IN VIVO BREAKDOWN OF TUBERCLE BACILLI BY HOST TISSUE'

B. GERSTL, C. C. DELWrICHE, W. E. DAVIS, JR., D. KIRSH, AND P. M. RAMORINO

Laboratory Service, Veterans Administration Hospital, Oakland, California; and M. T. KearneyFoundation for Futndamental Research in Soil Science, University of California,

Berkeley, California

Received for publication September 10, 1956

The forces representing resistance of a host toinfection may be grouped into factors limitingmultiplication of the parasite and those actuallydestroying the invader. If the former is the solemechanism taking place, or is associated withonly minimal destruction of the parasite, a pre-carious balance or symbiosis will prevail. Granu-lomatous processes such as tuberculosis andcoccidioidomycosis are good examples of this.Healing, however, requires destruction of theinvading microorganism.The destruction of tubercle bacilli by the host

as related to host resistance has been studied bybacteriological methods. The latter are predi-cated on the presumption that the number ofcolonies obtained indicate the number of livebacilli present in tissues (Lurie, 1928; Pierce andDubos, 1954).A biochemical study of the interaction of host

and invading organism is reported here. Proteinsand nucleoproteins are vital constituents of allliving organisms, multicellular or unicellular.Their degradation represents an essential step inthe subjugation of one organism by another.Hence, a study of the removal and catabolism ofthese constituents seemed to offer an approachto the problem of resistance in tuberculosis. Ifthe procedure selected proved feasible, compari-son of breakdown of attenuated or avirulentbacilli as compared to pathogenic ones by suscep-tible versus resistant strains and immunizedversus normal animals would extend our knowl-edge of host resistance.For the purpose of these studies labeled tu-

bercle bacilli were obtained by growing them on amedium wherein all sources of nitrogen contained

l This investigation was supported by the U. S.Veterans Administration and by grants from theCommittee on Medical Research of the AmericanTrudeau Society, Medical Section of the NationalTuberculosis Association, and from the CaliforniaTuberculosis and Health Association.

a high percentage of N"5. Since tubercle bacilli arealmost exclusively dependent upon their culturemedium for their nitrogen supply (Youmans andYoumans, 1954), their uptake of the nitrogenisotope should be proportional to that present inthe medium. Demonstration that the breakdownof the labeled substances occurred to an extentand at a rate sufficient to yield a favorableenough ratio of excess N15 in body fluids andorgan extracts was undertaken as the primarystep.The possibility was considered that the appear-

ance of isotopic nitrogen in the host mightrepresent an exchange of free amino acids fromthe organisms and would not actually requiredestruction of the bacilli themselves. Gale's(1947) investigation on gram positive organismssuggested that free amino acids may be presentwithin their cellular environment, and that nitro-gen could be transferred from them without indi-cating true catabolism. The estimation of freeamino acids in tubercle bacilli was, therefore,undertaken.A second source of error may arise from the

tubercle bacillus' own metabolism. Bernheimet al. (1953) reported that tubercle bacilli BCG8420, under certain conditions, discharge am-monia into the culture medium. Experiments totest this point with tubercle bacilli suspended inmouse plasma or saline were carried out. Anothergroup of control experiments was set up to deter-mine whether any disintegration of tuberclebacilli occurs under the mild conditions underwhich the organs were extracted, and whether theextracts obtained were free of intact tuberclebacilli.

MATERIALS AND METHODS

Tubercle bacilli. These were grown on a mediumrecommended by Dr. G. Middlebrook, with thefollowing composition: KH2P04, 1 g; Na2HP04-12H20, 6.3 g; NH4Cl, 0.6 g; Na3 citrate.2H20,

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0.1 g; FeSO4 7H20, 0.01 g; MgSO4 7H20, 0.05 g;CaCl2.2H20, 0.005 g; ZnSO4.7H20, 0.005 g;CuSO4*5H20, 0.005 g; Na lactate, 0.1 g; Napyruvate, 0.1 g; biotin, 0.1 mg; pyridoxine .HCl,1 mg; water, 1,000 ml; pH 6.6 to 6.7.Ammonium nitrate containing 60 atom per

cent excess N"1H4 concentration was obtainedfrom the Eastman Kodak Company and con-verted to ammonium chloride. For better growth,small amounts of mycobactin and of amino acidswere added to the culture medium. Crude myco-bactin was extracted as recommended by Franciset al. (1953) from Mycobacteruim phlei grown on asimilar medium with the ammonium chloridecontaining 30 atom per cent excess N'5. Labeledamino acids were secured by the procedure ofGreen and Anker (1954) from the fresh wateralga Scenedesmus B-3 grown in the presence ofN1"H4Cl.The strain BCG (A.T.C. 8420) (from Dr. H.

Pope, Duke University) was employed as theattenuated strain of tubercle bacilli. It has beenfound by Bernheim et al. (1953) not to produce ameasurable amount of ammonia on culture. As astrain pathogenic for mice Bovine strain 4228-5(from Dr. W. Steenken, Trudeau Laboratory,Saranac Lake, N. Y.) was selected because itreleased a minimal amount of nitrogen into thesuspending medium. The tubercle bacilli werewashed with saline and suspended in phosphatebuffered saline, pH 7.2. The suspensions wereadjusted, after preliminary estimations of weightper volume in Bauer-Schenck tubes, to give thedesired concentration of bacilli.The mouse was chosen as the experimental

animal. Suitable susceptible and resistant strainswere available, and the mouse's metabolism hasbeen extensively studied by Dewar and Newton(1948). The susceptibility of strain C57 Blackhas been reported by Pierce et al. (1947). Adultmale animals of 20 to 26 g weight were used. Allinoculations were made intraperitoneally withsuspensions containing 10 mg of bacilli per ml ofphosphate buffered saline.The animal under study was kept in an all-

glass metabolism cage, as described by Roth et al.(1948). Air introduced into the cage was ledthrough diluted sulfuric acid to remove ammonia,and dried. Ammonia of the air leaving the cagewas trapped in 1 N sulfuric acid. Mice were fedfox pellets (Purina), and water ad lib.

After death or sacrifice, the animal was dis-

sected and the organs weighed. When the organwas fractionated, a large aliquot was ground,after addition of buffered 0.14 M saline, for 3 minunder cooling in a micro attachment to a Waringblendor. The saline extract was centrifuged at10,000 rpm to remove tubercle bacilli possiblypresent therein. The proteins were precipitatedwith 5 per cent trichloracetic acid. The super-natant represented the nonprotein nitrogen(NPN) fraction. The nucleoproteins were ex-tracted from the residue of the ground tissuewith 1 M saline, the extract centrifuged as above,and then treated with hot 5 per cent trichloraceticacid to obtain the nucleic acid fraction, as recom-mended by Schneider (1945). Both sedimentsrepresented the "soluble proteins." Extracts pre-pared in this manner from mice inoculated withnonlabeled human tubercle bacilli were injectedinto guinea pigs to test for the presence of tuberelebacilli. None of the guinea pigs developed tuber-culosis, indicating that the NPN, soluble protein,and nucleic acid fractions (after centrifugation)did not contain any viable tubercle bacilli. Ali-quots of the sedimented "soluble proteins" weretransferred to slides and searched carefully foracid-fast organisms after staining by the Ziehl-Neelsen technique. None was observed.When mice were sacrificed, heparinized blood

was collected and also separated into protein andNPN fractions. Extraction of proteins from skinis notoriously difficult and was attempted by theabove method only on mouse No. 13. Advantagewas taken of the procedure described recently byUngar and Damgaard (1954) to obtain a betteryield of soluble material.

Urine was sedimented at 10,000 rpm to removeany tubercle bacilli possibly excreted and thesupernatant was digested.

Tuberele bacilli ground in the micro Waringblendor for 3 min did not give off nitrogenousproducts into the suspending saline.

Extracts and aliquots were digested with con-centrated sulfuric acid with Na2SeO4-CuS04 ascatalyst. Saline, sulfuric acid, and catalyst keptfor similar periods in the digestion rack served asblanks. Nitrogen was determined by the Kirk-Pregl micro-Kjeldahl method (Kirk, 1936). N"determinations were made on the nitrogen gasobtained by alkaline hypobromide oxidation ofthe ammonium ion from the micro-Kjeldahlprocedure. The instrument used was a consoli-dated Nier 21-201 mass spectrometer. Values

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above 0.010 atom per cent excess N'5 were con-sidered significant for quantitative evaluation.Atom per cent excess figures are referred to reagentammonium chloride and not to tank nitrogen(Abbott et al., 1953).

CONTROL EXPERIMENTS

Estimation offree amino acids within the cellularenvironment. The procedure was similar to theone employed by Gale (1947). Speck's (1949)method was used for a-amino acid nitrogendetermination. In this procedure the bacilli, afterremoval of amino acids adsorbed on the cell wall,are either boiled in water for 1 hr or shaken in1 per cent phenol. The former method was usedand the a-amino nitrogen in the suspendingmedium measured.Mutual effect of labeled tubercle bacilli and mouse

plasma. Under sterile conditions 1 ml mouseplasma was incubated with 0.5 mg labeled washedtubercle bacilli for 4 days, with slight, constantshaking. The following variants were set up: (1)plasma with live tubercle bacilli; (2) plasma withheat-killed tubercle bacilli; (3) suspension ofheat-killed tubercle bacilli plus saline; (4) suspen-sion of live tubercle bacilli plus saline. Bacterio-logical controls were done at the termination ofthe experiment and contaminated reaction mix-tures were discarded.

TABLE 1Distribution and excretion of bacillary nitrogen

by C57 mouse (mouse No. 13) inoculatedwith 20 mg live BCG

Specimen

Blood ...........Urine............Air outlet.......Lungs ...........Heart...........Spleen ...........Liver............Kidneys and

adrenals .....Stomach and in-

testines ......Skin.............Carcass (without

head) ........Feces ............

Total N ExN5ss

mg6.1

32.23.294.74.13.129.0

10.9

52.3188

21045.6

atom %

0.1900.0790.0850.0340.0750.1660.085

0.038

0.3120.033

0.0980.062

Amount ofN frombacilli

'Ag28*58.46.483.77.111.956.7

9.5

376143

47565.6

BacillaryN

Appearingin Fraction

2.34.800.530.300.700.984.6

0.78

3112

395.4

Total 1,241.38 100.32

Inoculated TBbacilli ....... 1.152 43.2

* See text.

RESULTS

The first experiment was set up to determinethe recoverability of N"5 and to obtain prelimi-nary data on the extent of labeling of host tissuesand excretory products. In this instance, theorgans of the mouse were digested in toto withoutfractionation.

Table 1 reports the data on a C57 mouse whichdied on the fourth day after inoculation with 20mg live BCG 8420. The recovery of bacillarynitrogen was complete. The 28 ,ug of bacillarynitrogen found in the blood represent only a frac-tion of the total, since perfusion of organs was

not carried out. If the total amount of blood and

bacillary nitrogen present therein are calculatedon the basis of the data published by Friedman

(1955), 210 Mug of bacillary nitrogen were presentin the blood.For the purpose of gaining a clue as to the rate of

breakdown of the tubercle bacilli, two C57 micewere inoculated, each with 20 mg heat-killed

BCG. One (No. 16) was sacrificed on the fourth,

the other (No. 17) on the fortieth day. Table 2gives the results. Of the bacillary nitrogen, 241 ,ugor 20.9 per cent were excreted in the urine within4 days, and 38.4 per cent in 40 days. Noteworthyalso are the relatively large amounts of N"present in liver and gastrointestinal tract at 4days when compared with the small amountspresent at 40 days. Furthermore, small butdefinite amounts of excess N" had entered theprotein and nucleic acid fractions of the variousorgans, and the blood proteins.Next we undertook a comparison of the break-

down of attenuated (BCG) bacilli with virulentbovine bacilli in the normal susceptible mouse.Each animal received 10 mg of living organisms.The data are shown in table 3. More than 15 percent of the injected nitrogen was found in theurine of mouse No. 20, which had received thebovine bacilli. This indicates a remarkable break-down of virulent bacilli by a normal susceptiblemouse in the short period of 4 days. Small butdefinite amounts of excess N" could be demon-

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TABLE 2In vivo removal and incorporation of bacillary nitrogen by C57 mice inoculated with 20 mg heat-killed BCG

Mouse No. 16; 4 Days Mouse No. 17; 40 Days

Specimen AmutBacillary Amut BacillaryTtlN Excess fmonf N appear- TtlN Excess

AonpereTotal N N of N from ing in Total N NS of N from N appear-

bacilli fraction bacilli fraction

mg atom % pg % ng atom% pg S

BloodSoluble protein .......... 20.0 0.036 16.58 1.44 7.0 0.044 7.08 0.61NPN............... 0.33 0.101 0.76 0.06 0.14 0.034 0.012 0.01

Urine .................... 66.7 0.157 241.0 20.9 833 0.023 442.0 38.37Air outlet .................. 0.38 0.185 1.62 0.14 22.6 0.031 16.1 1.40Lungs .................... 4.74 0.136 14.81 1.28 4.04 0.011 1.02 0.09Heart .................... 5.44 0.022 2.76 0.24 2.86 Trace Trace TraceSpleen .................... 5.57 0.152 19.4 1.69 4.38 0.026 2.53 0.22LiverTotal N ................ 46.0 0.154 163 14.17 20.6 0.054 25.6 2.22Soluble protein ........... 11.9 0.043 11.65 1.01 5.9 0.031 4.28 0.37NPN .................... 2.5 0.06 3.45 0.31 1.05 0.011 0.28 0.02Nucleic acid . ........... 1.77 Lost 0.71 0.168 2.76 0.24

Kidneys and adrenalsTotalN ..... 13.4 0.064 19.73 1.71 6.90 0.051 8.06 0.70Soluble protein .......... 3.59 0.065 4.26 0.37 2.38 0.045 2.49 0.22NPN........... 1.44 0.086 2.85 0.25 0.77 0.019 0.35 0.03Nucleic acid.........;. 0.074 0.59 0.99 0.09 0.18 0.239 0.99 0.09

Stomach and intestinesTotal N ................. 44.1 0.179 182 15.9 44.60 0.015 15.4 1.34Soluble protein .......... 5.1 0.064 7.5 0.65 5.51 0.060 7.62 0.66NPN.. 17.9 0.058 23.9 2.08 10.50 0.014 3.38 0.29Nucleic acid ............. 4.95 0.019 2.18 0.19 6.26 0.068 9.80 0.85

SkinTotalN ................. 197 0.016 72.5 6.29 194.5 0.014 62.6 4.17Soluble protein ......... * * * * 1.73 0.012 0.48 0.04NPN ...... ...... * * * * 22.7 0.020 11.55 1.01Nucleic acid ............. * * * * 0.72 0.019 0.32 0.03

Inoculated TB bacilli...... 1.152 43.2

* Not done.

strated in the soluble proteins of liver, kidney,and gastrointestinal tract, while the amount ofNPN N15 was greatest in the skin. It is also note-worthy that even the nucleic acid fractions con-tained a small amount of excess N15. Mouse No.23, which had received live BCG, unfortunatelydeveloped anuria soon after inoculation, probablyas a consequence of the very severe peritonitis.For this reason, a meaningful urinary excretionfigure could not be obtained. However, substan-tial amounts of nitrogen derived from bacilli werefound in the soluble proteins of liver, kidney, and,particularly, the gastrointestinal tract. Again,the NPN fraction of the skin contained a higheramount of N15 than did the corresponding frac-tions of other organs.

Results of control experiments. After 1 hr ofboiling in water, BCG 8420 revealed 26.58 ,ug offree a-amino nitrogen and bovine TB 4228-3,25.45 ,ug per 10 mg bacilli.The reaction mixtures where tubercle bacilli

and mouse plasma were incubated (table 4) con-tained appreciable quantities of N'5, suggesting asignificant cleavage of bacillary components byplasma enzymes. The bacillary metabolism evi-dently had little effect on this process, as indica-ted by the close correspondefice of the N15 dataobtained with heat-killed and live TB bacilli.

DISCUSSION

The significance of the results depends uponthe presumption that all or the major part of

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TABLE 3In vivo removal and incorporation of bacillary nitrogen by C57 mice inoculated with live tubercle bacilli

Mouse No. 20; 10 mg Bovine TB; 4 Days Mouse No. 23; 10 mg BCG; 4 Days

Specimen Amount Bacillary Amount BacillaryTotal N Excess of N N appear- Total N Excess of N N appear-TotalNaNilfrom ing in biN5from ing in

bacilli fraction bacilli fraction

mg atomn% g % mg atom % pg %Blood

Soluble protein ............ 15.90 0.009 Trace Trace 16.0 0.022 8.26 1.43NPN ..................... 0.35 0.018 0.14 0.02 .55 Trace Trace Trace

Urine ............. 129.10 0.030 90.0 15.62 1.12 0.0375 0.97 0.17

Air outlet 0.22 Trace Trace Trace 0.45 Trace Trace Trace

Lungs ...................... 4.95 0.022 2.51 0.44 1.44 0.023 0.77 0.13Heart ...................... 4.49 0.0176 1.82 0.32 4.19 0.021 1.98 0.34Spleen 3.51 0.120 9.75 1.69 2.23 0.254 13.02 2.26

LiverTotal N ................. 8.7 0.034 14.63 2.54 27.40 0.032 19.99 3.47

Soluble protein ............ 13.66 0.0165 5.18 0.90 11.70 0.039 10.55 1.83NPN ..................... 1.81 0.016 0.67 0.12 2.16 0.055 2.71 -0.47Nucleic acid ............... 1.59 0.014 0.51 0.09 1.42 0.050 1.63 0.28

Kidneys and adrenalsTotal N ................... 6.22 Lost Lost Lost 13.9 0.026 8.42 1.46Soluble protein ............ 2.29 0.0754 3.98 0.69 2.91 0.040 2.67 0.46NPN 0.31 0.173 1.22 0.21 1.0 0.046 1.06 0.18

Nucleic acid ............... 0.20 0.109 0.51 0.09 0.30 Trace Trace TraceStomach and intestinesTotal N ................... 50.4 0.025 28.98 5.03 55.26 0.032 40.90 7.10Soluble protein ............ 6.07 0.0234 3.27 0.57 12.60 0.065 18.75 3.26NPN ..................... 1.55 0.022 0.78 0.14 7.56 0.072 12.47 2.16

Nucleic acid ............... 7.44 0.024 4.12 0.72 4.56 0.087 9.15 1.59SkinTotal N ................... 219.1 0.028 141.04 24.48 197.6 0.013 59.09 10.26Soluble protein ............ 4.13 0.0196 1.86 0.32 3.03 0.050 3.50 0.61NPN ..................... 10.30 0.027 6.39 1.11 25.7 0.065 38.43 6.67

Nucleic acid ............... 0.266 0.028 0.16 0.03 1.81 Lost Lost LostInoculated TB bacilli....... 576 43.2*

* Estimated.

TABLE 4Mutual effect of mouse plasma and TB Bacillus; Ag of bacillary nitrogen appearing in fractions of 1 ml

mouse plasma after 96 hr incubation at 37 C

Bovine TB 4228 BCG 8420

Bacillary BacillaryNPN Protein Total Nappn NPN Protein Total

fraction fraction fraction fractionapernin plasma in plasmafractions fractions

Normal plasma + heat-killedTB ....................... 0.20 2.92 3.13 4.7 2.76 2.99 5.75 8.7

Normal plasma + live TB.. 0.219 3.89 4.12 6.2 2.05 3.52 5.57 8.3Buffered saline + heat-killedTB ..................... 0.432 0.184 0.437 0.66 0 0 0 0

Buffered saline + live TB. 0.55 0.016 0.565 0.85 0.41 0.05 0.46 0.69

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excess N15 found in body fluids and organ ex-tracts results from breakdown of the nitrogenouspart of the tubercle bacillus and not from leakingfiom, or presence of, whole bacilli. The results ofthe control experiments are, therefore, discussedfirst.The possibility of nitrogenous material being

removed from presumably intact tubercle bacilliwas investigated, first by estimating the totalamount of free amino acids within the cellularenvironment (Gale, 1947) that could be availablefor transfer, in the absence of enzymatic break-down of bacillary proteins. The presence of suchamino acids in tubercle bacilli has been reported,on the basis of qualitative determinations, byPauletta and Defranceschi (1952). The quantityof free amino nitrogen found in these experimentsmust be considered, however, in light of therigorous extraction procedure to which the bacilliwere subjected. Even if it were available fortransamination in its entirety in the instancewhere heat-killed bacilli were inoculated, theamount of bacillary nitrogen split from the micro-organisms was 10 times greater (table 2) than thenitrogen possibly contributed by free amino acids.Although it could be expected that the micro-

organisms would exhibit an exchange of theircellular nitrogen for that of the medium in whichthey are living, this exchange appears to be slowcompared with the destruction of cells by thehost. This is evidenced both by the close corre-spondence of data obtained with heat-killed andlive tuberele bacilli when incubated with plasma(table 4) and the comparatively rapid appearanceof bacillary nitrogen in various nitrogenous frac-tions of the living mouse.The possibility that the excess N15 found in

the organ extracts may be due to the presence ofintact bacilli, or to their disruption by the ex-traction procedure, was eliminated by the controlprocedures outlined under "Methods."

It is realized that not all nitrogen removed fromtubercle bacilli represents degradation of protein.Small amounts of nitrogen are present in phos-phatides and polysaccharides. Calculated on thebasis of Anderson's (1943) data these would be2.3 and 1.6 ,ug of nitrogen for the phosphatide ofthe human and bovine strains of tubercle bacilli,respectively, and 8.5 and 1.4 ,ug for the corre-sponding polysaccharides of the waxes, per 10 mgbacilli. Nitrogen derived from these two com-ponents, however, also would riepiresent break-down products.

The degradation of the proteinaceous compo-nent of the heat-killed bacilli proceeded rapidly;within 4 days mouse No. 16 (table 2) had re-moved approximately 321.7 ,g, or approximately28 per cent, an(l within 20 days animal No. 17removed 508.4 ,ug or 44 per cent of the bacillarynitrogen. These figures are given without con-sideration that they represent a minimum amountof nitrogen split off and probably should be cor-rected upward due to the incomplete elution ofproteins from the organs. There were most likelysubstantial amounts present in the skin of mouseNo. 16, as may be concluded from the figures forthe other mice, the corresponding data for mouseNo. 16 not being available. The prompt incorpo-ration of the isotope into the nucleic acid fractionis remarkable. Whether incorporation of thebacillary nitrogen took place in the form ofsimple or complex molecules is unknown. Thepossible significance of this nitrogen uptake forantibody formation remains a matter of con-jecture, but the fact deserves emphasis that after40 days the per cent of bacillary nitrogen wasdepleted in almost all fractions except the nucleicacid fraction, where it either remained the sameor increased. The slow uptake and retention ofradioisotopes by deoxyribonucleic acid has beenrepoited by Griffin et al. (1952).

Excretion of another isotope, p32, by guineapigs after inoculation of labeled tubercle bacillihas been reported by Strom and Rudback (1949).Urinary excretion of p32 was about 0.5 per centof the total inoculum during 54 hr in normalanimals and 3.1 per cent during 24 hr in immu-nized guinea pigs. The small amount of phos-phorus split off is surprising in view of the dem-onstrated ease by which it is removed fromtubercle phosphatide by enzymatic action invitro (Gerstl and Tennant, 1941). Strom andRudback's observations may be explained byassuming incorporation of the bacillary phos-phorus into the host's tissue, or difference inenzyme kinetics between mouse and guinea pigs,as reported earlier (Gerstl and Tennant, 1942).The quest for evidence of cleavage of nitroge-

nous material from live pathogenic tubercle bacilliby a susceptible host was the most interestingaspect of these studies. The data in table 3(mouse No. 23) reveal that more than 118 ug (or20 per cent) of the bacillary nitrogenous materialhad been removed from the injected live patho-genic bacilli within 4 days, and this, notwith-

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standing- the overwhelming peritonitis caused bythe inoculation of an enormous dose of bacilli.

Although the number of animals employed inthis study was small, the complexities of obtain-ing information on host resistance by the bio-chemical approach could be resolved. Formidableforces of the host in its fight against the invaderwere revealed that may well constitute an im-portant part of its resistance.

SUMMARY

Studies on the removal of nitrogen from viru-lent, avirulent, and attenuated strains of tuberclebacilli by the host (mouse) are reported.The rapid and substantial cleavage of bacillary

nitrogen and its urinary excretion indicate activedestruction of bacillary cells by the host. Controlexperiments revealing a slow rate of nitrogenseparation by plasma and a minimal amount ofnitrogen leaching from bacilli suspended in inor-ganic media support this interpretation.

Bacillary nitrogen appearing in the nucleic acidfractions of the mouse is considerable and, onceincorporated, is more stable than that in theprotein fractions.

Small amounts of free amino acids within thecellular environment of tubercle bacilli could bedemonstrated.

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