Vol. 24, No. 1
INFECTION AND IMMUNITY, Apr. 1979, p. 181-187 0019-9567/79/04-0181/07$02.00/0
Assessment of Protease (Elastase) as a Pseudomonas aeruginosa Virulence Factor in Experimental Mouse Burn Infection OLGERTS R. PAVLOVSKIS' * AND BENGT WRETLIND2 Department ofMicrobiology, Naval Medical Research Institute, Bethesda, Maryland 20014,' and Department of Clinical Microbiology, Karolinska Hospital, S-10401 Stockholm, Sweden' Received for publication 18 January 1979
The data presented indicate that in experimental Pseudomonas aeruginosa infection of mice, protease enhances the virulence of the organism. Anesthetized CBA/Lu mice were subjected to a 15-s flame burn and infected with a wild-type protease-producing strain and two of its protease-deficient mutants. The average bacterial cell mean lethal dose (LD5o) of 3.8 ± 0.3 standard deviation logoi) for mice infected with the protease-producing P. aeruginosa was at least 1 log lower than the LD5o of the protease-deficient mutants (0.02 > P > 0.01). The addition of purified protease to the infecting inoculum of protease-deficient strains reduced the LD50. Although the generation time in vitro was the same for all three bacterial strains used, there were consistently fewer viable bacteria in the blood of mice infected with protease-deficient strains than in those infected with the protease-producing strain. When a protease-deficient strain was mixed with the protease-producing wild-type strain, the number of protease-producing pseudomonas found in the blood remained constant, whereas the number of proteasedeficient organisms increased, suggesting that protease contributed to the invasiveness of the organisms. The survival of mice infected with protease-producing pseudomonas was enhanced by antiprotease serum. Antiprotease serum had no effect in mice infected with protease-deficient mutants. The pathogenesis of Pseudomonas aeruginosa infections has been investigated by examining a variety of extracellular products. Although exotoxin A (2, 3, 23) has been shown to be an important virulence factor in experimental infections (32, 33, 36, 37), other pseudomonas products have been implicated as well (21, 24, 30, 43). The proteolytic enzymes have aroused special interest since Carney et al. (4) presented evidence that they contribute to the invasiveness of the organisms. Despite the accumulating information on the biological activities of pseudomonas proteases (8, 10-12, 14, 15, 17, 18, 25), their roles in infections have not been clarified. Muszynski (31) found a correlation between pseudomonas protease production in vitro and virulence for mice. Others have shown that pseudomonas strains isolated from patients with septicemia produced larger amounts of protease than strains from other sources (A. Davis and J. R. Doubek, Abstr. 265, and A. L. Baltch, P. E. Griffin, and E. Malatino, Abstr. 266, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 15th, Washington, D. C., 1975). In contrast, Wretlind et al. (39) and Hedberg et al. (12) found no significant differences in production of
extracellular proteases between strains isolated from septicemic patients and other sources, nor did they find differences in virulence for mice between protease-producing and protease-deficient strains (40). Liu and Hsieh (22) found a negative correlation between in vitro protease production and virulence: high-titer protease producers were less virulent than low-titer protease producers. Several reasons may be suggested for the conflicting results. It has been shown, for example, that the amount of protease production is influenced by the experimental conditions (17, 42) and that there are differences in susceptibility to pseudomonas infection within the same species of animal (16; 0. R. Pavlovskis and B. Wretlind, unpublished data). However, the most probable explanation is a lack of a good infection model. Most experimental models used to study pseudomonas pathogenesis are inadequate substitutes for clinical infections, because unrealistically large doses of organisms or virulence-enhancing factors, such as mucin, are required to infect animals. Moreover, severely traumatized animals are difficult to use since the injury often contributes to their death. 181
182
PAVLOVSKIS AND WRETLIND
Recently, I. A. Holder (Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, B118, p. 35) and Pavlovskis and Wretlind (Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, B89, p. 28) have used a mouse burn infection model (13, 38) that resembles human burn wound sepsis to show that protease producers are more virulent than protease-deficient strains. In this model, the burn is nonlethal and infection is reproducibly produced by a small inoculum. Since the in vivo role of a given product of an organism can best be studied by using mutants lacking the ability to produce it, we examined the role of protease in pseudomonas infection by using a protease-producing wild type and its protease-deficient mutants. A similar approach has been used to define virulence factors of salmonella and Staphylococcus aureus in experimental animal infections (9, 19). By examining the protective effect of antiprotease serum in this model, we attempted to define the pathogenic role of protease as well as evaluate the immunoprophylactic potential of antiprotease serum. The results of this study indicate that protease enhances the virulence of pseudomonas and that its deleterious effects can be neutralized to some extent with antiprotease serum.
MATERIALS AND METHODS Bacterial strains. P. aeruginosa PAKS-1 (wild type) was isolated from a human urine specimen (42). In vitro the strain produced all three of the pseudomonas proteases described by Morihara and co-workers (27, 28) and Kreger and Griffin (18). The isoelectric points of proteases I, II, and III were 8.5, 6.6, and 4.5, respectively. Protease II possessing elastase activity (PR II) (29, 42) was responsible for more than 75% of the extracellular proteolytic activity (42). PAKS-1 also produced extracellular lipase, staphylolytic enzyme, hemolysin, and exotoxin A (Table 1). The protease-deficient mutant PAKS-17 was isolated according to the procedure described by Meynell and Meynell (26), using ethyl methane sulfonate as a mutagen. The bacteria were grown for 18 to 24 h on brain heart infusion (BHI) agar plates containing 15% skim milk. The protease-producing organisms gave clear zones around single colonies, in contrast to protease-deficient mutants. The protease-deficient strain PAKS-10 was a spontaneous mutant (41). Depending on experimental conditions, the proteolytic activity of the mutants varied in vitro from 1 to 10% of the activity of PAKS-1. The strains were prototrophic, and their in vitro generation time was the same as that of the wild type (41). The serotype, phage type, and antibiograms of PAKS-10 and PAKS-17 remained unchanged. PAKS-10 demonstrated slight elastase activity. PAKS-17 did not produce detectable amounts of PR II (elastase), as determined by elastin agar plates (39) and electrophoresis of culture supernatants, and only minute amounts of protease III (pI 4.5). The mutants were deficient also in lipase and staphylolytic enzyme, but produced exotoxin A (Table 1; 40, 41).
INFECT. IMMUN. TABLE 1. Properties of P. aeruginosa strain PAKS1 and its protease-deficient mutants Extracellular enzyme activities' (U/mg of cell [dry wt]) Strain Strain Pro-
StaphyStap .y
taelolytic tease enzyme
PAKS-1
9.4
Lipase
0.70
14.5
Bacterial LDM, in ExocycloA" phosphamideml) treated mice mice"
~~~~~toxin (LD50,/
(log,)
4
6.13
0.5 10 0 6.35 PAKS-10 0.4 2 0 0 6.19 PAKS-17 0.1 a Assays for staphylolytic enzyme and lipase have been described (41). 'Exotoxin A was determined in sterile, crude culture supernatants by mouse lethality tests. The organisms were grown in dialyzed Trypticase soy broth with nitrilotriacetate to inhibit protease production (3, 40). The lethal effect was completely inhibited by specific antitoxin A (3). c Determined by intraperitoneal challenge 4 days after injection of cyclophosphamide. Data from prior work (40).
Challenge inoculum. The inoculum was prepared as previously described (33).
Mouse burn infection model. The mouse burn infection model described by Holder and Jogan (13) and Stieritz and Holder (38) was used. Since the NIH/ NMRI strain available in Stockholm was significantly more resistant to pseudomonas infection than mice used previously (33), several other strains of mice were tested for susceptibility. Inbred (65th generation) CBA/LU mice from the Laboratory of Radiation Genetics, Stockholm (6), weighing 18 to 20 g, were selected for this study because of their high susceptibility. The backs of female mice were shaved, and the mice were anesthetized with methoxyflurane (Penthrane, Abbott Laboratories, North Chicago, Ill.). A template with a 2.5-by-2.5-cm opening (corresponding to approximately 15% of total body surface) was placed on the shaved area, covered with 0.5 ml of 95% ethanol, and flamed for 15 s. The burn was nonlethal and did penetrate the musculature slightly, but did not reach the abdominal organs (Pavlovskis and Wretlind, un-
published data; Holder, personal communication). Bacteria in logarithmic phase of growth were suspended shortly before use in phosphate-buffered saline (23"C) at a desired concentration and placed in an ice bath, and 0.5-ml volumes were injected subcutaneously in the burn area immediately after the trauma. Quantitation of bacteria in blood. Mice were anesthetized and blood was obtained from the brachial artery. The number of colony-forming units of P. aeruginosa per milliliter of blood was determined by serial dilution plate counts on BHI agar plates containing 15% skim milk. Mean values were determined from counts of three mice. PR II. PR II from P. aeruginosa PAKS-1 was purified by ammonium sulfate precipitation, isoelectric focusing, and gel chromatography (42). On disc electrophoresis and on conventional immunoelectrophoresis, the preparation showed one band and did not contain exotoxin A, hemolysin, or staphylolytic
P. AERUGINOSA PROTEASE
VOL. 24, 1979 activity. PR II appears to be identical to the protease (elastase) purified and characterized by Morihara et al. (29). The specific activity of PR II was 350 U per mg of protein; the mean intravenous lethal dose (LD50) for an 18- to 20-g mouse was 100 jig (35 U), in good agreement with the results obtained by Meinke et al. (25) and Kawaharajo et al. (17). The
enzyme
molecular weight of the enzyme was 23,000, and its isoelectric point was 6.6 ± 0.6. The toxicity of the PR II is lower than that of exotoxin A (LD50 = 60 to 80 ng) (3). Antisera. Anti-bovine serum albumin serum (antiBSA) was prepared in rabbits as reported (33). Antiprotease (anti-PR II) serum was prepared by injecting intramuscularly into male New Zealand white rabbits, weighing 3 to 5 kg, 200 jg of purified PR II mixed with 2 volumes of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.) and by injecting the same mixture subcutaneously twice at 3-week intervals. Both anti-BSA and anti-PR II were heat inactivated and absorbed with whole cells of the appropriate challenge strain to eliminate possible antibodies against external cell envelope components (33). After the final absorption, antisera were centrifuged at 105,000 x g for 4 to 6 h to remove contaminating cell fragments and lipopolysaccharide. By this treatment, unlike previous experience (33), we were able to inject intravenously at least 0.4 ml of serum without observing any adverse effects in the mice. The absorbed anti-PR II serum neutralized the activity of PR II (Fig. 1). Neither rabbit antitoxin A (33) nor anti-BSA sera neutralized the activity of PR II data not shown). Antibodies against exotoxin A could not be demonstrated in anti-BSA and anti-PR II sera used in the present work by passive hemagglutination (33), counterimmunoelectrophoresis (42), or neutralization of the lethal effect for mice (3). Assays. Protease activity was determined according to Wretlind and Wadstrom (42), using heat-denatured casein as substrate. Elastase activity was estimated by the elastin-congo red solubilization method of Sachar et al. (35); staphylolytic enzyme activity was estimated with heat-killed staphylococcal cells as the substrate; and lipase activity (substrate: p-nitrophenyl caprylate) was estimated as reported (42). loo r 0
183
Exotoxin A was determined by the mouse lethality test as described by Callahan (3), except that the preparations were given intraperitoneally. Statistical analysis. Survival distributions were compared by using the Wilcoxon rank sum test as described by Bradley (1). The P values were computed by using a normal approximation with continuity correction. The P values are consistent with those drawn from available Wilcoxon rank sum test tables. The mean lethal dose (LD5o) was calculated by the Spearman-Karber method described by Finney (7). Analysis of variance (5), a method for testing for significant differences among two or more means, was used to compare LD50 of the Pseudomonas strains. If means of subgroups are significantly different, the variance of the combined group is much larger than the variances of separate groups, and the analysis is based on this fact.
RESULTS Subcutaneous injections of viable PAKS-1, PAKS-10, or PAKS-17 in burn sites resulted in lethal infections associated with bacteremia and systemic invasion. Each of the three challenge strains was lethal at a dose characteristic for the individual strain (Table 2). The LD5o of the wildtype strain was 1 log lower than that of its protease-deficient mutants, PAKS-10 and PAKS-17. Analysis of variance (5) showed that the difference in virulence between the strains was significant [F(2/6 df) = 9.81; 0.02 > P > 0.01]. Postmortem examination of infected mice showed fatty infiltration of the liver (40) suggestive of exotoxin A (34), which they all produced (Table 1). No lesions were seen in the organs of burned, noninfected mice. Similar observations were made by Holder (personal communication). When mice were injected simultaneously with 1 LD50 of PAKS-10 and varying amounts of purified PR II, there was a corresponding decrease both in the percentage of mice surviving the infection and in the survival time (Fig. 2). Comparable results were obtained with PAKS-17 (data not shown). Both Holder (Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, B118, p. 35) and Snell et al. (37) also observed an increase
80
TABLE 2. LD50 of P. aeruginosa strain PAKS-1 and its protease-deficient mutants in burned micea
> 60
,
40
LDr,o logoi)
An 4
cr
Strain
Average ±
Expt no.
20
I 2
4
8 64 16 32 RECIPROCAL OF SERUM DILUTION
128
256
FIG. 1. Titer of PAKS-1 (whole cells)-absorbed anti-PR II serum as determined by its ability to inhibit PR H activity. Concentration ofprotease: 6.3 pg/ml.
II
III
SD"
PAKS-1 4.1 3.9 3.5 3.8 ± 0.3 5.1 PAKS-10 4.6 4.7 4.8 + 0.3 PAKS-17 5.3 4.7 4.6 4.9 ± 0.4 a Serial 10-fold dilutions of bacterial cells were used. Each dilution was injected into five mice. b SD, Standard deviation.
184
PAVLOVSKIS AND WRETLIND
INFECT. IMMUN.
exogenous protease. Burned, noninfected mice injected with 50 ,ug of PR II did not differ from burned, noninfected controls in either mortality (Fig. 2) or appearance. The above experiments suggested that elastase (PR II) contributes to the pathogenesis of the organism. To substantiate this observation, mice were passively immunized with anti-PR II serum before infection. In the first series of experiments mice were injected intravenously with 0.2 ml of anti-PR II serum, and 20 h later they were burned and infected with 1 LD50 of PAKS-10 and varying amounts of PR II (Fig. 3). Control mice were administered an equivalent volume of anti-BSA serum. The anti-PR II serum was able to neutralize to some extent the increased virulence of pseudomonas resulting
from low doses of exogenous protease (4.8 and 9.6 Mug) (P < 0.001); however, when mice were given excessive amounts of PR II (19.2 Mig), the anti-PR II had no effect (P > 0.05). Similar results were also obtained with PAKS-17 (data not shown). In the second series of experiments, groups of mice were administered 0.4 ml of anti-PR II or anti-BSA serum and then burned and infected 20 h later with 1 LD5o of PAKS-1, PAKS-10, or PAKS-7. The survival rates of the infected mice are shown in Fig. 4. Anti-PR II prolonged survival only in mice infected with the proteaseproducing strain, PAKS-1 (P < 0.001) (Fig. 4A), whereas it failed significantly to protect mice infected with protease-deficient strains (P > 0.05) (Fig. 4B and C). In fact, mice given antiPR II serum died more rapidly than control ,00 mice, but this difference was not significant. In both series of experiments (Fig. 3 and 4), antiso8 PR II was able to neutralize some of the effects of the protease and thus increase the survival of the infected mice. Thus the results indicate that I I o protease enhances the virulence of the organisms. Finally, burned mice were infected with 1 LDMm0 of one of three challenge strains and sacrificed sequentially 18 to 48 h later. Their blood , 0 20 40 100 60 I60 80 120 140 IS0 was cultured quantitatively on BHI-skim milk HOURS POST INFECTIM FIG. 2. Survival of burned mice infected with agar plates in order to distinguish between proPAKS-10, protease-deficient P. aeruginosa, and vary- tease-producing and protease-deficient colonies. ing amounts of PR I (0, no PR II; *, 6.0ug of PR II; The mice infected with protease-deficient strains A, 15.0 ,ug; A, 30.0 pg, x, 45.0 ug) and of burned, had consistently lower viable bacterial counts in noninfected mice injected with 50 pg PR II (O). Five blood than mice infected with PAKS-1 (Fig. 5A). mice were used for each determination. However, when burned mice were infected with --0 .............
-....
I
100 4,8 Lg PR I
0 *g PR II 80 60 40
20 r 140
I
o'
cr
CE
CL.
IC
9.6,sg 30
20 V
PR U
19.2 pLg PR
-
-
Fx 0
K
40
so
120
160
0
40
80
120
160
HOURS POST-INFECTION FIG. 3. Survival of mice pretreated with anti-PR II serum (0) or anti-BSA serum (0), burned, and infected with protease-deficient P. aeruginosa (PAKS-10) and varying amounts of PR II. Twenty mice were used per experiment: 10pretreated with anti-PR II serum and 10pretreated with anti-BSA serum.
P. AERUGINOSA PROTEASE
VOL. 24, 1979
C. PAKS-17
B. PAKS-1O
A. PAKS -I
185
°°0r 80
F
-j
I-
>0 4
w
0.1 20k I
l
40 80 120 HOURS POST- INFECTION
0
160
IlI 40 80 120
160
0
0
40
120
80
160
HOURS POST-INFECTION
HOURS POST-INFECTION
FIG. 4. Survival of burned, infected mice pretreated with anti-PR II serum (0) or anti-BSA serum (0). Forty mice were used for experiment A, and 20 mice each were used for experiments B and C. Halfof the mice in each group were pretreated with anti-PR II serum, and half were treated with anti-BSA serum. 0-PAKS 0 - PAKS 10 A -PAKS 17
A
.--
C
B
i/;
4
0
3E
3
cry CD) -
c#*
2 co 0 )-
C.) 40
MIXED INFECTIfN PAKS IS 10
SINGLE INFECTION
0
L-
I
I
I
o
20
40
60
fI 0
MIXED INFECTION PAKS I & 17
I
I
I
I
I
I
20
40
60
0
20
40
I~~_
60
HOOURS POST-INFECTION FIG. 5. Number ofpseudomonas colony-forming units ± standard error of the mean per milliliter of blood of burned infected mice. (A) Mice infected with either PAKS-i, PAKS-IO, or PAKS-i 7. (B) Mice infected with PAKS-I and PAKS-IO. (C) Mice infected with PAKS-i and PAKS-i 7.
186
PAVLOVSKIS AND WRETLIND
a mixture of PAKS-1 and PAKS-10 or PAKS-1 and PAKS-17 at a 1:1 ratio, the number of protease-deficient organisms in the blood was considerably higher (Fig. 5B and 5C) in the mixed infections than in the previous experiment (Fig. 5A). The results suggest that the in vivo production of protease by PAKS- 1 also enhances the invasiveness of the protease-deficient bacteria, PAKS-10 and PAKS-17.
INFECT. IMMUN.
mice. In the second model, the sensitivity of the host to the invading organism was significantly increased by the mucin. In our previous study on passive protection by antitoxin in experimental P. aeruginosa infections, it became obvious that exotoxin A was only one of a number of pathogenic factors involved in pseudomonas infections. Our study, as well as the work of Kawaharajo and Homma (10) and I. A. Holder, C. B. Saelinger, C. G. Haidaris, and M. Michael (Abstr. Annu. Meet. DISCUSSION Soc. Microbiol. 1978, B94, p. 29), who The mechanism by which protease exerts its Am. showed that mice immunized with elastase or its toxic action is not known. In septicemic infec- toxoid showed increased resistance to experition, protease may contribute to tissue damage mental P. aeruginosa infections, suggests that when it is produced locally by pseudomonas. B. this protease contributes to the pathogenesis of H. Iglewski, for example, found protease in tissue organism. Thus, it appears that protease is homogenates from pseudomonas-infected mice the rather than a major a virulence-enhancing (personal communication). Snell et al. (37) found virulence factor. The factor also indipresented data that the subcutaneous injection of purified pro- cate that to some extent these effects may be tease reduced elongation factor 2 activity in neutralized by antiprotease serum. These obsermouse liver. However, previous work with non- vations should provide a broader basis for studtoxigenic, protease-producing strains indicates leading to immunoprophylaxis and treatment that protein synthesis inhibition by protease ies infections. of pseudomonas during an infection is minimal (32) and that extremely high doses are required to kill mice as ACKNOWLEDGMENTS compared with toxigenic strains. It is unlikely, We thank E. Weiss, S. Joseph, and A. Eiche for their therefore, that protease by itself is responsible suggestions and comments throughout this study. We are also for the lethality of the organism. Our present grateful to K. Karlsson and G. Hare for their expert technical O.R.P. thanks the staff of the Department of data show an increase in the numbers of pseu- assistance. Clinical Microbiology at the Karolinska Hospital, where most domonas organisms in the blood of mice infected of this research was carried out. with protease-producing organisms (Fig. 5), sugThis study was supported by the U.S. Naval Research and gesting that protease may be important in ov- Development Command, Research Work Unit no. and the Research Institute of the Swedish ercoming the host's initial defense mechanisms. M0095PN001.0009, This may be accomplished either by proteolytic National Defense. action which provides additional nutrients, as LITERATURE CITED suggested by J. F. Cicmanec, C. G. Haidaris, and 1. Bradley, J. V. 1968. Distribution-free statistical tests, p. I. A. Holder (Abstr. Annu. Meet. Am. Soc. Mi105-114. Prentice-Hall, Inc., Englewood, N.J. crobiol. 1978, B90, p. 28), or by destruction of 2. Callahan, L. T., III. 1974. Purification and characterization of Pseudomonas aeruginosa exotoxin. Infect. Imanatomical barriers and more rapid invasion of mun. 9:113-118. the host. The latter possibility is indirectly sup- 3. Callahan, L. T., III. 1976. Pseudomonas aeruginosa ported by the work of Wretlind and Kronevi exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of highly specific (40), who infected cyclophosphamide-treated antitoxin serum. Infect. Immun. 14:55-61. mice with PAKS-1 or protease-deficient mu- 4. Carney, S. A., R. E. Dyster, and R. J. Jones. 1973. The tants. They found no difference in LD5,0 between invasion of burned skin by Pseudomonas aeruginosa. PAKS-1, PAKS-10, and PAKS-17. Neither did Br. J. Dermatol. 88:539-545. they find any difference in LD.% between PAKS- 5. Dixon, W. J., and F. J. Massey, Jr. 1951. Analysis of variance, p. 119-152. In Introduction to statistical anal1 and PAKS-10 when these strains were injected ysis. McGraw-Hill Book Co., New York. with mucin into normal mice. Both models were 6. Eiche, A. 1977. Effects of low dose X-irradiation on intrainitial defense host's designed to overcome the uterine death in mice subjected to exposure when young or at foetal stage. Hereditas 85:63-66. mechanisms, thus negating any advantage a proD. J. 1964. The Spearman-Karber method, p. tease-producing strain might have. In the first 7. Finney, 524-530. In Statistical methods in biological assay, 2nd model the host's initial defense mechanisms ed. Charles Griffin and Co., London. were already significantly lowered by the cyclo- 8. Fisher, E., Jr., and J. H. Allen. 1958. Mechanism of corneal destruction by Pseudomonas proteases. Am. J. phosphamide. In comparable experiments (PavOphthalmol. 46:249-254. lovskis and Wretlind, unpublished observa- 9. Forsgren, A. 1972. Pathogenicity of Staphylococcus autions), the leukocyte count dropped from apreus mutants in general and local infections. Acta Paproximately 8,000/mm3 for normal mice to less thol. Microbiol. Scand. Sect. B 80:564-570. than 500/mm3 for cyclophosphamide-treated 10. Gerke, J. R., and M. V. Magliocco. 1971. Experimental
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11. 12. 13. 14.
15.
16.
17.
18.
19.
20.
21. 22.
Pseudomonas aeruginosa infection of the mouse cornea. Infect. Immun. 3:209-216. Gray, L. D., and A. S. Kreger. 1975. Rabbit corneal damage produced by Pseudomonas aeruginosa infection. Infect. Immun. 12:419-432. Hedberg, M., J. K. Miller, and V. N. Tompkins. 1969. Elastase activity of Pseudomonas aeruginosa isolates from hospital patients. Am. J. Clin. Pathol. 52:631-633. Holder, I. A., and M. Jogan. 1971. Enhanced survival in burned mice treated with antiserum prepared against normal and burned skin. J. Trauma 11:1041-1046. Kawaharajo, K., C. Abe, J. Y. Homma, M. Kawano, E. Gotoh, N. Tanaka, and K. Morihara. 1974. Corneal ulcers caused by protease and elastase from Pseudomonas aeruginosa. Jpn. J. Exp. Med. 44:435-442. Kawaharajo, K., and J. Y. Homma. 1975. Pathogenesis of the mouse keratitis produced with Pseudomonas aeruginosa. Jpn. J. Exp. Med. 45:515-524. Kawaharajo, K., and J. Y. Homma. 1977. Effects of elastase, protease and common antigen (OEP) from Pseudomonas aeruginosa on protection against burns in mice. Jpn. J. Exp. Med. 47:495-500. Kawaharajo, K., J. Y. Homma, Y. Aoyama, and K. Morihara. 1975. In vivo studies on protease and elastase from Pseudomonas aeruginosa. Jpn. J. Exp. Med. 45:89-100. Kreger, A. S., and D. K. Griffin. 1974. Physiochemical fractionation of extracellular cornea-damaging proteases of Pseudomonas aeruginosa. Infect. Immun. 9: 828-834. Lindberg, A. A., L. T. Rosenberg, A. Ljunggren, P. J. Garegg, S. Svensson, and N.-H. Wallin. 1974. Effect of synthetic disaccharide-protein conjugate on an immunogen in Salmonella infection in mice. Infect. Immun. 10:541-545. Liu, P. V. 1966. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. III. Identity of the lethal toxins produced in vitro and in vivo. J. Infect. Dis. 166:481-489. Liu, P. V. 1974. Extracellular toxins of Pseudomonas aeruginosa. J. Infect. Dis. 130(Suppl.):S94-S99. Liu, P. V., and H. Hsieh. 1969. Inhibition of protease production of various bacteria by ammonium salts: its effect on toxin production and virulence. J. Bacteriol.
99:406-413. 23. Liu, P. V., S. Yoshii, and H. Hsieh. 1973. Exotoxins of
Pseudomonas aeruginosa. II. Concentration, purification, and characterization of exotoxin A. J. Infect. Dis. 128:514-519. 24. Lynn, M., J. W. Sensakovic, and P. F. Bartell. 1977. In vivo distribution of Pseudomonas aeruginosa slime glycolipoprotein: association with leukocytes. Infect. Immun. 15:109-114. 25. Meinke, G., J. Barum, B. Rosenberg, and R. Berk. 1970. In vivo studies with the partially purified protease (elastase) from Pseudomonas aeruginosa. Infect. Immun. 2:583-589. 26. Meynell, C. G., and E. Meynell (ed.). 1970. Genetic techniques, p. 256-294. In Theory and practice in ex-
perimental bacteriology, 2nd ed. Cambridge University Press, London. 27. Morihara, K. 1964. Production of elastase and proteinase by Pseudomonas aeruginosa. J. Bacteriol. 88:745-757.
187
28. Morihara, K., and H. Tsuzuki. 1977. Production of protease and elastase by Pseudomonas aeruginosa strains isolated from patients. Infect. Immun. 15:679685. 29. Morihara, K., H. Tsuzuki, T. Oka, H. Inoue, and M. Ebata. 1965. Pseudomonas aeruginosa elastase. Isolation, crystallization, and preliminary characterization. J. Biol. Chem. 240:3295-3302. 30. Mull, J. D., and W. S. Callahan. 1965. The role of elastase of Pseudomonas aeruginosa in experimental infections. Exp. Mol. Pathol. 4:567-575. 31. Muszynski, Z. 1973. Enzymatic and toxigenic activity of culture filtrates of high and low virulent strains of Pseudomonas aeruginosa in mice. Pathol. Microbiol. 39: 135-147. 32. Pavlovskis, O. R., B. H. Iglewski, and M. Pollack. 1978. Mechanism of action of Pseudomonas aeruginosa exotoxin A in experimental mouse infections: adenosine diphosphate ribosylation of elongation factor 2. Infect. Immun. 19:29-33. 33. Pavlovskis, 0. R., M. Pollack, L. T. Callahan III, and B. H. Iglewski. 1977. Passive protection by antitoxin in experimental Pseudomonas aeruginosa burn infections. Infect. Immun. 18:596-602. 34. Pavlovskis, 0. R., F. A. Voelker, and A. H. Shackelford. 1976. Pseudomonas aeruginosa exotoxin in mice: histopathology and serum enzyme changes. J. Infect. Dis. 133:253-259. 35. Sachar, L. A., K. K. Winter, N. Sicher, and S. Frankel. 1955. Photometric method for estimation of elastase activity. Proc. Soc. Exp. Biol. Med. 90:323-326. 36. Saelinger, C. B., K. Snell, and I. A. Holder. 1977. Experimental studies on pathogenesis of infections due to Pseudomonas aeruginosa: direct evidence for toxin production during pseudomonas infection of burned skin tissues. J. Infect. Dis. 136:555-561. 37. Snell, K., I. A. Holder, S. A. Leppla, and C. B. Saelinger. 1978. Role of exotoxin and protease as possible virulence factors in experimental infection with Pseudomonas aeruginosa. Infect. Immun. 19:839-845. 38. Stieritz, D. D., and I. A. Holder. 1975. Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: description of a burned mouse model. J. Infect. Dis. 131:688-691. 39. Wretlind, B., L. Heden, L. Sjoberg, and T. Wadstrom. 1973. Production of enzymes and toxins by hospital strains of Pseudomonas aeruginosa in relation to serotype and phage-typing pattern. J. Med. Microbiol. 6: 91-100. 40. Wretlind, B., and T. Kronevi. 1978. Experimental infections with protease-deficient mutants of Pseudomonas aeruginosa in mice. J. Med. Microbiol. 11:145-154. 41. Wretlind, B., L. Sjoberg, and T. Wadstrom. 1977. Protease-deficient mutants of Pseudomonas aeruginosa: pleiotropic changes in activity of other extracellular enzymes. J. Gen. Microbiol. 103:329-336. 42. Wretlind, B., and T. Wadstrom. 1977. Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa. J. Gen. Microbiol. 103:319327. 43. Young, L. S., and D. Armstrong. 1972. Pseudomonas aeruginosa infections. Crit. Rev. Clin. Lab. Sci. 3:291347.
INFECTION AND IMMUNITY, Apr. 1979, p. 181-187 0019-9567/79/04-0181/07$02.00/0
Assessment of Protease (Elastase) as a Pseudomonas aeruginosa Virulence Factor in Experimental Mouse Burn Infection OLGERTS R. PAVLOVSKIS' * AND BENGT WRETLIND2 Department ofMicrobiology, Naval Medical Research Institute, Bethesda, Maryland 20014,' and Department of Clinical Microbiology, Karolinska Hospital, S-10401 Stockholm, Sweden' Received for publication 18 January 1979
The data presented indicate that in experimental Pseudomonas aeruginosa infection of mice, protease enhances the virulence of the organism. Anesthetized CBA/Lu mice were subjected to a 15-s flame burn and infected with a wild-type protease-producing strain and two of its protease-deficient mutants. The average bacterial cell mean lethal dose (LD5o) of 3.8 ± 0.3 standard deviation logoi) for mice infected with the protease-producing P. aeruginosa was at least 1 log lower than the LD5o of the protease-deficient mutants (0.02 > P > 0.01). The addition of purified protease to the infecting inoculum of protease-deficient strains reduced the LD50. Although the generation time in vitro was the same for all three bacterial strains used, there were consistently fewer viable bacteria in the blood of mice infected with protease-deficient strains than in those infected with the protease-producing strain. When a protease-deficient strain was mixed with the protease-producing wild-type strain, the number of protease-producing pseudomonas found in the blood remained constant, whereas the number of proteasedeficient organisms increased, suggesting that protease contributed to the invasiveness of the organisms. The survival of mice infected with protease-producing pseudomonas was enhanced by antiprotease serum. Antiprotease serum had no effect in mice infected with protease-deficient mutants. The pathogenesis of Pseudomonas aeruginosa infections has been investigated by examining a variety of extracellular products. Although exotoxin A (2, 3, 23) has been shown to be an important virulence factor in experimental infections (32, 33, 36, 37), other pseudomonas products have been implicated as well (21, 24, 30, 43). The proteolytic enzymes have aroused special interest since Carney et al. (4) presented evidence that they contribute to the invasiveness of the organisms. Despite the accumulating information on the biological activities of pseudomonas proteases (8, 10-12, 14, 15, 17, 18, 25), their roles in infections have not been clarified. Muszynski (31) found a correlation between pseudomonas protease production in vitro and virulence for mice. Others have shown that pseudomonas strains isolated from patients with septicemia produced larger amounts of protease than strains from other sources (A. Davis and J. R. Doubek, Abstr. 265, and A. L. Baltch, P. E. Griffin, and E. Malatino, Abstr. 266, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 15th, Washington, D. C., 1975). In contrast, Wretlind et al. (39) and Hedberg et al. (12) found no significant differences in production of
extracellular proteases between strains isolated from septicemic patients and other sources, nor did they find differences in virulence for mice between protease-producing and protease-deficient strains (40). Liu and Hsieh (22) found a negative correlation between in vitro protease production and virulence: high-titer protease producers were less virulent than low-titer protease producers. Several reasons may be suggested for the conflicting results. It has been shown, for example, that the amount of protease production is influenced by the experimental conditions (17, 42) and that there are differences in susceptibility to pseudomonas infection within the same species of animal (16; 0. R. Pavlovskis and B. Wretlind, unpublished data). However, the most probable explanation is a lack of a good infection model. Most experimental models used to study pseudomonas pathogenesis are inadequate substitutes for clinical infections, because unrealistically large doses of organisms or virulence-enhancing factors, such as mucin, are required to infect animals. Moreover, severely traumatized animals are difficult to use since the injury often contributes to their death. 181
182
PAVLOVSKIS AND WRETLIND
Recently, I. A. Holder (Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, B118, p. 35) and Pavlovskis and Wretlind (Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, B89, p. 28) have used a mouse burn infection model (13, 38) that resembles human burn wound sepsis to show that protease producers are more virulent than protease-deficient strains. In this model, the burn is nonlethal and infection is reproducibly produced by a small inoculum. Since the in vivo role of a given product of an organism can best be studied by using mutants lacking the ability to produce it, we examined the role of protease in pseudomonas infection by using a protease-producing wild type and its protease-deficient mutants. A similar approach has been used to define virulence factors of salmonella and Staphylococcus aureus in experimental animal infections (9, 19). By examining the protective effect of antiprotease serum in this model, we attempted to define the pathogenic role of protease as well as evaluate the immunoprophylactic potential of antiprotease serum. The results of this study indicate that protease enhances the virulence of pseudomonas and that its deleterious effects can be neutralized to some extent with antiprotease serum.
MATERIALS AND METHODS Bacterial strains. P. aeruginosa PAKS-1 (wild type) was isolated from a human urine specimen (42). In vitro the strain produced all three of the pseudomonas proteases described by Morihara and co-workers (27, 28) and Kreger and Griffin (18). The isoelectric points of proteases I, II, and III were 8.5, 6.6, and 4.5, respectively. Protease II possessing elastase activity (PR II) (29, 42) was responsible for more than 75% of the extracellular proteolytic activity (42). PAKS-1 also produced extracellular lipase, staphylolytic enzyme, hemolysin, and exotoxin A (Table 1). The protease-deficient mutant PAKS-17 was isolated according to the procedure described by Meynell and Meynell (26), using ethyl methane sulfonate as a mutagen. The bacteria were grown for 18 to 24 h on brain heart infusion (BHI) agar plates containing 15% skim milk. The protease-producing organisms gave clear zones around single colonies, in contrast to protease-deficient mutants. The protease-deficient strain PAKS-10 was a spontaneous mutant (41). Depending on experimental conditions, the proteolytic activity of the mutants varied in vitro from 1 to 10% of the activity of PAKS-1. The strains were prototrophic, and their in vitro generation time was the same as that of the wild type (41). The serotype, phage type, and antibiograms of PAKS-10 and PAKS-17 remained unchanged. PAKS-10 demonstrated slight elastase activity. PAKS-17 did not produce detectable amounts of PR II (elastase), as determined by elastin agar plates (39) and electrophoresis of culture supernatants, and only minute amounts of protease III (pI 4.5). The mutants were deficient also in lipase and staphylolytic enzyme, but produced exotoxin A (Table 1; 40, 41).
INFECT. IMMUN. TABLE 1. Properties of P. aeruginosa strain PAKS1 and its protease-deficient mutants Extracellular enzyme activities' (U/mg of cell [dry wt]) Strain Strain Pro-
StaphyStap .y
taelolytic tease enzyme
PAKS-1
9.4
Lipase
0.70
14.5
Bacterial LDM, in ExocycloA" phosphamideml) treated mice mice"
~~~~~toxin (LD50,/
(log,)
4
6.13
0.5 10 0 6.35 PAKS-10 0.4 2 0 0 6.19 PAKS-17 0.1 a Assays for staphylolytic enzyme and lipase have been described (41). 'Exotoxin A was determined in sterile, crude culture supernatants by mouse lethality tests. The organisms were grown in dialyzed Trypticase soy broth with nitrilotriacetate to inhibit protease production (3, 40). The lethal effect was completely inhibited by specific antitoxin A (3). c Determined by intraperitoneal challenge 4 days after injection of cyclophosphamide. Data from prior work (40).
Challenge inoculum. The inoculum was prepared as previously described (33).
Mouse burn infection model. The mouse burn infection model described by Holder and Jogan (13) and Stieritz and Holder (38) was used. Since the NIH/ NMRI strain available in Stockholm was significantly more resistant to pseudomonas infection than mice used previously (33), several other strains of mice were tested for susceptibility. Inbred (65th generation) CBA/LU mice from the Laboratory of Radiation Genetics, Stockholm (6), weighing 18 to 20 g, were selected for this study because of their high susceptibility. The backs of female mice were shaved, and the mice were anesthetized with methoxyflurane (Penthrane, Abbott Laboratories, North Chicago, Ill.). A template with a 2.5-by-2.5-cm opening (corresponding to approximately 15% of total body surface) was placed on the shaved area, covered with 0.5 ml of 95% ethanol, and flamed for 15 s. The burn was nonlethal and did penetrate the musculature slightly, but did not reach the abdominal organs (Pavlovskis and Wretlind, un-
published data; Holder, personal communication). Bacteria in logarithmic phase of growth were suspended shortly before use in phosphate-buffered saline (23"C) at a desired concentration and placed in an ice bath, and 0.5-ml volumes were injected subcutaneously in the burn area immediately after the trauma. Quantitation of bacteria in blood. Mice were anesthetized and blood was obtained from the brachial artery. The number of colony-forming units of P. aeruginosa per milliliter of blood was determined by serial dilution plate counts on BHI agar plates containing 15% skim milk. Mean values were determined from counts of three mice. PR II. PR II from P. aeruginosa PAKS-1 was purified by ammonium sulfate precipitation, isoelectric focusing, and gel chromatography (42). On disc electrophoresis and on conventional immunoelectrophoresis, the preparation showed one band and did not contain exotoxin A, hemolysin, or staphylolytic
P. AERUGINOSA PROTEASE
VOL. 24, 1979 activity. PR II appears to be identical to the protease (elastase) purified and characterized by Morihara et al. (29). The specific activity of PR II was 350 U per mg of protein; the mean intravenous lethal dose (LD50) for an 18- to 20-g mouse was 100 jig (35 U), in good agreement with the results obtained by Meinke et al. (25) and Kawaharajo et al. (17). The
enzyme
molecular weight of the enzyme was 23,000, and its isoelectric point was 6.6 ± 0.6. The toxicity of the PR II is lower than that of exotoxin A (LD50 = 60 to 80 ng) (3). Antisera. Anti-bovine serum albumin serum (antiBSA) was prepared in rabbits as reported (33). Antiprotease (anti-PR II) serum was prepared by injecting intramuscularly into male New Zealand white rabbits, weighing 3 to 5 kg, 200 jg of purified PR II mixed with 2 volumes of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.) and by injecting the same mixture subcutaneously twice at 3-week intervals. Both anti-BSA and anti-PR II were heat inactivated and absorbed with whole cells of the appropriate challenge strain to eliminate possible antibodies against external cell envelope components (33). After the final absorption, antisera were centrifuged at 105,000 x g for 4 to 6 h to remove contaminating cell fragments and lipopolysaccharide. By this treatment, unlike previous experience (33), we were able to inject intravenously at least 0.4 ml of serum without observing any adverse effects in the mice. The absorbed anti-PR II serum neutralized the activity of PR II (Fig. 1). Neither rabbit antitoxin A (33) nor anti-BSA sera neutralized the activity of PR II data not shown). Antibodies against exotoxin A could not be demonstrated in anti-BSA and anti-PR II sera used in the present work by passive hemagglutination (33), counterimmunoelectrophoresis (42), or neutralization of the lethal effect for mice (3). Assays. Protease activity was determined according to Wretlind and Wadstrom (42), using heat-denatured casein as substrate. Elastase activity was estimated by the elastin-congo red solubilization method of Sachar et al. (35); staphylolytic enzyme activity was estimated with heat-killed staphylococcal cells as the substrate; and lipase activity (substrate: p-nitrophenyl caprylate) was estimated as reported (42). loo r 0
183
Exotoxin A was determined by the mouse lethality test as described by Callahan (3), except that the preparations were given intraperitoneally. Statistical analysis. Survival distributions were compared by using the Wilcoxon rank sum test as described by Bradley (1). The P values were computed by using a normal approximation with continuity correction. The P values are consistent with those drawn from available Wilcoxon rank sum test tables. The mean lethal dose (LD5o) was calculated by the Spearman-Karber method described by Finney (7). Analysis of variance (5), a method for testing for significant differences among two or more means, was used to compare LD50 of the Pseudomonas strains. If means of subgroups are significantly different, the variance of the combined group is much larger than the variances of separate groups, and the analysis is based on this fact.
RESULTS Subcutaneous injections of viable PAKS-1, PAKS-10, or PAKS-17 in burn sites resulted in lethal infections associated with bacteremia and systemic invasion. Each of the three challenge strains was lethal at a dose characteristic for the individual strain (Table 2). The LD5o of the wildtype strain was 1 log lower than that of its protease-deficient mutants, PAKS-10 and PAKS-17. Analysis of variance (5) showed that the difference in virulence between the strains was significant [F(2/6 df) = 9.81; 0.02 > P > 0.01]. Postmortem examination of infected mice showed fatty infiltration of the liver (40) suggestive of exotoxin A (34), which they all produced (Table 1). No lesions were seen in the organs of burned, noninfected mice. Similar observations were made by Holder (personal communication). When mice were injected simultaneously with 1 LD50 of PAKS-10 and varying amounts of purified PR II, there was a corresponding decrease both in the percentage of mice surviving the infection and in the survival time (Fig. 2). Comparable results were obtained with PAKS-17 (data not shown). Both Holder (Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, B118, p. 35) and Snell et al. (37) also observed an increase
80
TABLE 2. LD50 of P. aeruginosa strain PAKS-1 and its protease-deficient mutants in burned micea
> 60
,
40
LDr,o logoi)
An 4
cr
Strain
Average ±
Expt no.
20
I 2
4
8 64 16 32 RECIPROCAL OF SERUM DILUTION
128
256
FIG. 1. Titer of PAKS-1 (whole cells)-absorbed anti-PR II serum as determined by its ability to inhibit PR H activity. Concentration ofprotease: 6.3 pg/ml.
II
III
SD"
PAKS-1 4.1 3.9 3.5 3.8 ± 0.3 5.1 PAKS-10 4.6 4.7 4.8 + 0.3 PAKS-17 5.3 4.7 4.6 4.9 ± 0.4 a Serial 10-fold dilutions of bacterial cells were used. Each dilution was injected into five mice. b SD, Standard deviation.
184
PAVLOVSKIS AND WRETLIND
INFECT. IMMUN.
exogenous protease. Burned, noninfected mice injected with 50 ,ug of PR II did not differ from burned, noninfected controls in either mortality (Fig. 2) or appearance. The above experiments suggested that elastase (PR II) contributes to the pathogenesis of the organism. To substantiate this observation, mice were passively immunized with anti-PR II serum before infection. In the first series of experiments mice were injected intravenously with 0.2 ml of anti-PR II serum, and 20 h later they were burned and infected with 1 LD50 of PAKS-10 and varying amounts of PR II (Fig. 3). Control mice were administered an equivalent volume of anti-BSA serum. The anti-PR II serum was able to neutralize to some extent the increased virulence of pseudomonas resulting
from low doses of exogenous protease (4.8 and 9.6 Mug) (P < 0.001); however, when mice were given excessive amounts of PR II (19.2 Mig), the anti-PR II had no effect (P > 0.05). Similar results were also obtained with PAKS-17 (data not shown). In the second series of experiments, groups of mice were administered 0.4 ml of anti-PR II or anti-BSA serum and then burned and infected 20 h later with 1 LD5o of PAKS-1, PAKS-10, or PAKS-7. The survival rates of the infected mice are shown in Fig. 4. Anti-PR II prolonged survival only in mice infected with the proteaseproducing strain, PAKS-1 (P < 0.001) (Fig. 4A), whereas it failed significantly to protect mice infected with protease-deficient strains (P > 0.05) (Fig. 4B and C). In fact, mice given antiPR II serum died more rapidly than control ,00 mice, but this difference was not significant. In both series of experiments (Fig. 3 and 4), antiso8 PR II was able to neutralize some of the effects of the protease and thus increase the survival of the infected mice. Thus the results indicate that I I o protease enhances the virulence of the organisms. Finally, burned mice were infected with 1 LDMm0 of one of three challenge strains and sacrificed sequentially 18 to 48 h later. Their blood , 0 20 40 100 60 I60 80 120 140 IS0 was cultured quantitatively on BHI-skim milk HOURS POST INFECTIM FIG. 2. Survival of burned mice infected with agar plates in order to distinguish between proPAKS-10, protease-deficient P. aeruginosa, and vary- tease-producing and protease-deficient colonies. ing amounts of PR I (0, no PR II; *, 6.0ug of PR II; The mice infected with protease-deficient strains A, 15.0 ,ug; A, 30.0 pg, x, 45.0 ug) and of burned, had consistently lower viable bacterial counts in noninfected mice injected with 50 pg PR II (O). Five blood than mice infected with PAKS-1 (Fig. 5A). mice were used for each determination. However, when burned mice were infected with --0 .............
-....
I
100 4,8 Lg PR I
0 *g PR II 80 60 40
20 r 140
I
o'
cr
CE
CL.
IC
9.6,sg 30
20 V
PR U
19.2 pLg PR
-
-
Fx 0
K
40
so
120
160
0
40
80
120
160
HOURS POST-INFECTION FIG. 3. Survival of mice pretreated with anti-PR II serum (0) or anti-BSA serum (0), burned, and infected with protease-deficient P. aeruginosa (PAKS-10) and varying amounts of PR II. Twenty mice were used per experiment: 10pretreated with anti-PR II serum and 10pretreated with anti-BSA serum.
P. AERUGINOSA PROTEASE
VOL. 24, 1979
C. PAKS-17
B. PAKS-1O
A. PAKS -I
185
°°0r 80
F
-j
I-
>0 4
w
0.1 20k I
l
40 80 120 HOURS POST- INFECTION
0
160
IlI 40 80 120
160
0
0
40
120
80
160
HOURS POST-INFECTION
HOURS POST-INFECTION
FIG. 4. Survival of burned, infected mice pretreated with anti-PR II serum (0) or anti-BSA serum (0). Forty mice were used for experiment A, and 20 mice each were used for experiments B and C. Halfof the mice in each group were pretreated with anti-PR II serum, and half were treated with anti-BSA serum. 0-PAKS 0 - PAKS 10 A -PAKS 17
A
.--
C
B
i/;
4
0
3E
3
cry CD) -
c#*
2 co 0 )-
C.) 40
MIXED INFECTIfN PAKS IS 10
SINGLE INFECTION
0
L-
I
I
I
o
20
40
60
fI 0
MIXED INFECTION PAKS I & 17
I
I
I
I
I
I
20
40
60
0
20
40
I~~_
60
HOOURS POST-INFECTION FIG. 5. Number ofpseudomonas colony-forming units ± standard error of the mean per milliliter of blood of burned infected mice. (A) Mice infected with either PAKS-i, PAKS-IO, or PAKS-i 7. (B) Mice infected with PAKS-I and PAKS-IO. (C) Mice infected with PAKS-i and PAKS-i 7.
186
PAVLOVSKIS AND WRETLIND
a mixture of PAKS-1 and PAKS-10 or PAKS-1 and PAKS-17 at a 1:1 ratio, the number of protease-deficient organisms in the blood was considerably higher (Fig. 5B and 5C) in the mixed infections than in the previous experiment (Fig. 5A). The results suggest that the in vivo production of protease by PAKS- 1 also enhances the invasiveness of the protease-deficient bacteria, PAKS-10 and PAKS-17.
INFECT. IMMUN.
mice. In the second model, the sensitivity of the host to the invading organism was significantly increased by the mucin. In our previous study on passive protection by antitoxin in experimental P. aeruginosa infections, it became obvious that exotoxin A was only one of a number of pathogenic factors involved in pseudomonas infections. Our study, as well as the work of Kawaharajo and Homma (10) and I. A. Holder, C. B. Saelinger, C. G. Haidaris, and M. Michael (Abstr. Annu. Meet. DISCUSSION Soc. Microbiol. 1978, B94, p. 29), who The mechanism by which protease exerts its Am. showed that mice immunized with elastase or its toxic action is not known. In septicemic infec- toxoid showed increased resistance to experition, protease may contribute to tissue damage mental P. aeruginosa infections, suggests that when it is produced locally by pseudomonas. B. this protease contributes to the pathogenesis of H. Iglewski, for example, found protease in tissue organism. Thus, it appears that protease is homogenates from pseudomonas-infected mice the rather than a major a virulence-enhancing (personal communication). Snell et al. (37) found virulence factor. The factor also indipresented data that the subcutaneous injection of purified pro- cate that to some extent these effects may be tease reduced elongation factor 2 activity in neutralized by antiprotease serum. These obsermouse liver. However, previous work with non- vations should provide a broader basis for studtoxigenic, protease-producing strains indicates leading to immunoprophylaxis and treatment that protein synthesis inhibition by protease ies infections. of pseudomonas during an infection is minimal (32) and that extremely high doses are required to kill mice as ACKNOWLEDGMENTS compared with toxigenic strains. It is unlikely, We thank E. Weiss, S. Joseph, and A. Eiche for their therefore, that protease by itself is responsible suggestions and comments throughout this study. We are also for the lethality of the organism. Our present grateful to K. Karlsson and G. Hare for their expert technical O.R.P. thanks the staff of the Department of data show an increase in the numbers of pseu- assistance. Clinical Microbiology at the Karolinska Hospital, where most domonas organisms in the blood of mice infected of this research was carried out. with protease-producing organisms (Fig. 5), sugThis study was supported by the U.S. Naval Research and gesting that protease may be important in ov- Development Command, Research Work Unit no. and the Research Institute of the Swedish ercoming the host's initial defense mechanisms. M0095PN001.0009, This may be accomplished either by proteolytic National Defense. action which provides additional nutrients, as LITERATURE CITED suggested by J. F. Cicmanec, C. G. Haidaris, and 1. Bradley, J. V. 1968. Distribution-free statistical tests, p. I. A. Holder (Abstr. Annu. Meet. Am. Soc. Mi105-114. Prentice-Hall, Inc., Englewood, N.J. crobiol. 1978, B90, p. 28), or by destruction of 2. Callahan, L. T., III. 1974. Purification and characterization of Pseudomonas aeruginosa exotoxin. Infect. Imanatomical barriers and more rapid invasion of mun. 9:113-118. the host. The latter possibility is indirectly sup- 3. Callahan, L. T., III. 1976. Pseudomonas aeruginosa ported by the work of Wretlind and Kronevi exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of highly specific (40), who infected cyclophosphamide-treated antitoxin serum. Infect. Immun. 14:55-61. mice with PAKS-1 or protease-deficient mu- 4. Carney, S. A., R. E. Dyster, and R. J. Jones. 1973. The tants. They found no difference in LD5,0 between invasion of burned skin by Pseudomonas aeruginosa. PAKS-1, PAKS-10, and PAKS-17. Neither did Br. J. Dermatol. 88:539-545. they find any difference in LD.% between PAKS- 5. Dixon, W. J., and F. J. Massey, Jr. 1951. Analysis of variance, p. 119-152. In Introduction to statistical anal1 and PAKS-10 when these strains were injected ysis. McGraw-Hill Book Co., New York. with mucin into normal mice. Both models were 6. Eiche, A. 1977. Effects of low dose X-irradiation on intrainitial defense host's designed to overcome the uterine death in mice subjected to exposure when young or at foetal stage. Hereditas 85:63-66. mechanisms, thus negating any advantage a proD. J. 1964. The Spearman-Karber method, p. tease-producing strain might have. In the first 7. Finney, 524-530. In Statistical methods in biological assay, 2nd model the host's initial defense mechanisms ed. Charles Griffin and Co., London. were already significantly lowered by the cyclo- 8. Fisher, E., Jr., and J. H. Allen. 1958. Mechanism of corneal destruction by Pseudomonas proteases. Am. J. phosphamide. In comparable experiments (PavOphthalmol. 46:249-254. lovskis and Wretlind, unpublished observa- 9. Forsgren, A. 1972. Pathogenicity of Staphylococcus autions), the leukocyte count dropped from apreus mutants in general and local infections. Acta Paproximately 8,000/mm3 for normal mice to less thol. Microbiol. Scand. Sect. B 80:564-570. than 500/mm3 for cyclophosphamide-treated 10. Gerke, J. R., and M. V. Magliocco. 1971. Experimental
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11. 12. 13. 14.
15.
16.
17.
18.
19.
20.
21. 22.
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