Vol. 11, No. 3 Printed in U.S.A.
INFECTION AND IMMUNIry, Mar. 1975, p. 445-452 Copyright 0 1975 American Society for Microbiology
Genetic Basis of Toxin Production and Pathogenesis in Vibrio cholerae: Evidence Against Phage Conversion JOHN C. GERDES AND W. R. ROMIG* Department of Bacteriology, University of California, Los Angeles, California 90024 Received for publication 29 October 1974
The pathogenicity of Vibrio cholerae strains "cured" of "Kappa-type" phage not significantly altered relative to that of their "Kappa" lysogenic parental strains. Unlike Corynebacterium diphtheriae, the capacity of V. cholerae to produce exotoxin was not stimulated as a consequence of active phage multiplication. Toxin production in cultures in which Kappa-type phage multiplication was initiated either by inducing Kappa lysogens or by infecting naturally occurring or "cured" Kappa-sensitive strains was greatly reduced compared to normally growing control cultures. Kappa-sensitive El Tor strain Mak 757 and a Kappa lysogen derived from it did not differ in their capacity to colonize ligated rabbit ileal loops nor in their sensitivities to ultraviolet radiation, acidic pH, or osmotic shock. We conclude that Kappa-type phages do not directly affect the pathogenicity of these V. cholerae strains. was
The current cholera pandemic is unusual in that the etiological agent is the El Tor biotype of Vibrio cholerae, whereas the causative agent in all previous pandemics was attributed to classical biotype vibrios. Pathogenic El Tor strains isolated from the current pandemic are usually lysogenic for a bacteriophage designated by Takeya as a "Kappa-type" phage (27). It has been estimated that 85 to 95% of the El Tor strains isolated from clinical sources are lysogenic for Kappa (20, 26, 27), whereas asymptomatic or mildly pathogenic El Tor strains generally are not lysogenic for this phage (26). The prevalence of Kappa-type phage led Newman and Eisenstark (20) to suggest that phage conversion similar to that originally described for Corynebacteria and diphtheria toxin (11) might occur in V. cholerae. A phage or plasmid has been conclusively linked with the toxin production of several bacteria (30), including the enterotoxins of staphylococci (4) and Escherichia coli (24). The discovery of defective bacteriophage in classical biotype vibrios (22) and their subsequent identification as Kappa related (J. C. Gerdes and W. R. Romig, submitted for publication) suggested an unusual ubiquity of these phage in vibrios, regardless of biotype, and thereby further suggested a possible phage conversion. We report here the results of experiments designed to investigate the possibility of a causal relationship between Kappa-type bacteriophages and V. cholerae pathogenesis. Both El Tor and classical strains were investigated. 44j
The results do not support phage conversion in V. cholerae. V. cholerae therefore appears to be an exception to the general pattern of phage involvement in the production of bacterial exotoxins (30). MATERIALS AND METHODS Media. Viable counts were performed on the meat extract agar described previously (21). Cells were grown in TY broth (Gerdes and Romig, submitted for publication) or peptone saline which contained 2% peptone (Difco) and 0.5% NaCl adjusted to pH 8.0. Thy A medium was as described by Caster (5). Phage and bacterial strains. The bacterial strains have been described previously (22). The biotype and serotype of these strains is given in Table 2. Thymine auxotrophs were isolated using methotrexate selection as described by Caster (5). Kappa-related phages included VcA-1 and VcA-2 isolated from NIH 41, and the Kappa-type phage isolated from the El Tor strain HK-1. A clear-plaque mutant of VcA-2 designated VcA-2Cl was used for phage spraying. All of these bacteriophages were described previously (Gerdes and Romig, submitted for publication). Phage sensitivity and immunity. Phage sensitivity of bacterial strains to phage Kappa was determined by a modification of the phage spray technique of Stolp (25). Isolated colonies of the bacteria incubated 18 to 24 h at 30 C were sprayed with a suspension containing 2 x 10' phage VcA-2Cl/ml from a DeVilbiss atomizer. Plates were sprayed in an enclosed box so that only fine droplets hit the plates. After spraying, the colonies were further incubated for 18 to 24 h at 30 C before observing for the characteristic notching of VcA-2CI-sensitive clones (Fig. 1). Phage VcA-1 sensitivity was determined in a similar
446
GERDES AND ROMIG tion in the rabbit ileal loop
INFECT. IMMUN. as
described by De and
Chatterjee and by Kwan and Wishnow (8, 16). Logphase cultures were inoculated into 5-cm ligated sec-
.0
t
_
*:~ ~ ~ ~ ~ ~ ~ ...* ~~~~~~~~~~~~~~~~~. X
*; 0
1~~
.:
i.
.0 41
M.
.. ....
A...
**0: ......
q,_
FIG. 1. Phage sensitivity assay. Mak 757 (upper half) and Mak 757 (VcA-2) (lower half) 18 h after spraying with VcA-2CI phage. Note the obvious notching of Mak 757. manner, but since notching by phage VcA-1 is not nearly as pronounced, colonies were examined 6 to 10 h after spraying. Lysogens were derived by plating bacteria picked from plaque centers for isolated colonies and spraying as described above to detect phage-resistant clones. Resistant clones were confirmed as lysogens by their immunity when crossstreaked against homologous phage suspensions and by lytic activity of cultures grown 16 to 20 h in antiphage serum stabbed onto lawns of sensitive indicator. Thymine starvation. All thymine starvation experiments used mid-log-phase (108 cells/ml) thymine auxotrophs grown at 37 C with shaking at 250 rpm on a rotary shaker. Shifting from thymine-containing to thymine-deficient medium was accomplished by filtering the culture onto 0.22-gm membrane filters (Millipore Corp.). washing with five times the original volume of thymineless broth, and then resuspending in thymine-deficient broth. Phage induction and infection. The procedures for the induction of bacteriophages by ultraviolet light (UV) or mitomycin C and the production of high-titer lysates was as described previously (Gerdes and Romig, submitted for publication). Pathogenicity. Pathogenicity was defined by the ability of log-phase cultures to cause fluid accumula-
tions of' the ileum. Up to 16 loops with 2-cm intermediate loops interspersed were inoculated for each rabbit. As a general rule no more than 100 cm of' gut measuring from the appendix was used. Each rabbi. contained a negative (broth) and a positive (569B) control loop. Infective dose. The mean infective dose was determined in ligated loops of the rabbit ileum with a positive response defined as fluid accumulation of greater than 0.5 ml/cm 12 h after inoculation. Six loops were injected for each cell concentration. Toxigenicity. Toxin released into culture medium was assayed by passive hemagglutination inhibition (2, 14) and steroidogenesis assays (16). Steroidogenesis assays were kindly performed by C. Kwan, University of California at Los Angeles and Long Beach Veterans Administration Hospital. Cultures to be assayed for toxin were adjusted to pH 8.0 30 min before the removal of the cells to insure optimum release of toxin (2). All strains were injected and reisolated twice from rabbit ileal loops to insure optimum potential for toxin production before quantitative comparisons were made.
RESULTS Isolation of phage Kappa-sensitive strains. The phage-spraying technique allowed us readily to detect phage VcA-2-sensitive clones (Fig. 1). Phage-sensitive clones occurred spontaneously at a frequency of about 10 - 3for the El Tor strains Cq 1651 (RV 16) and HK-1 (RV 10) and for classical strain 569B (RV 5). Classical strain 569B also carries a defective VcA-1 bacteriophage, but no VcA-1-sensitive clones were detected among the 13,000 clones that were sur-
veyed. A variety of procedures reported to be effective in curing other bacteria of episomes or plasmids were utilized in attempts to cure 569B of defective phages dVcA-1 and dVcA-2. These methods included UV, mitomycin C or acridine orange treatment, and thymine starvation of thymine auxotrophs (3, 6, 9, 13). Of these, only thymine starvation proved successful in curing 569B of dVcA-2, whereas none of these methods was successful in curing dVcA-1. Thymine starvation increased the frequency of phage VcA-2-sensitive clones of 569B Thy -3
to as high as 8.59k (Table 1). The maximum number of phage-sensitive clones occurred at a time corresponding to the plateau observed in the optical density after the removal of thymine (Fig. 2). After this time, they decline in frequency. The defective phage in parental 569B were shown to be inducible after the addition of thymine to a thymine-deprived culture (Fig. 2). Evidence of complete curing of phage
PHAGE AND V. CHOLERAE PATHOGENICITY
VOL. 11, 1975
TABLE 1. Thymineless death and curing of 569B Thy-3 Length of starvat ion (min)
Survival
(P/pO)a
0 30 60 90 120 150 180 aSurviving fraction.
Curing ('N. VcA-
2Cl sensitive) 0.0 0.0
1.0 0.8 0.11 0.02 0.02 0.015 0.01
8.5
3.9 0. 0 1.3
o
p
Pa
_0.1
o.o0 L 0
50
100
150
200
0.01
MINUTES
FIG. 2. Thymineless death of 56'9B Thy-3. O.D., Optical density at 550 nm. Symbols: (@) 569B Thy-a grown in thymine-containing Thy. A medium; () 569B Thy-3 suspended in thymine!less medium at time zero; (V) 569B Thy-3 suspende medium at time zero and induced by the readditionof thymine at the time designated bi y the arrow; *, survival (PIP,) of 569B Thy- 3 after the removal of thymine at time zero.
Kappa-sensitive strains. WTe considered phage-sensitive strains to have c3ompletely lost phage if they met the following three criteria. (i) Phage sensitivity: All strains were selected on the basis of their sensitivity t4 o phage Kappa by the spraying technique, and thieir sensitivities were confirmed by their capaciity to support plaque formation by Kappa-tyj pe phages. (ii) Inducibility: El Tor strains seleacted as above were not inducible by either UV7 irradiation or mitomycin C treatment. Kappea-sensitive isolates of 569B, however, retainedItheir capacity
447
to be induced by these agents since all of them remained lysogenic for the "defective" VcA-1 prophage. (iii) Release of phage or phage-related particles: Upon induction of the El Tor phage-sensitive isolates there were no infective phage-detectable, and phage-related structures were not detected by electron microscopy of the pelleted lysates. Induced lysates of 569B VcA-2 sens-3 contained no tails of the VcA-2 length and subunit structure (Fig. 3), whereas approximately 10% of the tails found in supernatant fluids of induced parental 569B were of the VcA-2 type. Therefore, on the basis of the loss of the genetic functions of the phage repressor as well as physical phage-related structures, these strains were considered to be completely cured of their Kappa-type phage. Phage conversion by a phage-coded toxin structural gene. In Corynebacterium diphtheriae, the structural gene for toxin resides on the phage genome (28). Therefore, loss of the converting phage results in the concomitant loss of toxigenicity (12). Table 2 summarizes the results of pathogenicity comparisons of V. cholerae strains and their lysogenic or cured derivatives. Approximately 101 log-phase cells/ml were injected into ileal loops, and fluid accumulation was measured 18 to 24 h later. The cured derivatives of El Tor strains HK-1 and Cq 1651 and classical strain 569B, designated in Table 2 as HK-1 VcA-2 sens-1, Cq 1651 VcA-2 sens-1, and 569B VcA-2 sens-3, were not significantly altered in pathogenicity relative to the parental strains. El Tor strains that were not lysogenic for phage Kappa, such as Mak 757 or the weakly pathogenic "water vibrios" EW 6 or EW 1, were not altered in pathogenicity in the rabbit ileum when lysogenized with phages VcA-1, VcA-2, or Kappa-10 (Kappa isolated from RV 10). The amount of toxin accumulated by a lysogen or its corresponding nonlysogen was not significantly different after 12 h of growth at 30 C in peptone-saline (Table 3). EW 1 and EW 6 and their lysogens were not tested for toxin production by the PHI or steroidogenesis assays; however, the skin test assay (7) of unconcentrated supernatant fluids of all of these strains were uniformly negative for cholera toxin. Phage conversion involving a phage-coded toxin predicts that the synthesis of toxin in vitro parallels the replication of the bacteriophage. Thus diphtheria toxin is increased 15-fold (1) and streptococcal erythrogenic toxin 20-fold (29) upon induction of a converting phage lysogen. Similarly, infection of a sensitive Corynebacterium strain with a virulent mutant of
448
GERDES AND ROMIG
30 A
>, 20 z
Q LU
aD CY
wI 0
I
I I I
I
I
I I I I I I I 1
30 32 34 36 38 40 42 LENGTH (mm) 40 B
30t z w
a 20 w
10
0
I
1
30 32 34 36 38 40 LENGTH (mm)
42
I
I
I
I
I
FIG. 3. Frequency distribution of 569B phage tail lengths. (A) Tail length distribution of 100 random particles found in the pelleted lysate of 569B after induction by MC. (B) Tail length distribution of 100 random particles found in the pelleted lysate of the phage 2CI sensitive strain 569B VcA-2 sens-3 after induction by MC.
the converting phage resulted in extensive lysis of the culture but a millionfold increase in toxin
synthesis (18). Analogous experiments were carried out for V. cholerae strains (Table 3). Phage Kappa lysogens induced with UV or mitomycin C undergo lysis that can be followed by a decrease in optical density (Fig. 4). After
INFECT. IMMUN.
lysis was complete, the amount of toxin in the supernatant fluids of induced cultures relative to the uninduced controls was drastically reduced for 569B and not significantly altered for the El Tor strains (Table 3). El Tor vibrios produced much less toxin in vitro than the exceptionally toxigenic strain 569B (Table 3). This has been observed by others (23) and may be due to the fact that optimum conditions for toxin production have not been defined for these strains. We interpret the decrease in toxin release in infected or induced 569B cultures as a consequence of the death of cells that would otherwise have produced toxin. However, we cannot explain why a similar decrease was not observed in Mak 757 (Kappa-10) after induction. In any case, the production of extracellular toxin definitely does not parallel phage replication in the manner that has been reported in documented cases of phage conversion (30). Strains that were sensitive to phage Kappa were infected at a cell density of 108/ml with a multiplicity of infection of 1 of phage VcA-2Cl or VcA-1. Extensive lysis occurred 2 h after infection (Fig. 4), and the resulting lysates contained greater than 10'° phage/ml. Toxin measured in the lysates was again not significantly increased relative to the uninfected control (Table 3). Phage conversion by a phage-coded control gene. In Corynebacteria the level of expression of the phage toxin gene is regulated by host cell factors (18, 19). It therefore seemed possible that the regulator genes for toxin production in vibrios might be part of the phage genome. If that is the case, a quantitative difference in the level of toxin produced in a lysogen relative to that produced in a nonlysogen, should be observed. However, as recorded in Table 3, the amount of toxin in the supernatant fluids of phage Kappa lysogens or their corresponding cured derivatives was not significantly different. Therefore, we find no evidence for a causal relationship between phage Kappa and either qualitative or quantitative toxin production. Phage involvement in establishment in the host. For the host to develop the symptoms of cholera, V. cholerae must pass through the highly acidic stomach and establish itself in the small intestine. Therefore, the possibility of a role for phage Kappa in pH tolerance or colonization of the gut was investigated. The inactivations of Mak 757 and Mak 757 (Kappa) after dilution into pH 4.5 peptone-saline were not significantly different (see Fig. 6A), suggesting that phage Kappa probably plays no role in survival of vibrios in passing through the stom-
TABLE 2. Correlation of pathogenicity and lysogeny Avgs fluid accumulation
Biotype.serotypea
Lysogeny
Mak757 [RV79] Mak 757 (VcA-2) Mak 757 (VcA-1) Mak 757 (Kappa)
ET-O ET-O ET-O ET-O
VcA-2 VcA-1 Kappa-10
1.73 1.54 1.67 1.90
EW 1 [RV 26] EW 1 (Kappa)
ET-O ET-O
Kappa-10
0.1 [4; 8] 0.2 [4; 8]
EW6 [RV7] EW 6 (Kappa)
ET-O ET-O
Kappa-10
0.0 [3; 6] 0.0 [3; 6]
Cq 1651 [RV 16] Cq 1651 VcA-2 sens-1 Cq 1651 VcA-2 sens 1 (VcA-2) Cq 1651 VcA-2 sens-1 (VcA-1)
ET-I ET-I ET-I ET-I
Kappa-16 Cured VcA-2 VcA-1
1.71 1.95 1.74 1.92
HK-1 [RV 10] HK-1 VcA-2 sens-1 HK-1 VcA-2 sens-1 (VcA-2)
ET-O ET-O ET-O
Kappa- 10 Cured VcA-2
2.10 [4; 8] 2.00 [4; 8] 1.70 [4; 8]
C-I C-I C-I
dVcA-1,2 dVcA-1 dVcA-1
2.62 [7; 13] 1.62 [2; 3] 2.21 [1; 3]
Strain
569B [RV 5] 569B VcA-2 sens-3 569B VcA-2 sens-3 Thy
(ml/cm)"
[9; 23] [5; 15] [5; 15] [4; 8]
[3; [2; [2; [2;
6] 4] 4] 4]
ET, El Tor; C, classical; 0, Ogawa; I, Inaba. b Fluid accumulation 24 h after injection of approximately 10' log-phase cells/ml. Numbers in brackets refer to number of rabbits and number of ileal loops injected to arrive at the mean value given. a
TABLE 3. Correlation of toxigenicity and lysogeny Strain Strain
569B [RV5] 12-h culture 569B VcA-2 sens-3 12-h culture Mak 757 [RV 79] 12-h culture Mak 757 (Kappa 10) 12-h culture 569B [RV5] 569B UV induced 569B MC induced Mak 757 (Kappa- 10) Mak 757 (Kappa-10) UV induced Mak 757 (Kappa- 10) MC induced 569B VcA-2 sens-3 infected with VcA-2 Cq 1651 VcA-2 sens-1 Infected with VcA-1 Infected with VcA-2 Control
Toxin
(Cg/ml)
Amt" (,) Amt (Ad)
(3Round-up ~~~~~~(3h)'
Steroidc (24 h)
Estimated'd
ND
ND
ND
ND
20
ND
ND
ND
ND
20
50
+/-
36.0
0.5
INFECTION AND IMMUNIry, Mar. 1975, p. 445-452 Copyright 0 1975 American Society for Microbiology
Genetic Basis of Toxin Production and Pathogenesis in Vibrio cholerae: Evidence Against Phage Conversion JOHN C. GERDES AND W. R. ROMIG* Department of Bacteriology, University of California, Los Angeles, California 90024 Received for publication 29 October 1974
The pathogenicity of Vibrio cholerae strains "cured" of "Kappa-type" phage not significantly altered relative to that of their "Kappa" lysogenic parental strains. Unlike Corynebacterium diphtheriae, the capacity of V. cholerae to produce exotoxin was not stimulated as a consequence of active phage multiplication. Toxin production in cultures in which Kappa-type phage multiplication was initiated either by inducing Kappa lysogens or by infecting naturally occurring or "cured" Kappa-sensitive strains was greatly reduced compared to normally growing control cultures. Kappa-sensitive El Tor strain Mak 757 and a Kappa lysogen derived from it did not differ in their capacity to colonize ligated rabbit ileal loops nor in their sensitivities to ultraviolet radiation, acidic pH, or osmotic shock. We conclude that Kappa-type phages do not directly affect the pathogenicity of these V. cholerae strains. was
The current cholera pandemic is unusual in that the etiological agent is the El Tor biotype of Vibrio cholerae, whereas the causative agent in all previous pandemics was attributed to classical biotype vibrios. Pathogenic El Tor strains isolated from the current pandemic are usually lysogenic for a bacteriophage designated by Takeya as a "Kappa-type" phage (27). It has been estimated that 85 to 95% of the El Tor strains isolated from clinical sources are lysogenic for Kappa (20, 26, 27), whereas asymptomatic or mildly pathogenic El Tor strains generally are not lysogenic for this phage (26). The prevalence of Kappa-type phage led Newman and Eisenstark (20) to suggest that phage conversion similar to that originally described for Corynebacteria and diphtheria toxin (11) might occur in V. cholerae. A phage or plasmid has been conclusively linked with the toxin production of several bacteria (30), including the enterotoxins of staphylococci (4) and Escherichia coli (24). The discovery of defective bacteriophage in classical biotype vibrios (22) and their subsequent identification as Kappa related (J. C. Gerdes and W. R. Romig, submitted for publication) suggested an unusual ubiquity of these phage in vibrios, regardless of biotype, and thereby further suggested a possible phage conversion. We report here the results of experiments designed to investigate the possibility of a causal relationship between Kappa-type bacteriophages and V. cholerae pathogenesis. Both El Tor and classical strains were investigated. 44j
The results do not support phage conversion in V. cholerae. V. cholerae therefore appears to be an exception to the general pattern of phage involvement in the production of bacterial exotoxins (30). MATERIALS AND METHODS Media. Viable counts were performed on the meat extract agar described previously (21). Cells were grown in TY broth (Gerdes and Romig, submitted for publication) or peptone saline which contained 2% peptone (Difco) and 0.5% NaCl adjusted to pH 8.0. Thy A medium was as described by Caster (5). Phage and bacterial strains. The bacterial strains have been described previously (22). The biotype and serotype of these strains is given in Table 2. Thymine auxotrophs were isolated using methotrexate selection as described by Caster (5). Kappa-related phages included VcA-1 and VcA-2 isolated from NIH 41, and the Kappa-type phage isolated from the El Tor strain HK-1. A clear-plaque mutant of VcA-2 designated VcA-2Cl was used for phage spraying. All of these bacteriophages were described previously (Gerdes and Romig, submitted for publication). Phage sensitivity and immunity. Phage sensitivity of bacterial strains to phage Kappa was determined by a modification of the phage spray technique of Stolp (25). Isolated colonies of the bacteria incubated 18 to 24 h at 30 C were sprayed with a suspension containing 2 x 10' phage VcA-2Cl/ml from a DeVilbiss atomizer. Plates were sprayed in an enclosed box so that only fine droplets hit the plates. After spraying, the colonies were further incubated for 18 to 24 h at 30 C before observing for the characteristic notching of VcA-2CI-sensitive clones (Fig. 1). Phage VcA-1 sensitivity was determined in a similar
446
GERDES AND ROMIG tion in the rabbit ileal loop
INFECT. IMMUN. as
described by De and
Chatterjee and by Kwan and Wishnow (8, 16). Logphase cultures were inoculated into 5-cm ligated sec-
.0
t
_
*:~ ~ ~ ~ ~ ~ ~ ...* ~~~~~~~~~~~~~~~~~. X
*; 0
1~~
.:
i.
.0 41
M.
.. ....
A...
**0: ......
q,_
FIG. 1. Phage sensitivity assay. Mak 757 (upper half) and Mak 757 (VcA-2) (lower half) 18 h after spraying with VcA-2CI phage. Note the obvious notching of Mak 757. manner, but since notching by phage VcA-1 is not nearly as pronounced, colonies were examined 6 to 10 h after spraying. Lysogens were derived by plating bacteria picked from plaque centers for isolated colonies and spraying as described above to detect phage-resistant clones. Resistant clones were confirmed as lysogens by their immunity when crossstreaked against homologous phage suspensions and by lytic activity of cultures grown 16 to 20 h in antiphage serum stabbed onto lawns of sensitive indicator. Thymine starvation. All thymine starvation experiments used mid-log-phase (108 cells/ml) thymine auxotrophs grown at 37 C with shaking at 250 rpm on a rotary shaker. Shifting from thymine-containing to thymine-deficient medium was accomplished by filtering the culture onto 0.22-gm membrane filters (Millipore Corp.). washing with five times the original volume of thymineless broth, and then resuspending in thymine-deficient broth. Phage induction and infection. The procedures for the induction of bacteriophages by ultraviolet light (UV) or mitomycin C and the production of high-titer lysates was as described previously (Gerdes and Romig, submitted for publication). Pathogenicity. Pathogenicity was defined by the ability of log-phase cultures to cause fluid accumula-
tions of' the ileum. Up to 16 loops with 2-cm intermediate loops interspersed were inoculated for each rabbit. As a general rule no more than 100 cm of' gut measuring from the appendix was used. Each rabbi. contained a negative (broth) and a positive (569B) control loop. Infective dose. The mean infective dose was determined in ligated loops of the rabbit ileum with a positive response defined as fluid accumulation of greater than 0.5 ml/cm 12 h after inoculation. Six loops were injected for each cell concentration. Toxigenicity. Toxin released into culture medium was assayed by passive hemagglutination inhibition (2, 14) and steroidogenesis assays (16). Steroidogenesis assays were kindly performed by C. Kwan, University of California at Los Angeles and Long Beach Veterans Administration Hospital. Cultures to be assayed for toxin were adjusted to pH 8.0 30 min before the removal of the cells to insure optimum release of toxin (2). All strains were injected and reisolated twice from rabbit ileal loops to insure optimum potential for toxin production before quantitative comparisons were made.
RESULTS Isolation of phage Kappa-sensitive strains. The phage-spraying technique allowed us readily to detect phage VcA-2-sensitive clones (Fig. 1). Phage-sensitive clones occurred spontaneously at a frequency of about 10 - 3for the El Tor strains Cq 1651 (RV 16) and HK-1 (RV 10) and for classical strain 569B (RV 5). Classical strain 569B also carries a defective VcA-1 bacteriophage, but no VcA-1-sensitive clones were detected among the 13,000 clones that were sur-
veyed. A variety of procedures reported to be effective in curing other bacteria of episomes or plasmids were utilized in attempts to cure 569B of defective phages dVcA-1 and dVcA-2. These methods included UV, mitomycin C or acridine orange treatment, and thymine starvation of thymine auxotrophs (3, 6, 9, 13). Of these, only thymine starvation proved successful in curing 569B of dVcA-2, whereas none of these methods was successful in curing dVcA-1. Thymine starvation increased the frequency of phage VcA-2-sensitive clones of 569B Thy -3
to as high as 8.59k (Table 1). The maximum number of phage-sensitive clones occurred at a time corresponding to the plateau observed in the optical density after the removal of thymine (Fig. 2). After this time, they decline in frequency. The defective phage in parental 569B were shown to be inducible after the addition of thymine to a thymine-deprived culture (Fig. 2). Evidence of complete curing of phage
PHAGE AND V. CHOLERAE PATHOGENICITY
VOL. 11, 1975
TABLE 1. Thymineless death and curing of 569B Thy-3 Length of starvat ion (min)
Survival
(P/pO)a
0 30 60 90 120 150 180 aSurviving fraction.
Curing ('N. VcA-
2Cl sensitive) 0.0 0.0
1.0 0.8 0.11 0.02 0.02 0.015 0.01
8.5
3.9 0. 0 1.3
o
p
Pa
_0.1
o.o0 L 0
50
100
150
200
0.01
MINUTES
FIG. 2. Thymineless death of 56'9B Thy-3. O.D., Optical density at 550 nm. Symbols: (@) 569B Thy-a grown in thymine-containing Thy. A medium; () 569B Thy-3 suspended in thymine!less medium at time zero; (V) 569B Thy-3 suspende medium at time zero and induced by the readditionof thymine at the time designated bi y the arrow; *, survival (PIP,) of 569B Thy- 3 after the removal of thymine at time zero.
Kappa-sensitive strains. WTe considered phage-sensitive strains to have c3ompletely lost phage if they met the following three criteria. (i) Phage sensitivity: All strains were selected on the basis of their sensitivity t4 o phage Kappa by the spraying technique, and thieir sensitivities were confirmed by their capaciity to support plaque formation by Kappa-tyj pe phages. (ii) Inducibility: El Tor strains seleacted as above were not inducible by either UV7 irradiation or mitomycin C treatment. Kappea-sensitive isolates of 569B, however, retainedItheir capacity
447
to be induced by these agents since all of them remained lysogenic for the "defective" VcA-1 prophage. (iii) Release of phage or phage-related particles: Upon induction of the El Tor phage-sensitive isolates there were no infective phage-detectable, and phage-related structures were not detected by electron microscopy of the pelleted lysates. Induced lysates of 569B VcA-2 sens-3 contained no tails of the VcA-2 length and subunit structure (Fig. 3), whereas approximately 10% of the tails found in supernatant fluids of induced parental 569B were of the VcA-2 type. Therefore, on the basis of the loss of the genetic functions of the phage repressor as well as physical phage-related structures, these strains were considered to be completely cured of their Kappa-type phage. Phage conversion by a phage-coded toxin structural gene. In Corynebacterium diphtheriae, the structural gene for toxin resides on the phage genome (28). Therefore, loss of the converting phage results in the concomitant loss of toxigenicity (12). Table 2 summarizes the results of pathogenicity comparisons of V. cholerae strains and their lysogenic or cured derivatives. Approximately 101 log-phase cells/ml were injected into ileal loops, and fluid accumulation was measured 18 to 24 h later. The cured derivatives of El Tor strains HK-1 and Cq 1651 and classical strain 569B, designated in Table 2 as HK-1 VcA-2 sens-1, Cq 1651 VcA-2 sens-1, and 569B VcA-2 sens-3, were not significantly altered in pathogenicity relative to the parental strains. El Tor strains that were not lysogenic for phage Kappa, such as Mak 757 or the weakly pathogenic "water vibrios" EW 6 or EW 1, were not altered in pathogenicity in the rabbit ileum when lysogenized with phages VcA-1, VcA-2, or Kappa-10 (Kappa isolated from RV 10). The amount of toxin accumulated by a lysogen or its corresponding nonlysogen was not significantly different after 12 h of growth at 30 C in peptone-saline (Table 3). EW 1 and EW 6 and their lysogens were not tested for toxin production by the PHI or steroidogenesis assays; however, the skin test assay (7) of unconcentrated supernatant fluids of all of these strains were uniformly negative for cholera toxin. Phage conversion involving a phage-coded toxin predicts that the synthesis of toxin in vitro parallels the replication of the bacteriophage. Thus diphtheria toxin is increased 15-fold (1) and streptococcal erythrogenic toxin 20-fold (29) upon induction of a converting phage lysogen. Similarly, infection of a sensitive Corynebacterium strain with a virulent mutant of
448
GERDES AND ROMIG
30 A
>, 20 z
Q LU
aD CY
wI 0
I
I I I
I
I
I I I I I I I 1
30 32 34 36 38 40 42 LENGTH (mm) 40 B
30t z w
a 20 w
10
0
I
1
30 32 34 36 38 40 LENGTH (mm)
42
I
I
I
I
I
FIG. 3. Frequency distribution of 569B phage tail lengths. (A) Tail length distribution of 100 random particles found in the pelleted lysate of 569B after induction by MC. (B) Tail length distribution of 100 random particles found in the pelleted lysate of the phage 2CI sensitive strain 569B VcA-2 sens-3 after induction by MC.
the converting phage resulted in extensive lysis of the culture but a millionfold increase in toxin
synthesis (18). Analogous experiments were carried out for V. cholerae strains (Table 3). Phage Kappa lysogens induced with UV or mitomycin C undergo lysis that can be followed by a decrease in optical density (Fig. 4). After
INFECT. IMMUN.
lysis was complete, the amount of toxin in the supernatant fluids of induced cultures relative to the uninduced controls was drastically reduced for 569B and not significantly altered for the El Tor strains (Table 3). El Tor vibrios produced much less toxin in vitro than the exceptionally toxigenic strain 569B (Table 3). This has been observed by others (23) and may be due to the fact that optimum conditions for toxin production have not been defined for these strains. We interpret the decrease in toxin release in infected or induced 569B cultures as a consequence of the death of cells that would otherwise have produced toxin. However, we cannot explain why a similar decrease was not observed in Mak 757 (Kappa-10) after induction. In any case, the production of extracellular toxin definitely does not parallel phage replication in the manner that has been reported in documented cases of phage conversion (30). Strains that were sensitive to phage Kappa were infected at a cell density of 108/ml with a multiplicity of infection of 1 of phage VcA-2Cl or VcA-1. Extensive lysis occurred 2 h after infection (Fig. 4), and the resulting lysates contained greater than 10'° phage/ml. Toxin measured in the lysates was again not significantly increased relative to the uninfected control (Table 3). Phage conversion by a phage-coded control gene. In Corynebacteria the level of expression of the phage toxin gene is regulated by host cell factors (18, 19). It therefore seemed possible that the regulator genes for toxin production in vibrios might be part of the phage genome. If that is the case, a quantitative difference in the level of toxin produced in a lysogen relative to that produced in a nonlysogen, should be observed. However, as recorded in Table 3, the amount of toxin in the supernatant fluids of phage Kappa lysogens or their corresponding cured derivatives was not significantly different. Therefore, we find no evidence for a causal relationship between phage Kappa and either qualitative or quantitative toxin production. Phage involvement in establishment in the host. For the host to develop the symptoms of cholera, V. cholerae must pass through the highly acidic stomach and establish itself in the small intestine. Therefore, the possibility of a role for phage Kappa in pH tolerance or colonization of the gut was investigated. The inactivations of Mak 757 and Mak 757 (Kappa) after dilution into pH 4.5 peptone-saline were not significantly different (see Fig. 6A), suggesting that phage Kappa probably plays no role in survival of vibrios in passing through the stom-
TABLE 2. Correlation of pathogenicity and lysogeny Avgs fluid accumulation
Biotype.serotypea
Lysogeny
Mak757 [RV79] Mak 757 (VcA-2) Mak 757 (VcA-1) Mak 757 (Kappa)
ET-O ET-O ET-O ET-O
VcA-2 VcA-1 Kappa-10
1.73 1.54 1.67 1.90
EW 1 [RV 26] EW 1 (Kappa)
ET-O ET-O
Kappa-10
0.1 [4; 8] 0.2 [4; 8]
EW6 [RV7] EW 6 (Kappa)
ET-O ET-O
Kappa-10
0.0 [3; 6] 0.0 [3; 6]
Cq 1651 [RV 16] Cq 1651 VcA-2 sens-1 Cq 1651 VcA-2 sens 1 (VcA-2) Cq 1651 VcA-2 sens-1 (VcA-1)
ET-I ET-I ET-I ET-I
Kappa-16 Cured VcA-2 VcA-1
1.71 1.95 1.74 1.92
HK-1 [RV 10] HK-1 VcA-2 sens-1 HK-1 VcA-2 sens-1 (VcA-2)
ET-O ET-O ET-O
Kappa- 10 Cured VcA-2
2.10 [4; 8] 2.00 [4; 8] 1.70 [4; 8]
C-I C-I C-I
dVcA-1,2 dVcA-1 dVcA-1
2.62 [7; 13] 1.62 [2; 3] 2.21 [1; 3]
Strain
569B [RV 5] 569B VcA-2 sens-3 569B VcA-2 sens-3 Thy
(ml/cm)"
[9; 23] [5; 15] [5; 15] [4; 8]
[3; [2; [2; [2;
6] 4] 4] 4]
ET, El Tor; C, classical; 0, Ogawa; I, Inaba. b Fluid accumulation 24 h after injection of approximately 10' log-phase cells/ml. Numbers in brackets refer to number of rabbits and number of ileal loops injected to arrive at the mean value given. a
TABLE 3. Correlation of toxigenicity and lysogeny Strain Strain
569B [RV5] 12-h culture 569B VcA-2 sens-3 12-h culture Mak 757 [RV 79] 12-h culture Mak 757 (Kappa 10) 12-h culture 569B [RV5] 569B UV induced 569B MC induced Mak 757 (Kappa- 10) Mak 757 (Kappa-10) UV induced Mak 757 (Kappa- 10) MC induced 569B VcA-2 sens-3 infected with VcA-2 Cq 1651 VcA-2 sens-1 Infected with VcA-1 Infected with VcA-2 Control
Toxin
(Cg/ml)
Amt" (,) Amt (Ad)
(3Round-up ~~~~~~(3h)'
Steroidc (24 h)
Estimated'd
ND
ND
ND
ND
20
ND
ND
ND
ND
20
50
+/-
36.0
0.5
