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Reversible and irreversible adsorption of T1 phage to water-insoluble polar lipids
VIROLOGY
9,
151-167 (1959)
Reversible
and Irreversible to Water-insoluble
Adsorption of Tl Phage Polar Lipids’
LE:O ZELKOWITZ~ AKD HANS ?JOLL" Department
of Microbiology, School of Medicine, Pittsburgh, Pennsylvania
University
of Pittsburgh,
Accepted June 19, 1959 The intera&ions of bacteriophage Tl with water-insolube polar lipids were studied, using the previously developed methods of water-lipid partitioning. The results of experiments on the adsorption kinetics, on the factors determining adsorption efficiency, and on the reversibility of the virus-lipid bond closely parallel the findings reported for lipophilic animal viruses. Phage adsorbed to cholesterol or stearic acid dissociates readily upon dilution during plating, as indicated by the quantitative recovery of infectivity. whereas adsorption of Tl to hexadecylamine causes irreversible inactivation. Adsorption to a cholesterol column followed by ether-w-ater partit,ioning proved to be a convenient met,hod for t,hc rapid removal of the major portion of nonviral protein. Passage of 10 ml of high titer anti-T1 rabbit serum t,hrough a cholcstcrol column saturat,ed with phage rcsultcd in t,he adsorption of 88 ‘yO of specific antibody. INTRODUCTION
Preliminary experiments, reported previously (Youngner and ISoll, 1958), indicated that bacteriophages Tl and T2, like lipophilic animal viruses, had a high adsorption affinity for cholesterol columns. In subsequent adsorption experiments with bacteriophage Tl, however, considerable variations in adsorption efficiencies were observed. A systematic study of the variables influencing the adsorption efficiency was, therefore, undertaken. The experiments reported in this paper show 1 This investigation was carried on Influenza of the Armed Forces the Office of the Surgeon General, 2 Aided by rt Student Fellowship mittee. 3 Recipient of Senior Research Public Health Service.
out under the sponsorship of the Commission Epidemiological Board, and was supported by Department of the Army. from the Tobacco lndustry Research ComFellowship 151
No. SF-274 from the United
States
152
ZELKOWITZ
ANI)
KOLL
t,hat, under properly controlled conditions, highly reproducible results are obt,ained. The data on the kinetics of the interaction of the bacterial virus Tl wit,h various lipids closely parallel the findings obtained with lipophilic animal viruses. This further strengthens our previous conclusions concerning the mechanism of these interactions. Moreover, compared wit,h many animal viruses, t’he coliphages were found to be better suited for such studies, because of the more sensit’ivc and precise methods available for t,heir assay. MATERIALS
AiXl>
METHODS
coli was grown in nutriPreparation oj phage. Strain R of Escherichia ent broth (7.5 mg/ml) to a concentrat,ion of approximately lo9 cells per millilit’ers. Aft.er infection with about 10’” plaque-forming units (PFU) of the Tl strain, incubat8ion was continued uruil clear lysates containing between 10’” and 10” l’I’U/ml were obtained. The crude lysat’es were centrifuged at 3000 rpm for 30 minutes in a refrigerated cent’rifuge, the supernatant collected and stored at 4” until used. Assag of phagr was carried out by the agar layer method as described by Adams (1950). All samples were plat,ed in duplicat,e over at least a hundredfold concentration range t,o producr: an optimal number of countable plaques. Spccijic anfi-7’1 w-urn was prepared by repeated injection of high t,it,er lysatcs int,o five rabhit,s. The pooled serum was stored at - 10” and yielded a K-value of 100. Protein was determined according to Lowry et al. (1951). Absorption of phage to lipids was carried out’ at, room temperature by c&her the column or batch method following the techniques described in detail in the preceding paper (Sol1 and Youngner, 1959).
Crude lysates of coliphage Tl were passed through 2-g cholesterol columns. The number of phage particles adsorbed to the column was obtained (I) by determining the difference between the totma number of PPU in the influent volume and the collected effluent fractions and (2) by plating dilutions of the resuspended cholesterol. All columns were washed with at’ least 30 ml of phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954). A number of typical experiments are summarized in Table 1, which
ADSORPTlON
ADSORPTION OF Tl
Column
1 2 3 4 5
OF
Total
-
9 1.0 2.76 5 2
9 9.5 1.14 1.5 2.0
(LComparison of adsorption ference and by direct plating b Plaque-forming units.
PFU on column
By difference
109 10’0 IO’O 10’0 10’2
153
PHAGE
TABLE 1 TO 2.~ CHOLESTEROL COLTJMNP
Total PFU* input
x x X x x
Tl
x 109 x 109 x 10’0 x 10’0 x 10”
data obtained of resuspended
By direct plating 9 9.5 1.06 1.5 1.8 by determining lipid.
x x X x x
I>eviation (76)
109 10” 10’” 10’” 10” input -output
0 0 8 0 10 dif-
shows that the phage removed from the influent is quantitatively rccovered from the resuspended adsorbent. In most cases the suspension method of assay was preferred because of its greater accuracy and ximplicity. Eficiency
of Column Operation
As a result of numerous adsorption experiments with uniform 2-g cholesterol columns and varying quantities and concentrations of phage, it was found that the adsorption efficiency, i.e., t’he percentage of totma phage input adsorbed, was determined mainly by t,he adsorption capacity of the column and the rate of equilibration. This is illustrated in Fig. 1, which shows that phage was adsorbed wit,h 95-100 ‘3%efficiency as long as the input did not exceed a total of about 1 X 10’” part’iclcs. Further increase of the input resulted in rapid saturat,ion of t,he columns, as indicated by the sharp decrease in adsorption efficiency. Thus, loadings of 2 X 10” PFU/2 g cholesterol, e.g., could only be achieved at the expense of a 90 % loss of phage in the effluents. The quantity of phage adsorbable to cholesterol columns was also found to be limited by the practical difficulties encountered in passing large fluid volumes through the columns. After collecting about 300 ml, t’he flow rates became impractically slow, because of the packing effect produced by the pressure applied to the top of the column. Since the influent concentrations in the experiments represented in Fig. 1 varied, the adsorption efficiencies were plotted as a function of the
154
AK;D NOLL
ZELKOWITZ
TOTAL l?F.U. PASSED THROUGH COLUMN
FIG. 1. Adsorption efficiency as a function passed through 2-g cholesterol column. TABLF: INFLUENCE
C0hln
1
2 3 4 5
of total
number
of Tl
particles
2
OF PHAGE CONCENTRATIOX ON EFFICIEKCY OF ADSORPTION TO 2-c: CHOLESTEROL COLLJMNS USING COXSTANT PHACE INPIJT Influent cont. (PFU/ml)
3 9 1.5 4.3 1
x x x X x
10’ 107 108 lo8 10”
Volumeof phage suspension passed through column (ml)
300 100 70 25 10
Total PFU input
9 9 1.05 1.07 1
x 109 x 109 x 10’0 x 10’0 x 101”
~-____
Total PFU
Adsorbed
9 9 9 8.5 9.5
x x x x x
Per cent
109 109 109 109 109
100 100 89 83 95
tot’s1 number of particles passed through the columns. This procedure seems to be justified by the finding, illustrated in Table 2, that the adsorption efficiency was independent, of the concentration of the phage suspensions passed through t’he column over the thirtyfold range tested. Faster flow rates obtained at higher pressures, however, reduced the adsorption efficiency considerably by prevent,ing efficient equilibration, as shown in Table 3. Kinetics
of Column
Saturation
For a furt(her characterization of the adsorption process, the kinetics of column saturat’ion were determined as described by Youngner and
ADSORPTION
Tl
OF
TABLE
PHAGE
155
3
INFLUENCE OF FLOW RATE ON ADSORVTIOX EFFICIENCY OF Tl CHOLESTEROL COLUMN Column 1 2
Flow rate (ml/min)
Total PFU input
0.5 5.0
5 x 5 x
Total PFU adsorbed
10’0 10’0
1.5 x 7.3 x
204oBoBo
IO0
. ......
1 I20
EFFLUENT
FIG. 2. Adsorption lesterol column.
isoplane
describing
I I40 VOLUME
I IS0
4
I IBO
TO
2-c
% Adsorbed
lOLO 108
WASHING w.7
0
PHAGE
30 1.2
BEGUN \
I 200
r 220
I 240
(ml.)
the interaction
of phage Tl
with
cho-
No11 (1958). The results represented in Fig. 2 closely paralleled those obtained with influenza virus. A 190-ml aliquot of a diluted crude lysate containing 1.5 X lo8 PFU per milliliter were passed through a 2-g cholesterol column under standard conditions. The column was then washed with 50 ml of PBS. A total of 121 effluent fractions, ranging from 0.4 to 10 ml were collected and their phage concentrations determined. The break-through curve in Fig. 2 (effluent cont. X lOO/ in fl uent cont., plotted against volume passed through column) shows that saturation is reached rapidly between 80 and 100 ml. It is interesting that the slope increases continuously,
156
ZELKOX-ITZ
AND
I\‘OLL
m . . .
\
mr
.
, IO
I 20
! 30
7 40
r 50
FIG. 3. Kiudics of s:it.uration of rlution of ph:agc recoverable
3 60
+J
N 80 90 IdO I@0 I90 EFFLUENT VOLUME (ml.)
of cholesterol hy washing.
260
2Kl
2bO
Zjo
.
240
column wit,h phagr Tl and kinetics
giving rise t,o a diAnctly asymmetrical break-t,hrough curve. This asymmetry is reflected most st’rikingly in the observsCon that’ t,he effluent, concentration rises abruptly from 07 to 100 ‘1, of the influent concent~ration wit,hin a Z-ml increment of effluent collected. This asymmetry holds true, even if a more gradual change is assumed (Fig. 2 : broken line), since the accuracy of t,he assay met,hod was greater t’han It10 %. A semilogarithmio plot of the break-t,hrough curve (Fig. 3) shows t’hat leakage was very small (less t,hun 0.001 ‘3) during the collection of the first 35 ml of effluent. It rose rapidly during t’he next, 20 ml, reaching 1 !% in the fifty-second milliliter of effluent. After washing was st,arted, the phage concentration in the effluent declined exponent8ially, as evident from t,he straight, line in t,he semilog plot of I;ig. 3. This indicates t,hat, a portion of the virus is less firmly associated with t,he lipid and recoverable by washing, an observation which is similar to the one described for influenza virus (Youngner and X011, 1958). This decrease in virus concent,rat,ion follows the equation :
ADSORPTION
OF
TABLE IIISTRIBUTI~N
0~
PHAGE"
OS
Tl 4 2-c
CHOLESTEROL
Column Section
I. Top 6 mm II. Middle 6 mm III. Bottom 6 mm I f
ing
II + III
Bcforc washing 59.1 21.6 19.3 __ lO@Z
u Percentage values are based on total = 1.29 X 1O’O PFU = 100 %.
157
PHAGIC
CULLJMS
,4
Washed out
After washing
Column B after washing
0.1 3.2 9.6
59.0 18.4 9.7
58.5 20.1 9.6
12.9
87.1
88.2
phage adsorbed
to column
before
wnsh-
where v = effluent volume (in milliliters) ; o. = effluent volume at beginning of washing; c = phage concentration (I’FU/ml) corresponding t’o v - VO;CO= initial concentration of phage recoverable by washing (at v = vu). The constant lc is det’ermined from Fig. 3, using t’he pararneters corresponding to half-concentration:
The total amount of phage recoverable by washing ((I,) was calculated by integration of Eliy. (1) between v. (= 191 ml) and u (= 250 ml), giving C, = 1.5 X log PFU/2 g of cholesterol. Det’ermination of total phage remaining on the washed column gave 1.06 X 10’” PFU by direct plating, 1.14 X 10LC’l’E’1!/2 g cholesterol by input-output difference. In order to determine the distribut,ion of the firmly adsorbed phagc on the washed column, the adsorbent was divided into three equal sections of 6 mm and each section assayed. The distribution, shown in Table 4 (column B), closely resembles the concentration gradient found with influenza virus on a cholesterol column (Toungner and X011, 1958). In order to compare the concentration gradient of firmly adsorbed phage with the distribution of phage recoverable by washing, a second cholesterol column was saturated with phage and sectioned without washing. The cholesterol corresponding to each section was resuspended in 10 ml of PBS and O.l-ml aliyuok were withdrawn for assay by direct’ plating. The suspensions were then filtered wit’h What’man Ko. 11H
158
ZELKOWITZ
.&XI)
NOLL
paper and each cholesterol portion was washed with another 10 ml of PBS. Both the filtrates and the remaining cholesterol were again assayed for phage. The results in Table 4 (column A) clearly show an inverse relationship bet,ween the concentration gradients of elutable and firmly adsorbed phage, i.e., the former increasing from top to bottom by a factor of 100, the latter decreasing by a factor of 3. It can also be seen that identical concentrat,ion gradients of firmly bound phage were obt,ained regardless of whether washing was carried out’ before or aft#er sectioning (column B). Although a yuant.itativc kinetic analysis and interpretation of these concentration gradients is complex and beyond the scope of this study, the results clearly rule out the possibilit#y that the observed effects were due to filt,ration. In a qualitative way, however, it seems plausible that, in a statistmica dist,ribution, the less firmly bound phage part,icles would tend to become concentrated toward the bottom of t#hecolumn. Praparation
of
Purijicd
Phagc
bq Bth,cr-Water
Partitioning
OJ Lipid-
A study of t,he conditjionn which would permit the dissociation of the phage from the lipid was of imerest because of the information that would be gained about, t,hc mechanism of adsorption and also because of the possible usefuhiess of t,his method for phage purificat~ion. As wit,h the other viruses tested (l-oungner and n-011, 1958), washing of t,he columns with solutions of \-arying ionic strength failed to elute the firmly adsorbed particles. K&her did washing with undiluted normal rabbit serum cause any elut,ion. On the other hand, t#he previously described met,hod of ether-water partitioning proved to be an efficient method for the recovery of infectious phage. Two grams of cholesterol from a column containing 1.5 X 10’” 1’l’U was suspended into a volume of 25 ml with PBS. The cholesterol was extracted with three 50-ml portions of ether at 4”. The insoluble material accumulat,ing at, the interphase was left with the aqueous portion during the first t,wo extractions and discarded after the third. Ktrogen was then bubbled for 3 minutes through the turbid solution at room temperature to remove residual ether. This treatment gave a clear solution containing a flocculent precipitate which was removed by gravity filtration through Whatman Ko. 41H filter paper. The resulting phage suspension was clear and colorless and comained 1.35 X 1O’OPI’U, or 90 % of the original infectivity. The protein concen-
ADSORPTION
OF
Tl
PHAGE
159
tration of this solution was too low to be determined by the Lowry method, i.e., less than approximately 10 pg/ml as compared with 7.5 mg/ml before adsorption, indicating that a considerable purification can be achieved by this method. In contrast to the lipophilic animal viruses studied previously, the adsorption to cholesterol and subsequent separation with ether seems to offer a generally useful method for the concentration and purification of phage, because of the remarkable resistance of phage particles to inactivation by organic solvents. InJluence of Purification Cholesterol
and Ether Treatment on Adsorption of Phage to
In view of the possibility that the adsorption of phage to cholesterol was mediated by lipophilic substances present in lysates, it seemed interesting to determine the adsorbability of phage which had been purified by the method of adsorption and water-ether partitioning described in the previous section. When such a purified phage solution containing 9 X log PFU was passed through a 2-g cholesterol column under standard conditions, 99 % of the phage was adsorbed to the lipid. The unchanged adsorption characteristics of this highly purified phage preparation indicates that the adsorption affinity for lipid is due to ether-resistant lipophilic components of the phage surface. Adsorption of Tl Phage to Lipids Other Than Cholestero(l In a previous paper (No11 and Younger, 1959), it has been shown that a great variety of water-insoluble polar lipids are capable of adsorbing lipophilic viruses. Saturation studies with influenza virus and the lipids, cholesterol, palmitic acid, and hexadecylamine (HDA) showed considerable variations in adsorption activities and capacities of these different lipids. In order to study the affinity of Tl phage for different lipids, adsorption experiments with stearic acid and HDA were carried out. A 2-g stearic acid column was prepared and lO*OPFU passed through. After washing, the phage both in the effluents and the column was determined. It was found on plating of the resuspended lipid that only 1 % of the phage particles passed through the column were firmly adsorbed to the stearic acid column. This result is in contrast to similar experiments with lipophilic animal viruses which, under comparable conditions, all showed a stronger affinity for long-chain fatty acids than for the neutral lipid cholesterol.
160
ZELKOWITZ
AND
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TABLE15 ADSORPTION EFFICIENCIES AND RECOVERY OF I~VFECTIVITY AND HEUDECYLAMISE RY BATCH STEARIC ACID,
Adsorbent (100 mu)
Cholest,erol
St earic ncitl HI)A
Tota\fzyj
1.75 x 3 x
input
10’” 10”
1.8 x 10’” 3 x 5 x 1.8
x
Total PFU in filtrate
1.75 x 3 x
10’” 109
1.8 x IO’”
10”
3 x
109
10’” 10’0
5 x
10”
3.5 x
10”
WITH CHOLESTEROL, ADSORPTION
Total PFU in resuspended lipid
1X108 1X10’ 3 x lo* 6 x 10’ 1 x 9 x
10” 107
% Adsorbed
99.9 >99.9
y& PFU recovered in filtrate + iTSUSpended lipid
100 100 100 100
Adsorption experiments with HDA were carried out, by the bat’ch method because of t,he previously explained difficulties in preparing columns with this lipid (No11 and Youngner, 1959). A4 total of 100 mg of HDh was mixed wit,h 5 ml of crude lyeat#e by grinding in a mortar, filtered and washed with PBS as described in t’he preceding paper. For wmparison, analogous batch adsorption experiment,s were carried out, with cholesterol and st,earic acid. The result)s of typical experiments, summarized in Table 5, show that’ less than 2 ‘X8 of the phage was adsorbed to cholesterol or stearic acid. This corresponds t#o about, 10 ‘2 of t,hc efficiency obt~ained by column ndsorpt,ion nft,er correcting for the smaller amounts of lipid used in these bat,ch adsorpt,ion experimcnt,s. Similar differences in adsorption efficiencies between ba.tch and column operaCon are commonly observed ill chemisorpt,ion studies wi:ith ion exchangers and other udsorhents. In contrast, to cholest8erol and st,earic acid, which failed t,o cause a dete&blc reduction of 1’1~1:‘s in t,he filtrat,es, the basic lipid hexadecylamine adsorbed close to 100 ‘X of the phage particles under t#he same conditions, as evident from t,he lob-fold reduction of infectivity in the filtrates. Yet, less than 1 % of the phage originally present in the adsorption mixture could be demonstrat,ed by plntCng the resuspended lipid. The loss of infectivity observed with HDX could be due to &her of the following mechanisms: (1) t,he phage combines irreversibly with the lipid particle in a way which makes it,s tail st’ruct’ure inaccessible t)o the receptor areas of the bacterial surface; (‘2) combination of HDX and phage results in secondary structural changes of t,he latter [cl.g., deoxyribonucleic acid (DIY.4) eject,ion] fol-
ADSORPTION
OF
‘r1
PHAGE
161
lowed by dissociation of the inactivated phage; (3) phage is inactivated by a detergent effect of HDA present in filtrable molecular-dispersed form. The last possibility was ruled out by a separat’e experiment. HDA (100 mg) was ground with 5 ml of PBS in a mortar and filtered t,hrough Whatman 41H filter paper, as usual. One milliliter of lysate containing 6 X log PFU was added to the filtrate. After 2 hours of mechanical agitation, the solution was assayed. Phage was recovered quantitatively. The present experiments fail to distinguish between the first t#wo mechanisms. Analogous results obtained with influenza \?rus, however, indicated that inactivation is caused by irreversible adsorption of the virus to HDA, since it was possible to demonstrate t#he presence of adsorbed viral antigen by assay of antibodies produced by guinea pigs which had been immunized w&h the washed influenza virus-HDA suspension (No11 and Toungner, 1959). Conversely, the low adsorpt’ion efficiencies and complete recoveries of infectious phage from resuspended cholesterol and stearic acid are consistent with the assumption of an equilibrium adsorption in the case of these latter lipids. Accordingly, dilution of the virus-lipid complex would cause dissociation of t,he infective particle. The results of infectivity titrations with influenza virus adsorbed to cholesterol and determinations of the particle size of the lipid suspension support this conclusion (No11 and Youngner, 1959). Interaction
of Antibody with Phage Adsorbed to Chobcsterol Column
It has been reported previously (Youngner and Koll, 1958) t’hat the passage of antiserum through a cholesterol column, to which influenza virus had been adsorbed, resulted in the specific removal of homologous antibodies and that this method offered a promising tool for the purification of antibody protein. The following experiments show that the same technique can be applied also to the separat,ion of antibodies against bacteriophage TI . Antibody was assayed according to Hershey (1941), using the equation log p,/p = K tj2.3 D, where ~0 = phage assay at zero time, p = phage assay at time t minutes, D = final dilution of serum in the phage-serum mixture, K = velocity constant. The velocity constant K was determined from the slope of the linear portion of the graph obtained by plotting the inactivation values log pa/p, corresponding to various serum dilutions, against t/2.3 D using a constant inactivaGon time of t = 30 minutes. The rabbit immune serum used in t,hese experiments gave a value of K = 100. The ant,ibody concenkations of effluent fractions
162
ZELKOWITZ
AND
NOLL
collected from the column were assayed with the aid of the standard curve prepared for the determination of K. If necessary, the serum samples were diluted so as to give inactivation values falling within the linear range of the standard curve. All antibody concentrations were expressed as percentages of t’he antibody concentration in the original serum. Two 2-g cholest,erol columns were saturated with phage. After washing with PBS, each column was found to contain 3.2 X 10” Tl particles. Ten milliliters of anti-T1 serum was passed through one column, and the Hame quantity of normal rabbit serum through the other. As a further control, an identical amount’ of immune serum was passed through a t,hird column which contained no phage. The effluents were collected in fractions and assayed for protein and antibody. Figure -l shows the distributions of protein and antibody concentrat#ions in the effluent, fractions obtained by passing antiserum through t#he column saturated with phage (il) and the control column without phage (R). For comparison, both the protein and the antibody concentrations (represented graphically by t’he height of the differently shaded areas over the corresponding effluent fractions) are expressed as percentages of their original influent concentrations. The protein concentrations in the effluent fract#ions of both columns rapidly rose to t’he level of t’he influent, followed by an equally rapid fall wit’h t,he beginning of washing, indicat,ing that most of t,he serum proteins failed to interact with the cholesterol. A st,riking difference, however, was observed in t#he quantities and distribut’ion of antibody collected from the two columns. The first two effluent fractions from the column sat,urated with phage (n), although containing 60 %I of the input serum protein, yielded less t,han 2 %I of tot’al antibody. After collecting 8 ml of effluent, the antibody concentration rose, indicating that the available antigenic surface of t,he phage was approaching saturation. Analysis of the effluent,s from the control column without phage (B), on the other hand, showed that protein and antibody were eluted at the same rate. The recoveries of protein and antibody in the effluent from the two columns (equivalent in Fig. 4 t’o the areas under the broken and solid lines, respectively) are summarized in Table 6. It can be seen that the phage column adsorbed 88 5%of the phage-inactivating antibodies present in 10 ml of serum, whereas the control column removed neither protein nor antibody t#o any measurable ext’ent. Essentially complete recovery of protein was also obtained when normal rabbit, serum (K = 0) was passed through a
ADSORPTION
OF
Tl
163
PHAGE
PROTEIN
ANTIBODY
0 4
Iv----” I L.---J
TOTAL PROTEIN IN EFFLUENTS
m
TOTAL ANTIBODY IN EFFWENTS
io
6 12 I6 20 24 26 32 0 4 6 12 16 EFFLUENT
VOLUME
(ml.1
FIG. 4. Adsorption of antibody to phage-sat,urated cholesterol column. Distribution of protein and neutralizing antibody in effluent fractions during passage of 10 ml of anti-T1 rabbit serum through 2-g cholesterol columns with and without phage. (A): Column saturated with Tl; (B) : Control column without phage. TABLE
6
ADSORPTION OF NORMAL AND ANTI-Tl SERT:M TO CHOLESTEROL COLUMNS WITH AND WITHOUT ADSORBED PHAGE: RECOVERY OF YROTEIN, ANTIBODY, AND INFECTIVITY FROM EFFLUENTS AND COLUMNS K-Value of
serum Serumprotein Antibody Column Total PFU adsorbed t~~~$h recovered in recovered in COllDlo effluents (70) emuents (%) (10 ml) i
3.2 X0 10”
100
>99 >95
11.9 >99
C
3.2 X 10”
0
>99
0
Protein recovered fromcolwnn Total PFU recovered after ether- from resuspended water parcholesterol titioning (w) 5.0 7.2
6 X0 IO8 3.2 X 10”
cholesterol column saturated with phage. Table 6 further shows that the amount of antibody protein present in 10 ml of a high titer anti-T1 phage rabbit serum is too small (less than 5 % or approximately 40 mg) to be measured by differences in protein content between influent and effluent. A direct determination of the protein on the column saturated with phage and the corresponding phageless column was therefore carried out by assaying the aqueous phase obtained after extracting the lipid with ether. Determination by the Lowry method, using bovine albumin as a standard, gave values of 7.2 and 5.0 mg of total protein, respectively. It is obvious from the relatively high-protein blank of the control
164
ZELKOWITZ
AICD
NOLL
column that the prot,ein recovered from the phage-saturated column cannot simply be equated with antibody. In the absence of more experimental determinations, it is also doubtful that the difference between the two values (i.e., 2.2 mg) can serve as a valid basis for an accurate correlation between antibody and protein. Even if this value is taken merely as an indication of the approximate range, it would appear far too high, since it, would follow that each phage particle on the column combined m&h an average of 26,000 antibody molecules. Discounting these prot,ein values as a basis for an estimation of antibody concentration, however, does not invalidate t’he unexpected, though well documented, finding t#hat the relatively small quant’ity of 3.2 X 1OlLphage particles was capable of adsorbing more than 99 % of antibody contained in 8.8 ml of undihued high t’it#er (K = 100) antiserum. This corresponds to a maximal serum-blocking power of 2.75 X 10” ml per Tl particle. In contrast to this, Hershey et al. (1943) found a value of 3.3 X 10-l” ml per T2 particle under conditions of antigen saturation and using a serum of K = 360. The large discrepancy between Hershey’s and our resuhs is difhcult to explain and requires further investigation. The possibility exists t,hat the column process permits more efficient saturat’ion of the antigenic surface of the phage with antibody than do precipit,aCons from free solut,ions. It is of particular interest to not,e that the experiments reported here are concerned with neutralizing antibodies which are presumably directed against t’he phage tail (Lanni and Lanni, 1953). This would support t,he notion that phage is adsorbed to cholesterol by its head, leaving the tail st,ructure freely accessible to antibody. This would be consistent with the finding t,hat, phage adsorbed to cholesterol can be recovered in infectious form. Since inactivation of phage with HDA, on the ot,her hand, suggest’rd ut8tuchmcnt, to the lipid by the tail, it would be intercsting t,o t,est, whether neutralizing amibodies are capable of interacting with phage adsorbed t#oHDA. 1)1SCUSS10N
In a preceding paper (Sol1 and Youngner, 1959), the mechanism of adsorption of lipophilic viruses t,o various lipids and the biological significance of such interactions for the attachment of viruses to cells have been discussed in detail. On t’he basis of their interactions with lipids, viruses were classified as lipophilic or hydrophilic. The hypothesis was proposed that, in the adsorption of the former group of viruses to
ADSORPTION
OF
Tl
PHAGE
165
their host cells, lipid-lipid interactions of the van der Waals’ type may play an important role. The high lipid content of the surfaces of both cells and lipophilic viruses was thought to provide strong support in favor of this concept. The interactions of the bacterial virus Tl with polar lipids described in this paper suggest a similar interpretation. The high lipid content of receptor active compounds, isolated by chemical dissection of cell walls of phage-sensitive E. co& is indicative of a possible participation of lipids in the adsorption of phage to it,s host. Extensive chemical studies have shown that all of these preparations which are capable of irreversibly inactivating phage under physiological conditions in vitro are particulate macromolecules consisting of lipids combined with specific carbohydrates and/or proteins (Weidel, 1958). In the case of T4, the essential function of the lipid moiety for phage inactivation has been clearly demonstrated by Jesaitis and Goebel (1955). These workers isolat’ed sonnei a lipocarbohydrate which specifically from phase II Shigella inactivated T4 by rapid irreversible formation of a noninfect,ious complex, followed by the slower disintegrat,ion of phage and release of DNA. Extraction of lipid from the lipocarbohydrate with 70 % ethauol destroyed receptor act,ivity; it was restored, however, on readdition of the extracted lipid which by it,self was inactive. The carbohydrate moiety was responsible for the specificity whereas t,he lipid part could be replaced by a number of long-chain fat,ty acids (C,,-C,,). Maximal activity of the reconstituted system was dependent, on the ratio of fat’ty acid to ext,racted lipocarbohydrate, indicating format’ion of a st’oichiometric complex between the two components. Although the authors did not comment on this point, it seems likely that the fatt’y acids combined with basic amino groups of t,he carbohydrate moiety which was shown to cont,ain hexosamine. This would be in agreement wit’h the failure of fatty acid esters to restore activity. Comparison of the important observations of Jesaitis and Goebel with our results reveals certain similarities which may be integrated into a hypothesis of the mechanism of adsorption of phages to bacterial cells. It has been found that Tl combines reversibly with neutral and acid lipids (cholesterol, stearic acid), presumably by relatively weak van der Waals’ interactions. However, if the lipid carries a positively charged group (as in the case of hexadecylamine), irreversible binding occurs resulting in loss of infectivity. This may be attributed to reinforcement of the binding by the additional electrostatic attraction between the
166
ZELKOWITZ
AXD
NOLL
basic group of the lipid and negatively charged groups at the tip of the phage tail. Thus, hexadecylamine may be regarded as the simplest chemical model of a cellular receptor, since it combines in one molecule the dual functions characteristic of all natural receptor substances. In the natural receptor material isolated by Jesaitis and Goebel, these two functional entities are represented by extractable lipid and complex carbohydrate, and their functional cooperation is strikingly illust,rated by the loss of receptor activity upon chemical separation. Our model (based on the interaction of Tl with HDA) indicates an irreversible reaction as the primary st’ep in the attachment of phage to host cell and dispenses with the necessity of invoking a reversible initial reaction as has been postulated from the exclusive consideration of ionic interactions (Puck, 1953; Tolmach, 1957). It also becomes unnecessary t’o postulate subsequent enzymat,ic react,ions as a means of achieving irreversible binding. Weidel (1958) has in fact’ shown that bacterial receptor subst’ances combine irreversibly wit,h phage and are distinct from the substrate of t,he phage enzyme which, in addition, lacks type specificity. It should be pointed out in this connection t,hat the failure t,o observe irreversible atjtachment of Tl under conditions which prevent, infection has been used as a strong argument in favor of reversible attachment as an obligatory step in t,he process of infection (Puck et al., 1951). A crit,ical review of the pertinent fact,s and interpret8ations, ou t,he other hand, led Hershey (1957) to conclude t’hat reversible adsorption observed under these conditions may represent, a different, kind of reaction involving recept,ors which have no relation to the infective process and t#hnt, in the case “of t,he interesting kind, namely infection, t,here is little evidence for a charact8eristic reversible st,ep.” He also point,ed out that) “the existing information about, isolated receptor substances comes from experiments using irreversible inactivation of phage as a test for receptor act,ivit,y.” It is of part#icular interest, therefore, t,hat hexadecylamine has been found to be the first receptorlike substance capable of irreversible combinat,ion with Tl . Previous failures to isolate Tl receptor substances from the host’ cells may have been due t,o the disruption of the essential link between the st,ructures carrying the lipophilic and ionizable basic groups. No claim is made that t,he proposed model is able to explain and unify all t,he complex aspect,s of phage attachment. Together wit,h certain new viewpoints it also int’roduces assumptions which will have to be tested by further experiments. Thus, it was postulated that, t,he phage
ADSORPTION
OF
Tl
PHAGE
167
attaches to hexadecylamine by its tail and that the tail contains the required complementary combination of lipid and negatively charged groups. By electronmicroscopy it should be possible to decide the first question but the problem of the lipid content of the phage tail may present difficult analytical problems. Experiments are also planned to find out whether inactivation of Tl by adsorption to hexadecylamine is accompanied by the ejection of DNA. ACKNOWLEDGMENTS The authors areindebt)ed helpful discussions.
to Drs. J. S. Youngner
and M. H. Weiner
for numerous
REFERENCES ADAMS, M. H. (1950). In Methods in Medical Research (J. H. Comroe, Jr., ed.), Vol. 2, pp. l-73. Year Book Publishers, Chicago, Illinois. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines of poliomyelitis viruses. J. Exptl. Med. 99, 167-182. HERSHEY, A. D. (1941). The absolute rate of the phage-antiphage reaction. J. Immunol. 41, 299-319. HERSHEY, A. D. (1957). Bacteriophages as genetic and biochemical systems. Advances in Virus Research 4, 25-61. HERSHEY, A. D.,KALMANSOPI',G., and BRONFENRRENNER, J. (1943).Quantitative relationships in the phage-antiphage reaction: Unity and homogeneity of the reactants. J. Immunol. 46, 281-299. JESAITIS, M. A,, and GOEREL, W. F. (1955). Lysis of T4 phage by the specific lipocarbohydrate of Phase II Shigella sonnei. J. Exptl. Med. 102, 733-752. LANNI, F., and LANNI, Y. T. (1953). Antigenic structure of bacteriophage. Cold Spring Harbor Symposia Quant. Biol. 18, 159-168. LOWRY,~. H., ROSEBROCGH, K. J., FARR, A. L.,and RANDALL, R.J. (1951).Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. ?JOLL, H., and YOCNGNER, J. S. (1959). Virus-lipid interactions. II. The mechanism of adsorption of lipophilic viruses t,o water-insoluble polar lipids. Virology 8, 319-343. PIJCK, T. T. (1953). The first steps of virus invasion. Cold Spring Harbor Symposia Quant. Biol. 18, 149-154. PUCK, T. T., GAREN, A., and CLINE, J. (1951). The mechanism of virus attachment to host cells. I. The role of ions in the primary reaction. J. Exptl. Med. 93,65-88. TOLMACH, L. J. (1957). Attachment and penetration of cells by viruses. Advances in Virus Research 4, 63-110. WEIDEL, W. (1958). Bacterial Viruses (with particular reference to adsorption/ penetration). Ann. Rev. Microbial. 12, 27-48. YOUNGNER, J. S., and NOLL, H. (1958). Virus-lipid interactions. I. Concentration and purification of viruses by adsorption to a cholesterol column and studies of the biological properties of lipid-adsorbed virus. Virology 6. 157-180.
9,
151-167 (1959)
Reversible
and Irreversible to Water-insoluble
Adsorption of Tl Phage Polar Lipids’
LE:O ZELKOWITZ~ AKD HANS ?JOLL" Department
of Microbiology, School of Medicine, Pittsburgh, Pennsylvania
University
of Pittsburgh,
Accepted June 19, 1959 The intera&ions of bacteriophage Tl with water-insolube polar lipids were studied, using the previously developed methods of water-lipid partitioning. The results of experiments on the adsorption kinetics, on the factors determining adsorption efficiency, and on the reversibility of the virus-lipid bond closely parallel the findings reported for lipophilic animal viruses. Phage adsorbed to cholesterol or stearic acid dissociates readily upon dilution during plating, as indicated by the quantitative recovery of infectivity. whereas adsorption of Tl to hexadecylamine causes irreversible inactivation. Adsorption to a cholesterol column followed by ether-w-ater partit,ioning proved to be a convenient met,hod for t,hc rapid removal of the major portion of nonviral protein. Passage of 10 ml of high titer anti-T1 rabbit serum t,hrough a cholcstcrol column saturat,ed with phage rcsultcd in t,he adsorption of 88 ‘yO of specific antibody. INTRODUCTION
Preliminary experiments, reported previously (Youngner and ISoll, 1958), indicated that bacteriophages Tl and T2, like lipophilic animal viruses, had a high adsorption affinity for cholesterol columns. In subsequent adsorption experiments with bacteriophage Tl, however, considerable variations in adsorption efficiencies were observed. A systematic study of the variables influencing the adsorption efficiency was, therefore, undertaken. The experiments reported in this paper show 1 This investigation was carried on Influenza of the Armed Forces the Office of the Surgeon General, 2 Aided by rt Student Fellowship mittee. 3 Recipient of Senior Research Public Health Service.
out under the sponsorship of the Commission Epidemiological Board, and was supported by Department of the Army. from the Tobacco lndustry Research ComFellowship 151
No. SF-274 from the United
States
152
ZELKOWITZ
ANI)
KOLL
t,hat, under properly controlled conditions, highly reproducible results are obt,ained. The data on the kinetics of the interaction of the bacterial virus Tl wit,h various lipids closely parallel the findings obtained with lipophilic animal viruses. This further strengthens our previous conclusions concerning the mechanism of these interactions. Moreover, compared wit,h many animal viruses, t’he coliphages were found to be better suited for such studies, because of the more sensit’ivc and precise methods available for t,heir assay. MATERIALS
AiXl>
METHODS
coli was grown in nutriPreparation oj phage. Strain R of Escherichia ent broth (7.5 mg/ml) to a concentrat,ion of approximately lo9 cells per millilit’ers. Aft.er infection with about 10’” plaque-forming units (PFU) of the Tl strain, incubat8ion was continued uruil clear lysates containing between 10’” and 10” l’I’U/ml were obtained. The crude lysat’es were centrifuged at 3000 rpm for 30 minutes in a refrigerated cent’rifuge, the supernatant collected and stored at 4” until used. Assag of phagr was carried out by the agar layer method as described by Adams (1950). All samples were plat,ed in duplicat,e over at least a hundredfold concentration range t,o producr: an optimal number of countable plaques. Spccijic anfi-7’1 w-urn was prepared by repeated injection of high t,it,er lysatcs int,o five rabhit,s. The pooled serum was stored at - 10” and yielded a K-value of 100. Protein was determined according to Lowry et al. (1951). Absorption of phage to lipids was carried out’ at, room temperature by c&her the column or batch method following the techniques described in detail in the preceding paper (Sol1 and Youngner, 1959).
Crude lysates of coliphage Tl were passed through 2-g cholesterol columns. The number of phage particles adsorbed to the column was obtained (I) by determining the difference between the totma number of PPU in the influent volume and the collected effluent fractions and (2) by plating dilutions of the resuspended cholesterol. All columns were washed with at’ least 30 ml of phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954). A number of typical experiments are summarized in Table 1, which
ADSORPTlON
ADSORPTION OF Tl
Column
1 2 3 4 5
OF
Total
-
9 1.0 2.76 5 2
9 9.5 1.14 1.5 2.0
(LComparison of adsorption ference and by direct plating b Plaque-forming units.
PFU on column
By difference
109 10’0 IO’O 10’0 10’2
153
PHAGE
TABLE 1 TO 2.~ CHOLESTEROL COLTJMNP
Total PFU* input
x x X x x
Tl
x 109 x 109 x 10’0 x 10’0 x 10”
data obtained of resuspended
By direct plating 9 9.5 1.06 1.5 1.8 by determining lipid.
x x X x x
I>eviation (76)
109 10” 10’” 10’” 10” input -output
0 0 8 0 10 dif-
shows that the phage removed from the influent is quantitatively rccovered from the resuspended adsorbent. In most cases the suspension method of assay was preferred because of its greater accuracy and ximplicity. Eficiency
of Column Operation
As a result of numerous adsorption experiments with uniform 2-g cholesterol columns and varying quantities and concentrations of phage, it was found that the adsorption efficiency, i.e., t’he percentage of totma phage input adsorbed, was determined mainly by t,he adsorption capacity of the column and the rate of equilibration. This is illustrated in Fig. 1, which shows that phage was adsorbed wit,h 95-100 ‘3%efficiency as long as the input did not exceed a total of about 1 X 10’” part’iclcs. Further increase of the input resulted in rapid saturat,ion of t,he columns, as indicated by the sharp decrease in adsorption efficiency. Thus, loadings of 2 X 10” PFU/2 g cholesterol, e.g., could only be achieved at the expense of a 90 % loss of phage in the effluents. The quantity of phage adsorbable to cholesterol columns was also found to be limited by the practical difficulties encountered in passing large fluid volumes through the columns. After collecting about 300 ml, t’he flow rates became impractically slow, because of the packing effect produced by the pressure applied to the top of the column. Since the influent concentrations in the experiments represented in Fig. 1 varied, the adsorption efficiencies were plotted as a function of the
154
AK;D NOLL
ZELKOWITZ
TOTAL l?F.U. PASSED THROUGH COLUMN
FIG. 1. Adsorption efficiency as a function passed through 2-g cholesterol column. TABLF: INFLUENCE
C0hln
1
2 3 4 5
of total
number
of Tl
particles
2
OF PHAGE CONCENTRATIOX ON EFFICIEKCY OF ADSORPTION TO 2-c: CHOLESTEROL COLLJMNS USING COXSTANT PHACE INPIJT Influent cont. (PFU/ml)
3 9 1.5 4.3 1
x x x X x
10’ 107 108 lo8 10”
Volumeof phage suspension passed through column (ml)
300 100 70 25 10
Total PFU input
9 9 1.05 1.07 1
x 109 x 109 x 10’0 x 10’0 x 101”
~-____
Total PFU
Adsorbed
9 9 9 8.5 9.5
x x x x x
Per cent
109 109 109 109 109
100 100 89 83 95
tot’s1 number of particles passed through the columns. This procedure seems to be justified by the finding, illustrated in Table 2, that the adsorption efficiency was independent, of the concentration of the phage suspensions passed through t’he column over the thirtyfold range tested. Faster flow rates obtained at higher pressures, however, reduced the adsorption efficiency considerably by prevent,ing efficient equilibration, as shown in Table 3. Kinetics
of Column
Saturation
For a furt(her characterization of the adsorption process, the kinetics of column saturat’ion were determined as described by Youngner and
ADSORPTION
Tl
OF
TABLE
PHAGE
155
3
INFLUENCE OF FLOW RATE ON ADSORVTIOX EFFICIENCY OF Tl CHOLESTEROL COLUMN Column 1 2
Flow rate (ml/min)
Total PFU input
0.5 5.0
5 x 5 x
Total PFU adsorbed
10’0 10’0
1.5 x 7.3 x
204oBoBo
IO0
. ......
1 I20
EFFLUENT
FIG. 2. Adsorption lesterol column.
isoplane
describing
I I40 VOLUME
I IS0
4
I IBO
TO
2-c
% Adsorbed
lOLO 108
WASHING w.7
0
PHAGE
30 1.2
BEGUN \
I 200
r 220
I 240
(ml.)
the interaction
of phage Tl
with
cho-
No11 (1958). The results represented in Fig. 2 closely paralleled those obtained with influenza virus. A 190-ml aliquot of a diluted crude lysate containing 1.5 X lo8 PFU per milliliter were passed through a 2-g cholesterol column under standard conditions. The column was then washed with 50 ml of PBS. A total of 121 effluent fractions, ranging from 0.4 to 10 ml were collected and their phage concentrations determined. The break-through curve in Fig. 2 (effluent cont. X lOO/ in fl uent cont., plotted against volume passed through column) shows that saturation is reached rapidly between 80 and 100 ml. It is interesting that the slope increases continuously,
156
ZELKOX-ITZ
AND
I\‘OLL
m . . .
\
mr
.
, IO
I 20
! 30
7 40
r 50
FIG. 3. Kiudics of s:it.uration of rlution of ph:agc recoverable
3 60
+J
N 80 90 IdO I@0 I90 EFFLUENT VOLUME (ml.)
of cholesterol hy washing.
260
2Kl
2bO
Zjo
.
240
column wit,h phagr Tl and kinetics
giving rise t,o a diAnctly asymmetrical break-t,hrough curve. This asymmetry is reflected most st’rikingly in the observsCon that’ t,he effluent, concentration rises abruptly from 07 to 100 ‘1, of the influent concent~ration wit,hin a Z-ml increment of effluent collected. This asymmetry holds true, even if a more gradual change is assumed (Fig. 2 : broken line), since the accuracy of t,he assay met,hod was greater t’han It10 %. A semilogarithmio plot of the break-t,hrough curve (Fig. 3) shows t’hat leakage was very small (less t,hun 0.001 ‘3) during the collection of the first 35 ml of effluent. It rose rapidly during t’he next, 20 ml, reaching 1 !% in the fifty-second milliliter of effluent. After washing was st,arted, the phage concentration in the effluent declined exponent8ially, as evident from t,he straight, line in t,he semilog plot of I;ig. 3. This indicates t,hat, a portion of the virus is less firmly associated with t,he lipid and recoverable by washing, an observation which is similar to the one described for influenza virus (Youngner and X011, 1958). This decrease in virus concent,rat,ion follows the equation :
ADSORPTION
OF
TABLE IIISTRIBUTI~N
0~
PHAGE"
OS
Tl 4 2-c
CHOLESTEROL
Column Section
I. Top 6 mm II. Middle 6 mm III. Bottom 6 mm I f
ing
II + III
Bcforc washing 59.1 21.6 19.3 __ lO@Z
u Percentage values are based on total = 1.29 X 1O’O PFU = 100 %.
157
PHAGIC
CULLJMS
,4
Washed out
After washing
Column B after washing
0.1 3.2 9.6
59.0 18.4 9.7
58.5 20.1 9.6
12.9
87.1
88.2
phage adsorbed
to column
before
wnsh-
where v = effluent volume (in milliliters) ; o. = effluent volume at beginning of washing; c = phage concentration (I’FU/ml) corresponding t’o v - VO;CO= initial concentration of phage recoverable by washing (at v = vu). The constant lc is det’ermined from Fig. 3, using t’he pararneters corresponding to half-concentration:
The total amount of phage recoverable by washing ((I,) was calculated by integration of Eliy. (1) between v. (= 191 ml) and u (= 250 ml), giving C, = 1.5 X log PFU/2 g of cholesterol. Det’ermination of total phage remaining on the washed column gave 1.06 X 10’” PFU by direct plating, 1.14 X 10LC’l’E’1!/2 g cholesterol by input-output difference. In order to determine the distribut,ion of the firmly adsorbed phagc on the washed column, the adsorbent was divided into three equal sections of 6 mm and each section assayed. The distribution, shown in Table 4 (column B), closely resembles the concentration gradient found with influenza virus on a cholesterol column (Toungner and X011, 1958). In order to compare the concentration gradient of firmly adsorbed phage with the distribution of phage recoverable by washing, a second cholesterol column was saturated with phage and sectioned without washing. The cholesterol corresponding to each section was resuspended in 10 ml of PBS and O.l-ml aliyuok were withdrawn for assay by direct’ plating. The suspensions were then filtered wit’h What’man Ko. 11H
158
ZELKOWITZ
.&XI)
NOLL
paper and each cholesterol portion was washed with another 10 ml of PBS. Both the filtrates and the remaining cholesterol were again assayed for phage. The results in Table 4 (column A) clearly show an inverse relationship bet,ween the concentration gradients of elutable and firmly adsorbed phage, i.e., the former increasing from top to bottom by a factor of 100, the latter decreasing by a factor of 3. It can also be seen that identical concentrat,ion gradients of firmly bound phage were obt,ained regardless of whether washing was carried out’ before or aft#er sectioning (column B). Although a yuant.itativc kinetic analysis and interpretation of these concentration gradients is complex and beyond the scope of this study, the results clearly rule out the possibilit#y that the observed effects were due to filt,ration. In a qualitative way, however, it seems plausible that, in a statistmica dist,ribution, the less firmly bound phage part,icles would tend to become concentrated toward the bottom of t#hecolumn. Praparation
of
Purijicd
Phagc
bq Bth,cr-Water
Partitioning
OJ Lipid-
A study of t,he conditjionn which would permit the dissociation of the phage from the lipid was of imerest because of the information that would be gained about, t,hc mechanism of adsorption and also because of the possible usefuhiess of t,his method for phage purificat~ion. As wit,h the other viruses tested (l-oungner and n-011, 1958), washing of t,he columns with solutions of \-arying ionic strength failed to elute the firmly adsorbed particles. K&her did washing with undiluted normal rabbit serum cause any elut,ion. On the other hand, t#he previously described met,hod of ether-water partitioning proved to be an efficient method for the recovery of infectious phage. Two grams of cholesterol from a column containing 1.5 X 10’” 1’l’U was suspended into a volume of 25 ml with PBS. The cholesterol was extracted with three 50-ml portions of ether at 4”. The insoluble material accumulat,ing at, the interphase was left with the aqueous portion during the first t,wo extractions and discarded after the third. Ktrogen was then bubbled for 3 minutes through the turbid solution at room temperature to remove residual ether. This treatment gave a clear solution containing a flocculent precipitate which was removed by gravity filtration through Whatman Ko. 41H filter paper. The resulting phage suspension was clear and colorless and comained 1.35 X 1O’OPI’U, or 90 % of the original infectivity. The protein concen-
ADSORPTION
OF
Tl
PHAGE
159
tration of this solution was too low to be determined by the Lowry method, i.e., less than approximately 10 pg/ml as compared with 7.5 mg/ml before adsorption, indicating that a considerable purification can be achieved by this method. In contrast to the lipophilic animal viruses studied previously, the adsorption to cholesterol and subsequent separation with ether seems to offer a generally useful method for the concentration and purification of phage, because of the remarkable resistance of phage particles to inactivation by organic solvents. InJluence of Purification Cholesterol
and Ether Treatment on Adsorption of Phage to
In view of the possibility that the adsorption of phage to cholesterol was mediated by lipophilic substances present in lysates, it seemed interesting to determine the adsorbability of phage which had been purified by the method of adsorption and water-ether partitioning described in the previous section. When such a purified phage solution containing 9 X log PFU was passed through a 2-g cholesterol column under standard conditions, 99 % of the phage was adsorbed to the lipid. The unchanged adsorption characteristics of this highly purified phage preparation indicates that the adsorption affinity for lipid is due to ether-resistant lipophilic components of the phage surface. Adsorption of Tl Phage to Lipids Other Than Cholestero(l In a previous paper (No11 and Younger, 1959), it has been shown that a great variety of water-insoluble polar lipids are capable of adsorbing lipophilic viruses. Saturation studies with influenza virus and the lipids, cholesterol, palmitic acid, and hexadecylamine (HDA) showed considerable variations in adsorption activities and capacities of these different lipids. In order to study the affinity of Tl phage for different lipids, adsorption experiments with stearic acid and HDA were carried out. A 2-g stearic acid column was prepared and lO*OPFU passed through. After washing, the phage both in the effluents and the column was determined. It was found on plating of the resuspended lipid that only 1 % of the phage particles passed through the column were firmly adsorbed to the stearic acid column. This result is in contrast to similar experiments with lipophilic animal viruses which, under comparable conditions, all showed a stronger affinity for long-chain fatty acids than for the neutral lipid cholesterol.
160
ZELKOWITZ
AND
P;OLL
TABLE15 ADSORPTION EFFICIENCIES AND RECOVERY OF I~VFECTIVITY AND HEUDECYLAMISE RY BATCH STEARIC ACID,
Adsorbent (100 mu)
Cholest,erol
St earic ncitl HI)A
Tota\fzyj
1.75 x 3 x
input
10’” 10”
1.8 x 10’” 3 x 5 x 1.8
x
Total PFU in filtrate
1.75 x 3 x
10’” 109
1.8 x IO’”
10”
3 x
109
10’” 10’0
5 x
10”
3.5 x
10”
WITH CHOLESTEROL, ADSORPTION
Total PFU in resuspended lipid
1X108 1X10’ 3 x lo* 6 x 10’ 1 x 9 x
10” 107
% Adsorbed
99.9 >99.9
y& PFU recovered in filtrate + iTSUSpended lipid
100 100 100 100
Adsorption experiments with HDA were carried out, by the bat’ch method because of t,he previously explained difficulties in preparing columns with this lipid (No11 and Youngner, 1959). A4 total of 100 mg of HDh was mixed wit,h 5 ml of crude lyeat#e by grinding in a mortar, filtered and washed with PBS as described in t’he preceding paper. For wmparison, analogous batch adsorption experiment,s were carried out, with cholesterol and st,earic acid. The result)s of typical experiments, summarized in Table 5, show that’ less than 2 ‘X8 of the phage was adsorbed to cholesterol or stearic acid. This corresponds t#o about, 10 ‘2 of t,hc efficiency obt~ained by column ndsorpt,ion nft,er correcting for the smaller amounts of lipid used in these bat,ch adsorpt,ion experimcnt,s. Similar differences in adsorption efficiencies between ba.tch and column operaCon are commonly observed ill chemisorpt,ion studies wi:ith ion exchangers and other udsorhents. In contrast, to cholest8erol and st,earic acid, which failed t,o cause a dete&blc reduction of 1’1~1:‘s in t,he filtrat,es, the basic lipid hexadecylamine adsorbed close to 100 ‘X of the phage particles under t#he same conditions, as evident from t,he lob-fold reduction of infectivity in the filtrates. Yet, less than 1 % of the phage originally present in the adsorption mixture could be demonstrat,ed by plntCng the resuspended lipid. The loss of infectivity observed with HDX could be due to &her of the following mechanisms: (1) t,he phage combines irreversibly with the lipid particle in a way which makes it,s tail st’ruct’ure inaccessible t)o the receptor areas of the bacterial surface; (‘2) combination of HDX and phage results in secondary structural changes of t,he latter [cl.g., deoxyribonucleic acid (DIY.4) eject,ion] fol-
ADSORPTION
OF
‘r1
PHAGE
161
lowed by dissociation of the inactivated phage; (3) phage is inactivated by a detergent effect of HDA present in filtrable molecular-dispersed form. The last possibility was ruled out by a separat’e experiment. HDA (100 mg) was ground with 5 ml of PBS in a mortar and filtered t,hrough Whatman 41H filter paper, as usual. One milliliter of lysate containing 6 X log PFU was added to the filtrate. After 2 hours of mechanical agitation, the solution was assayed. Phage was recovered quantitatively. The present experiments fail to distinguish between the first t#wo mechanisms. Analogous results obtained with influenza \?rus, however, indicated that inactivation is caused by irreversible adsorption of the virus to HDA, since it was possible to demonstrate t#he presence of adsorbed viral antigen by assay of antibodies produced by guinea pigs which had been immunized w&h the washed influenza virus-HDA suspension (No11 and Toungner, 1959). Conversely, the low adsorpt’ion efficiencies and complete recoveries of infectious phage from resuspended cholesterol and stearic acid are consistent with the assumption of an equilibrium adsorption in the case of these latter lipids. Accordingly, dilution of the virus-lipid complex would cause dissociation of t,he infective particle. The results of infectivity titrations with influenza virus adsorbed to cholesterol and determinations of the particle size of the lipid suspension support this conclusion (No11 and Youngner, 1959). Interaction
of Antibody with Phage Adsorbed to Chobcsterol Column
It has been reported previously (Youngner and Koll, 1958) t’hat the passage of antiserum through a cholesterol column, to which influenza virus had been adsorbed, resulted in the specific removal of homologous antibodies and that this method offered a promising tool for the purification of antibody protein. The following experiments show that the same technique can be applied also to the separat,ion of antibodies against bacteriophage TI . Antibody was assayed according to Hershey (1941), using the equation log p,/p = K tj2.3 D, where ~0 = phage assay at zero time, p = phage assay at time t minutes, D = final dilution of serum in the phage-serum mixture, K = velocity constant. The velocity constant K was determined from the slope of the linear portion of the graph obtained by plotting the inactivation values log pa/p, corresponding to various serum dilutions, against t/2.3 D using a constant inactivaGon time of t = 30 minutes. The rabbit immune serum used in t,hese experiments gave a value of K = 100. The ant,ibody concenkations of effluent fractions
162
ZELKOWITZ
AND
NOLL
collected from the column were assayed with the aid of the standard curve prepared for the determination of K. If necessary, the serum samples were diluted so as to give inactivation values falling within the linear range of the standard curve. All antibody concentrations were expressed as percentages of t’he antibody concentration in the original serum. Two 2-g cholest,erol columns were saturated with phage. After washing with PBS, each column was found to contain 3.2 X 10” Tl particles. Ten milliliters of anti-T1 serum was passed through one column, and the Hame quantity of normal rabbit serum through the other. As a further control, an identical amount’ of immune serum was passed through a t,hird column which contained no phage. The effluents were collected in fractions and assayed for protein and antibody. Figure -l shows the distributions of protein and antibody concentrat#ions in the effluent, fractions obtained by passing antiserum through t#he column saturated with phage (il) and the control column without phage (R). For comparison, both the protein and the antibody concentrations (represented graphically by t’he height of the differently shaded areas over the corresponding effluent fractions) are expressed as percentages of their original influent concentrations. The protein concentrations in the effluent fract#ions of both columns rapidly rose to t’he level of t’he influent, followed by an equally rapid fall wit’h t,he beginning of washing, indicat,ing that most of t,he serum proteins failed to interact with the cholesterol. A st,riking difference, however, was observed in t#he quantities and distribut’ion of antibody collected from the two columns. The first two effluent fractions from the column sat,urated with phage (n), although containing 60 %I of the input serum protein, yielded less t,han 2 %I of tot’al antibody. After collecting 8 ml of effluent, the antibody concentration rose, indicating that the available antigenic surface of t,he phage was approaching saturation. Analysis of the effluent,s from the control column without phage (B), on the other hand, showed that protein and antibody were eluted at the same rate. The recoveries of protein and antibody in the effluent from the two columns (equivalent in Fig. 4 t’o the areas under the broken and solid lines, respectively) are summarized in Table 6. It can be seen that the phage column adsorbed 88 5%of the phage-inactivating antibodies present in 10 ml of serum, whereas the control column removed neither protein nor antibody t#o any measurable ext’ent. Essentially complete recovery of protein was also obtained when normal rabbit, serum (K = 0) was passed through a
ADSORPTION
OF
Tl
163
PHAGE
PROTEIN
ANTIBODY
0 4
Iv----” I L.---J
TOTAL PROTEIN IN EFFLUENTS
m
TOTAL ANTIBODY IN EFFWENTS
io
6 12 I6 20 24 26 32 0 4 6 12 16 EFFLUENT
VOLUME
(ml.1
FIG. 4. Adsorption of antibody to phage-sat,urated cholesterol column. Distribution of protein and neutralizing antibody in effluent fractions during passage of 10 ml of anti-T1 rabbit serum through 2-g cholesterol columns with and without phage. (A): Column saturated with Tl; (B) : Control column without phage. TABLE
6
ADSORPTION OF NORMAL AND ANTI-Tl SERT:M TO CHOLESTEROL COLUMNS WITH AND WITHOUT ADSORBED PHAGE: RECOVERY OF YROTEIN, ANTIBODY, AND INFECTIVITY FROM EFFLUENTS AND COLUMNS K-Value of
serum Serumprotein Antibody Column Total PFU adsorbed t~~~$h recovered in recovered in COllDlo effluents (70) emuents (%) (10 ml) i
3.2 X0 10”
100
>99 >95
11.9 >99
C
3.2 X 10”
0
>99
0
Protein recovered fromcolwnn Total PFU recovered after ether- from resuspended water parcholesterol titioning (w) 5.0 7.2
6 X0 IO8 3.2 X 10”
cholesterol column saturated with phage. Table 6 further shows that the amount of antibody protein present in 10 ml of a high titer anti-T1 phage rabbit serum is too small (less than 5 % or approximately 40 mg) to be measured by differences in protein content between influent and effluent. A direct determination of the protein on the column saturated with phage and the corresponding phageless column was therefore carried out by assaying the aqueous phase obtained after extracting the lipid with ether. Determination by the Lowry method, using bovine albumin as a standard, gave values of 7.2 and 5.0 mg of total protein, respectively. It is obvious from the relatively high-protein blank of the control
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column that the prot,ein recovered from the phage-saturated column cannot simply be equated with antibody. In the absence of more experimental determinations, it is also doubtful that the difference between the two values (i.e., 2.2 mg) can serve as a valid basis for an accurate correlation between antibody and protein. Even if this value is taken merely as an indication of the approximate range, it would appear far too high, since it, would follow that each phage particle on the column combined m&h an average of 26,000 antibody molecules. Discounting these prot,ein values as a basis for an estimation of antibody concentration, however, does not invalidate t’he unexpected, though well documented, finding t#hat the relatively small quant’ity of 3.2 X 1OlLphage particles was capable of adsorbing more than 99 % of antibody contained in 8.8 ml of undihued high t’it#er (K = 100) antiserum. This corresponds to a maximal serum-blocking power of 2.75 X 10” ml per Tl particle. In contrast to this, Hershey et al. (1943) found a value of 3.3 X 10-l” ml per T2 particle under conditions of antigen saturation and using a serum of K = 360. The large discrepancy between Hershey’s and our resuhs is difhcult to explain and requires further investigation. The possibility exists t,hat the column process permits more efficient saturat’ion of the antigenic surface of the phage with antibody than do precipit,aCons from free solut,ions. It is of particular interest to not,e that the experiments reported here are concerned with neutralizing antibodies which are presumably directed against t’he phage tail (Lanni and Lanni, 1953). This would support t,he notion that phage is adsorbed to cholesterol by its head, leaving the tail st,ructure freely accessible to antibody. This would be consistent with the finding t,hat, phage adsorbed to cholesterol can be recovered in infectious form. Since inactivation of phage with HDA, on the ot,her hand, suggest’rd ut8tuchmcnt, to the lipid by the tail, it would be intercsting t,o t,est, whether neutralizing amibodies are capable of interacting with phage adsorbed t#oHDA. 1)1SCUSS10N
In a preceding paper (Sol1 and Youngner, 1959), the mechanism of adsorption of lipophilic viruses t,o various lipids and the biological significance of such interactions for the attachment of viruses to cells have been discussed in detail. On t’he basis of their interactions with lipids, viruses were classified as lipophilic or hydrophilic. The hypothesis was proposed that, in the adsorption of the former group of viruses to
ADSORPTION
OF
Tl
PHAGE
165
their host cells, lipid-lipid interactions of the van der Waals’ type may play an important role. The high lipid content of the surfaces of both cells and lipophilic viruses was thought to provide strong support in favor of this concept. The interactions of the bacterial virus Tl with polar lipids described in this paper suggest a similar interpretation. The high lipid content of receptor active compounds, isolated by chemical dissection of cell walls of phage-sensitive E. co& is indicative of a possible participation of lipids in the adsorption of phage to it,s host. Extensive chemical studies have shown that all of these preparations which are capable of irreversibly inactivating phage under physiological conditions in vitro are particulate macromolecules consisting of lipids combined with specific carbohydrates and/or proteins (Weidel, 1958). In the case of T4, the essential function of the lipid moiety for phage inactivation has been clearly demonstrated by Jesaitis and Goebel (1955). These workers isolat’ed sonnei a lipocarbohydrate which specifically from phase II Shigella inactivated T4 by rapid irreversible formation of a noninfect,ious complex, followed by the slower disintegrat,ion of phage and release of DNA. Extraction of lipid from the lipocarbohydrate with 70 % ethauol destroyed receptor act,ivity; it was restored, however, on readdition of the extracted lipid which by it,self was inactive. The carbohydrate moiety was responsible for the specificity whereas t,he lipid part could be replaced by a number of long-chain fat,ty acids (C,,-C,,). Maximal activity of the reconstituted system was dependent, on the ratio of fat’ty acid to ext,racted lipocarbohydrate, indicating format’ion of a st’oichiometric complex between the two components. Although the authors did not comment on this point, it seems likely that the fatt’y acids combined with basic amino groups of t,he carbohydrate moiety which was shown to cont,ain hexosamine. This would be in agreement wit’h the failure of fatty acid esters to restore activity. Comparison of the important observations of Jesaitis and Goebel with our results reveals certain similarities which may be integrated into a hypothesis of the mechanism of adsorption of phages to bacterial cells. It has been found that Tl combines reversibly with neutral and acid lipids (cholesterol, stearic acid), presumably by relatively weak van der Waals’ interactions. However, if the lipid carries a positively charged group (as in the case of hexadecylamine), irreversible binding occurs resulting in loss of infectivity. This may be attributed to reinforcement of the binding by the additional electrostatic attraction between the
166
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basic group of the lipid and negatively charged groups at the tip of the phage tail. Thus, hexadecylamine may be regarded as the simplest chemical model of a cellular receptor, since it combines in one molecule the dual functions characteristic of all natural receptor substances. In the natural receptor material isolated by Jesaitis and Goebel, these two functional entities are represented by extractable lipid and complex carbohydrate, and their functional cooperation is strikingly illust,rated by the loss of receptor activity upon chemical separation. Our model (based on the interaction of Tl with HDA) indicates an irreversible reaction as the primary st’ep in the attachment of phage to host cell and dispenses with the necessity of invoking a reversible initial reaction as has been postulated from the exclusive consideration of ionic interactions (Puck, 1953; Tolmach, 1957). It also becomes unnecessary t’o postulate subsequent enzymat,ic react,ions as a means of achieving irreversible binding. Weidel (1958) has in fact’ shown that bacterial receptor subst’ances combine irreversibly wit,h phage and are distinct from the substrate of t,he phage enzyme which, in addition, lacks type specificity. It should be pointed out in this connection t,hat the failure t,o observe irreversible atjtachment of Tl under conditions which prevent, infection has been used as a strong argument in favor of reversible attachment as an obligatory step in t,he process of infection (Puck et al., 1951). A crit,ical review of the pertinent fact,s and interpret8ations, ou t,he other hand, led Hershey (1957) to conclude t’hat reversible adsorption observed under these conditions may represent, a different, kind of reaction involving recept,ors which have no relation to the infective process and t#hnt, in the case “of t,he interesting kind, namely infection, t,here is little evidence for a charact8eristic reversible st,ep.” He also point,ed out that) “the existing information about, isolated receptor substances comes from experiments using irreversible inactivation of phage as a test for receptor act,ivit,y.” It is of part#icular interest, therefore, t,hat hexadecylamine has been found to be the first receptorlike substance capable of irreversible combinat,ion with Tl . Previous failures to isolate Tl receptor substances from the host’ cells may have been due t,o the disruption of the essential link between the st,ructures carrying the lipophilic and ionizable basic groups. No claim is made that t,he proposed model is able to explain and unify all t,he complex aspect,s of phage attachment. Together wit,h certain new viewpoints it also int’roduces assumptions which will have to be tested by further experiments. Thus, it was postulated that, t,he phage
ADSORPTION
OF
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167
attaches to hexadecylamine by its tail and that the tail contains the required complementary combination of lipid and negatively charged groups. By electronmicroscopy it should be possible to decide the first question but the problem of the lipid content of the phage tail may present difficult analytical problems. Experiments are also planned to find out whether inactivation of Tl by adsorption to hexadecylamine is accompanied by the ejection of DNA. ACKNOWLEDGMENTS The authors areindebt)ed helpful discussions.
to Drs. J. S. Youngner
and M. H. Weiner
for numerous
REFERENCES ADAMS, M. H. (1950). In Methods in Medical Research (J. H. Comroe, Jr., ed.), Vol. 2, pp. l-73. Year Book Publishers, Chicago, Illinois. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines of poliomyelitis viruses. J. Exptl. Med. 99, 167-182. HERSHEY, A. D. (1941). The absolute rate of the phage-antiphage reaction. J. Immunol. 41, 299-319. HERSHEY, A. D. (1957). Bacteriophages as genetic and biochemical systems. Advances in Virus Research 4, 25-61. HERSHEY, A. D.,KALMANSOPI',G., and BRONFENRRENNER, J. (1943).Quantitative relationships in the phage-antiphage reaction: Unity and homogeneity of the reactants. J. Immunol. 46, 281-299. JESAITIS, M. A,, and GOEREL, W. F. (1955). Lysis of T4 phage by the specific lipocarbohydrate of Phase II Shigella sonnei. J. Exptl. Med. 102, 733-752. LANNI, F., and LANNI, Y. T. (1953). Antigenic structure of bacteriophage. Cold Spring Harbor Symposia Quant. Biol. 18, 159-168. LOWRY,~. H., ROSEBROCGH, K. J., FARR, A. L.,and RANDALL, R.J. (1951).Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. ?JOLL, H., and YOCNGNER, J. S. (1959). Virus-lipid interactions. II. The mechanism of adsorption of lipophilic viruses t,o water-insoluble polar lipids. Virology 8, 319-343. PIJCK, T. T. (1953). The first steps of virus invasion. Cold Spring Harbor Symposia Quant. Biol. 18, 149-154. PUCK, T. T., GAREN, A., and CLINE, J. (1951). The mechanism of virus attachment to host cells. I. The role of ions in the primary reaction. J. Exptl. Med. 93,65-88. TOLMACH, L. J. (1957). Attachment and penetration of cells by viruses. Advances in Virus Research 4, 63-110. WEIDEL, W. (1958). Bacterial Viruses (with particular reference to adsorption/ penetration). Ann. Rev. Microbial. 12, 27-48. YOUNGNER, J. S., and NOLL, H. (1958). Virus-lipid interactions. I. Concentration and purification of viruses by adsorption to a cholesterol column and studies of the biological properties of lipid-adsorbed virus. Virology 6. 157-180.