever-sal of Rvr

nh-p-ami- sal ofIh-noben- Inhi- acetyl-bition zoic bition salicylic

acd acidaidhbi inhibi-tion to

p-Aminobenzoic acid.... + +p-Hydroxybenzoic acid. + - -Salicylic acid........... * +Acetylsalicylic acid _ +Anthranilic acid ........p-Aminosalicylic acid...-p-Nitrobenzoic acid..... * _ _ *Shikimic acid...........Nicotinic acid ..........Pantothenic acid .......Glutamic acid ..........Pteroylglutamic acid. -Leucovorin ..........Stilfadiazine ..........

* Very slight inhibition.

TABLE 4Effect of salicylic acid on rickettsial strains

Chick Embryos

Increased survivalStrain Suvvl(en (mean days)Survival (mean Treated Gumo1es/egg)

Untreated29 43 58

Parent 5.8-6.6 0.0 0.0 -0.1pr 5.9-6.6 0.6 1.3 2.7prr 5.7-6.6 1.0 1.5 2.3

TABLE 5Effect of acetylsalicylic acid on rickettsial strains

Chick Embryos

Increased survivalStrain Survival (mean (mean days)

days) Treated (jumoles/egg)Untreated

33 67

Parent 5.8-6.0 0.0 0.7p8r 6.1 1.5pr 5.8-6.6 1.6 2.5-3.7prr 5.5-6.1 2.0 4.1

more convincing than in the case of salicylicacid. Because of the higher inhibitory effect ofacetylsalicylic acid and its lower toxicity for thechick embryo, it was used for most of thie sub-sequent experiments in preference to salicylicacid.The p-aminobenzoic acid-resistant strains did

not acquire a susceptibility to a number of otherrelated compounds, listed in table 3. However,p-nitrobenzoic acid inhibited all strains tested,including the parent, to a very slight degree.

Strains Qr and pQr were also tested for thesusceptibility to acetylsalicylic acid. The formerappeared to be resistant, the latter susceptible,providing further confirmation that acetylsali-cylic acid susceptibility was associated with thep-aminobenzoic acid marker.

Reversal of acetylsalicylic acid inhibition byp-aminobenzoic acid. The effect of various com-pounds on inhibition of the rickettsial strains byacetylsalicylic acid was investigated. The stur-prising results which were obtained are shown intable 3, figure 1, and table 6. Of the compoundstested only p-aminobenzoic acid reversed theinhibition by acetylsalicylic acid. Figure 1 depicts

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L4 10 -0~~~~~~~~~~~~~

d ASA pAB WITH CONSTANT AMOUNT OFASAPARENT--.-A23 -0 r r~~~~~~~~~~

C (N pB Pi"F a. R

0~~~~~~~~~

(NOSAr (N S

of acetylsalicylic acid alone is shown at the left, of the maximum amount of p-aminobenzoic acid usedis shown at the right, and of the mixture of the two drugs is shown at the center. Note that in the ex-periments with the three resistant strains, the following amounts were used: acetylsalicylic acid, 67jAmoles; p-aminobenzoic acid, 2-fold increases from approximately 1 or 2,uAmoles (as shown) to 33,umoles.With the parent strain the amounts were: acetylsalicylic acid, 100,uAmoles; p-aminobenzoic acid, 4-foldincreases from approximately 0.4 to 25,4moles. See text.

experiments with four strains in which a constantamount of acetylsalicylic acid was tested againstvarying concentrations of p-aminobenzoic acid.The inverse relationship between acetylsalicylicand p-aminobenzoic acid susceptibility of thefour strains is quite apparent. p-Aminobenzoicacid, added in increasing concentrations, reducedand finally eliminated the inhibition of prr byacetylsalicylic acid. The effect on pr is verysimilar, except that the highest concentration ofp-aminobenzoic acid used alone or in combinationwith acetylsalicylic acid is, possibly, slightlyinhibitory. p8r is inhibited to a small, but signifi-cant, extent by both acetylsalicylic and p-amino-benzoic acid and reversal can be shown whensmall amounts of the latter drug are used in com-bination with large concentrations of the former.With large concentrations of both drugs thisstrain is inhibited, but apparently there is no

summation of the inhibitory effects of acetyl-salicylic and p-aminobenzoic acid. The parentstrain is not generally inhibited by the amount ofacetylsalicylic acid (67 ,umoles) used with theother strains, but an appreciable inhibition wasobtained with 100l,moles. The results shown infigure 1 suggest that this inhibition is reversedby p-aminobenzoic acid within a very narrowrange of concentration. As the concentration ofthis drug is increased it rapidly becomes inhibi-tory. When the four experiments are compared,they indicate that the amount of p-aminobenzoicacid required for reversal increases with the de-gree of susceptibility of the strain to acetyl-salicylic acid.

Table 6 illustrates several attempts to deter-mine whether the reversal of acetylsalicylic acidinhibition of prr by p-aminobenzoic acid is com-petitive or noncompetitive. The results include

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TABLE 6Reversal of acetylsalicylic acid inhibition by

p-aminobenzoic acid (Prr strain)Chick Embryos

p-Aminobenzoic Increased survival (mean days*)Acid (umoles/

egg) Acetylsalicylic acid (,umoles/egg)

25 33 50 50 67 100

0 0.9 1.8 3.2 3.0 3.8 5.50.5 0.5t 1.2 1.8 1.2 4.51 0.0t 0.7 4.0 2.9 5.32 0.2 0.5 1.5 1.1 2.6 3.94 -0.1 0.6 1.4 1.0 1.2 4.08 -0.2 0.6 0.4 0.1 0.6 1.716 0.5 0.8 0.333 0.2

* Untreated embryos died 5.6 to 6.0 days afterrickettsial inoculation.

t Results which seem to indicate partial or com-plete reversal of acetylsalicylic acid inhibitionwith smallest amounts of p-aminobenzoic acid areindicated in italics or boldface type, respectively.

discrepancies which cannot be satisfactorily ex-plained. For example, in two separate experimentswith 50 ,umoles of acetylsalicylic acid it appearedthat inhibition was partially reversed by 0.5 butnot 1 ,umole of p-aminobenzoic acid. If theseresults are disregarded, a competitive relation-ship is suggested. The reaction seems to fit thekinetics of a second order reaction. For example,the effect of 0.5,Amoles of p-aminobenzoic acidon 25 jAmoles of acetylsalicylic acid can best becompared to the effect of 2 on 50 ,umoles or 8 on100 ,moles.

Salicylic acid inhibition of pr and prr was alsoreversed by p-aminobenzoic acid.Antagonism between p-hydroxybenzoic and p-

aminobenzoic acid in the reversal of acetylsalicylicacid inhibition. Table 7 illustrates findings whichhave been most surprising. The first three linesshow results which are entirely analogous to thoseobtained in previous experiments: prr was in-hibited by acetylsalicylic acid and its inhibitionwas reversed by an adequate amount of p-amino-benzoic acid. The other lines show the effect ofadding increasing amounts of p-hydroxybenzoicacid to the mixture of the two compounds. Asthe concentration of p-hydroxybenzoic acid wasincreased, rickettsial inhibition reappeared. Since

p-hydroxybenzoic acid has no direct effect on prr(table 2), it must be assumed that it antagonizedthe reversing effect of p-aminobenzoic acid. Theresults shown in table 7 indicate that partial andnearly complete antagonisms were obtained withratios (p-hydroxybenzoic to p-aminobenzoic acid)of 1:8 and 1 :4, respectively. These ratios do notdiffer substantially from those required for thereversal of the inhibitory effect of p-aminobenzoicacid of the parent strain.The specificity of the reaction is attested by

the fact that anthranilic acid and sulfadiazinefailed to replace p-hydroxybenzoic acid in therestoration of acetylsalicylic acid inhibition.

Similar results were obtained with pr.Isolation of strain Ar. prr was passed serially

in eggs in the presence of increasing concentra-tions of acetylsalicylic acid. Resistance to thiscompound rapidly developed and a limit-dilutionisolate was obtained following 9 drug passages.This strain, called Ar, was not appreciably af-fected by as much as 100 ,umoles of acetylsalicylicacid per egg. A test of the susceptibility of thisstrain to p-aminobenzoic and p-hydroxybenzoicacid is shown in table 8. It is clear from this test,and several others which are not shown, that theresistance of Ar to p-aminobenzoic acid was some-what reduced. The results presented in table 8furthermore suggest that the slight susceptibilityof Ar to p-aminobenzoic acid was reversed byp-hydroxybenzoic acid.

TABLE 7Effect of p-hydroxybenzoic acid on the reversal ofacetylsalicylic acid inhibition by p-aminobenzoic

acid (Prr strain)


TreatedAcetylsalicylic p-Amino- p-Hydroxy- Increased sur-

acid benzoic acid benzoic acid vival (meandays)

0 0 0 -67 0 0 4.767 16 0 0.367 16 0.5 0.667 16 1 0.567 16 2 1.067 16 4 2.767 16 8 2.367 16 16 3.8

* The mean survival time of untreated embryoswas 6.1 days.

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TABLE 8Effect of p-aminobenzoic and p-hydroxybenzoic

acid on the Ar strain


p-Hydroxbenzoic Treatedp-Aminobenzoic acid p-Hydroxybenzoic Increased survivalacid ~~(mean days)

o 016 0 0.932 0 1.30 16 0.20 32 0.316 16 0.332 16 0.4

* The mean survival time of untreated embryoswas 5.2 days.

TABLE 9Conmparison of p-aminobenzoic acid susceptibility

of rickettsial strains*

Chick Embryos

Strain Treated (73Untreated ,umoles/egg)survival increased

(mean days) survival(mean days)

Ar 6.7 1.36.2 1.2

Pr 5.9 1.25.9 1.8

prr isolate 1 6.1 0.55.9 0.6

Prr isolate 2 6.6 1.16.5 1.1

Prr isolate 3 5.7 0.16.1 0.8

prr isolate 4 6.0 0.86.1 0.8

* Two independentin each case.

determinations were made

It was assumed that the small loss of p-amino-benzoic acid resistance occurring during thechange from prr to Ar was due to the acquisitionof acetylsalicylic acid resistance. An experimentoriginally intended for a different purpose servedas a check for this assumption. A mixture of prrand Qr was passed serially in eggs 10 times. Alllimit-dilution isolates obtained after the tenthpassage appeared to be of the prr type. Deter-minations were made of p-aminobenzoic acid

susceptibility of 4 isolates along with similartests on pr and Ar. The results are shown intable 9. They are in agreement with those shownin table 8 and indicate that the levels of p-amino-benzoic acid susceptibility of Ar and pr werecomparable. The tests with the isolates, however,cast some doubt on the stability of the thirdstep (from pr to prr) of p-aminobenzoic acidresistance or its phenotypic expression. There-fore, the development of acetylsalicylic acid re-sistance cannot be held entirely responsible forthe reduction in p-aminobenzoic acid resistanceof Ar.

DISCUSSION

Table 3 lists the effects of compounds that weretested in attempts to link the observations de-scribed in this paper with metabolic steps thathave been found to be involved, directly orindirectly, with the action of p-aminobenzoic acidon other microorganisms. Although superficialsimilarities were found, the experiments suggestsome unique features in the metabolism ofrickettsiae. A few illustrations of the differencesencountered will be given.Emerson and Cushing (1946) and Emerson

(1947) isolated a strain of Neurospora, in whichthe roles of sulfanilamide and p-aminobenzoicacid were reversed, the former compound beingthe one required and the latter the competitiveinhibitor. Zalokar's (1948) experiments indicatedthat this strain produced more than the toleratedamount of p-aminobenzoic acid and requiredsulfanilamide as a detoxicant. Table 3 offersevidence that such a mechanism did not operatein our rickettsial strains. Sulfadiazine did notinhibit either strain or reverse the inhibition ofthe parent strain by p-aminobenzoic acid. Itshould be noted, however, that the sulfonamideto p-aminobenzoic acid molar ratio of 100:1required for the reversal of either inhibition inNeurospora was not quite achieved in our tests.The discovery of the tuberculostatic activity of

p-aminosalicylic acid (Lehman, 1946) was aidedby the report by Bernheim (1941) that sodiumsalicylate increased the oxygen uptake of tuberclebacilli. However, Youmans et al. (1947) foundthat p-aminobenzoic acid, and not salicylate,reversed the effect of p-aminosalicylic acid. Asshown in table 3, p-aminosalicylic acid had noeffect on the rickettsial strains, despite its closestructural resemblance to the two active com-pounds, salicylic and p-aminobenzoic acids.

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The inhibition of microorganisms by salicylicand acetylsalicylic acids has been attributed tointerference in a step in pantothenic acid me-tabolism (IvAnovics, 1942a, b; Maas, 1952). Ourfinding that pantothenic acid did not reverseacetylsalicylic acid inhibition was not surprisingin view of the report by Kleinschmidt et al.(1956) that purified rickettsiae do not containsignificant amounts of pantothenic acid.Davis (1951a) obtained a strain of Escherichia

coli with a multiple requirement for tyrosine,phenylalanine, tryptophan, p-aminobenzoic acid,and partial requirement for p-hydroxybenzoicacid. Shikimic acid substituted for the quintuplerequirement of this strain, apparently functioningas an early metabolic intermediate. In our studies,as shown in table 3, shikimic acid did not replaceeither p-hydroxy- or p-aminobenzoic acid in theirrespective roles as reversers of inhibition.The work of Morgan (1948, 1952) offers some

interesting points of comparison, because hismethods were almost identical to ours. Morganshowed that inhibition of psittacosis virus bysulfadiazine was reversed competitively byp-aminobenzoic acid and noncompetitively bypteroylglutamic acid or citrovorum factor. Colonanid Moulder (1958) obtained conclusive evidence

that viruses of the psittacosis group contain folicacid. They have also demonstrated a parallelismbetween loss of infectivity and loss of folic acidin purified virus suspensions and a markedlyslower decline in infectivity in the presence ofpteroylglutamic acid in the suspending medium.In our experiments with rickettsiae, pteroyl-glutamic acid and leucovorin did not replacep-aminobenzoic acid in the reversal of acetyl-salicylic acid inhibition. This would indicate thatp-aminobenzoic acid is not involved in folic acidmetabolism of rickettsiae or that a folic aciddifferent from pteroylglutamic acid or leucovorinis produced. The results of Kleinschmidt et al.(1956) which indicate that rickettsiae do notcontain significant amounts of folic acid tend tosupport the first explanation.Davis (1951b) and Davis and Maas (1952)

furnished the most satisfactory bacterial modelwhich can be used as the starting point for anexplanation of competitive inhibition in rickett-siae. They isolated a strain of E. coli which re-quired both p-amino- and p-hydroxybenzoicacids. Large concentrations of p-aminobenzoicacid interfered with the utilization of p-hydroxy-benzoic acid, but the competition between thetwo metabolites was not symmetrical. Even large

SPECIFIC TRANSPORT SYSTEMS

PARENT prr


concentrations of p-hydroxybenzoic acid were notinhibitory. p-Nitrobenzoic acid interfered withthe utilization of both compounds, being reversedby p-hydroxybenzoic acid plus p-aminobenzoicacid, but not by either compound alone. Thereare several points of similarity between theseresults and ours with rickettsiae, including re-sistance to p-hydroxybenzoic acid and very slightsusceptibility to p-nitrobenzoic acid of bothparent and prr strains.

Figure 2 is an attempt to represent our resultsbased on the above described bacterial model. Ittakes into consideration the findings of Mathiesonand Catcheside (1955), who have shown thatcompetition may occur not only at the enzymaticsite, but also at the site of specific transport.Parent as well as drug-resistant strains areassumed to have (a) an enzymatic site for theattachment and utilization of p-hydroxybenzoicacid, which site also has some affinity for com-peting p-aminobenzoic acid; (b) a similar site forp-aminobenzoic acid, with affinity for competingacetylsalicylic acid; (c) specific transport systemsfor p-hydroxybenzoic, p-aminobenzoic, and ace-tylsalicylic acid.

Strain characteristics are influenced by therelative strengths of the two enzymatic sites. Asin the case of the experiments of Mathieson andCatcheside (1955) with Neurospora crassa, it isnot necessary to assume that a change has takenplace at the sites of specific transport. Accordingto this schema, the enzymatic p-hydroxybenzoicacid site of the parent strain has high affinity forits metabolite and relatively high affinity forcompeting p-aminobenzoic acid. As the strainmutates to p-aminobenzoic acid resistance, thissite loses affinity for its metabolite and competi-tive inhibitor. This change is accompanied by anincrease in affinity of the p-aminobenzoic acidenzymatic site for its metabolite and a corre-sponding increase for its competitive inhibitor,acetylsalicylic acid. Ar can be visualized as astrain which has lost some affinity at both enzy-matic sites.Drug inhibition is produced by two types of

competitions: p-hydroxybenzoic- p-aminobenzoicacid and p-aminobenzoic-acetylsalicylic acid.Attempts to show a direct interaction betweenp-hydroxybenzoic and acetylsalicylic acid havenot been successful. However, an indirect actionwas shown in the presence of p-aminobenzoicacid (see table 7). It is necessary to assume thatcompetition may occur at both enzymatic and

specific transport sites. Thus, a high concentra-tion of p-aminobenzoic acid interferes withp-hydroxybenzoic acid utilization by the parentstrain. Similarly, a high concentration of acetyl-salicylic acid interferes with the utilization ofp-aminobenzoic acid by prr. p-Hydroxybenzoicacid, although very effectively eliminatingp-aminobenzoic inhibition of the parent strain atits own site, has no affinity for the p-aminoben-zoic acid site and cannot directly inhibit prr.However, it can compete with p-aminobenzoicacid at the specific transport site, and restoreacetylsalicylic acid inhibition when p-amino-benzoic acid acts as a reverser of this inhibition.The above representation of our results is the

simplest of several that we have attempted. Itsadequacy must be tested in further experiments.

SUMMARY

Changes associated with the p-aminobenzoicacid resistance of three Madrid E strains ofRickettsia prowazekii, psr pr, and prr, represent-ing first-, second-, and third-step mutants, re-spectively, were analyzed. Tests were carried outwith 7- to 8-day-old chick embryos and inhibitionof rickettsial growth was measured by the in-crease in survival time of the embryos. Previousfindings that the parent strain is highly suscep-tible to p-aminobenzoic acid and that the inhibi-tion by this drug is competitively reversed byp-hydroxybenzoic acid were confirmed. p-Hy-droxybenzoic acid, which by itself did not inhibitany of the strains, also reversed the slight inhibi-tion by p-aminobenzoic acid of psr and pr. Thedrug-resistant strains were inhibited by salicylicacid and acetylsalicylic acid to a larger extentthan the parent strain and this drug susceptibilityappeared to increase with resistance to p-amino-benzoic acid. Inhibition by acetylsalicylic acidwas competitively reversed by p-aminobenzoicacid. The addition of p-hydroxybenzoic acid to amixture of p-aminobenzoic acid and acetylsali-cylic acid reestablished the inhibitory effect ofacetylsalicylic acid. By 9 serial egg passages ofprr in the presence of acetylsalicylic acid, astrain resistant to this compound (Ar) was ob-tained, which retained most, but not all, of itsresistance to p-aminobenzoic acid. Ten additionalcompounds, chemically related to p-aminobenzoicacid or which have been involved in the p-amino-benzoic acid metabolism of other microorganisms,were tested for their effects on the rickettsialstrains. They were found to be ineffective when

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used either alone or in combination with p-amino-benzoic acid or acetylsalicylic acid. An interpre-tation of the changes involved in the mutantstrains, based on specifically balanced p-hydroxy-benzoic and p-aminobenzoic acid requirements,and on competitions occurring at the enzymaticand specific transport sites, has been offered.

REFERENCES

BERNHEIM, F. 1941 The effect of various sub-stances on the oxygen uptake of the tuberclebacillus. J. Bacteriol., 41, 387-395.

BOVARNICK, M. R., MILLER, J. C., AND SNYDER,J. C. 1950 The influence of certain salts,amino acids, sugars, and proteins on thestability of rickettsiae. J. Bacteriol., 59,509-522.

COL6N, J. I. AND MOULDER, J. W. 1958 Folicacid in purified preparations of members ofthe psittacosis group of micro-organisms. J.Infectious Diseases, 103, 109-119.

DAVIS, B. D. 1951a Aromatic biosynthesis. I.The role of shikimic acid. J. Biol. Chem.,191, 315-325.

DAVIS, B. D. 1951b Inhibition of Escherichiacoli by p-aminobenzoic acid and its reversalby p-hydroxybenzoic acid. J. Exptl. Med.,74, 243-254.

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