Vol. 59, No. 2

INFECTION AND IMMUNITY, Feb. 1991, p. 708-711

0019-9567/91/020708-04$02.00/0 Copyright © 1991, American Society for Microbiology

Isolation and Characterization of a Transposon-Induced CytotoxinDeficient Mutant of Pseudomonas aeruginosa LAWRENCE H. BOPP,lt* ALDONA L. BALTCH,2 MARK C. HAMMER,2 MARY A. FRANKE,2 RAYMOND P. SMITH,2 AND FRIEDER LUTZ3 Laboratories for Bacteriology, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-05091; Infectious Disease Section, Department of Veterans Affairs Medical Center, and Albany Medical College, Albany, New York 122082; and Institute of Pharmacology and

Toxicology, Justus-Liebig University, D-6300 Giessen, Germany3 Received 10 May 1990/Accepted 12 November 1990

In order to provide a better system for investigating the role of cytotoxin in pathogenesis, we mutated wild-type Pseudomonas aeruginosa PA158 by introducing a transposon. The resulting pool of mutants was screened for cytotoxin-deficient strains. One mutant strain, PA114F5, was compared with PA158. Except for cytotoxin production and antibiotic resistance (specified by the transposon), the two strains appear isogenic. This mutant strain should be useful in further clarifying the role of cytotoxin in pathogenesis.

Pseudomonas aeruginosa is a ubiquitous, gram-negative bacterium commonly found in soil, water, and sewage (26) as well as in numerous locations in the hospital environment (26, 29). It is generally viewed as an opportunistic pathogen and has become increasingly important in nosocomial infections, especially in immunosuppressed or otherwise debilitated patients (1, 10, 11, 30). Despite aggressive medical management, the mortality rate associated with bacteremia continues to be high (2, 4, 7, 48). P. aeruginosa makes numerous products that may be important in the pathogenesis of human infections (25, 30). The roles of several of these virulence factors in pathogenesis have been studied extensively, both in vitro and in vivo (19, 20), but little is known about the importance of cytotoxin. Cytotoxin is a protein toxin of P. aeruginosa and is localized in the periplasmic space (24). It was first described by Scharmann (39) and originally called leukocidin because it had cytotoxic effects on leukocytes (40). It has since been shown to cause cytopathic effects in most types of eukaryotic cells by interacting with cell membranes (3, 6, 15, 16, 41, 45). A survey of P. aeruginosa clinical isolates showed that all isolates produce cytotoxin (5), and its importance in pathogenesis is suggested by its cytotoxicity and the observation that patients with chronic P. aeruginosa infections have elevated levels of anticytotoxin antibodies (Sa). A powerful method for assessing the importance of putative virulence factors in pathogenicity is to compare the effects in model systems of strains that produce or fail to produce the factor in question but are otherwise isogenic. There are reports of such comparative studies involving mutants of toxin A (32, 33, 46), exoenzyme S (31, 32), elastase (33, 46), and other virulence factors (18, 20). However, no paired strains suitable for studying the role of cytotoxin in the pathogenicity of P. aeruginosa infections exist. The purpose of this study was to generate, isolate, and characterize cytotoxin-deficient mutants of P. aeruginosa PA158. Transpositional mutagenesis was used because of its *

inherent advantages over chemical mutagenesis in the generation and maintenance of bacterial mutants. Furthermore, the effectiveness of transpositional mutagenesis has been shown in several species of Pseudomonas, including P. aeruginosa (28, 31, 44, 49, 50). Bacterial strains are listed in Table 1. Trypticase soy agar, Vogel-Bonner minimal salts medium (VB), and Luria (L) medium were prepared as described previously (8, 37) and supplemented as indicated below. A suicide vector (pRKTV14) containing the transposon construct Tn7::TnS was used for mutagenesis. Cells from overnight cultures of PA158 and Escherichia coli NECO100(pRKTV14) were placed on sterile 0.45-pim-pore-size membrane filters. The filters with the mating cells were then incubated on L agar plates for 6 h at 37°C. After incubation, cells were plated on VB agar containing 1,000 pLg of kanamycin per ml to select for transpositional mutants of PA158. Growth of E. coli NECO100(pRKTV14) was suppressed because none of the nutritional supplements that it requires was present. Subsequently, it was demonstrated that PA158 produces a diffusible substance that is rapidly lethal to NECO100. Thus, PA158 colonies that arose on VB-kanamycin agar did not require further purification. Kanamycin-resistant (Kmr) PA158 transpositional mutants arose at a frequency of 2 x 10-6 per recipient cell, indicating that transfer of pRKTV14 and transposition of TnS or Tn7::TnS worked well. Individual colonies from the pools of mutants were transferred to nitrocellulose membranes and screened for the presence of cytotoxin. This was accomplished by probing with affinity-purified rabbit anticytotoxin antibody (5) and then visualizing cytotoxin-producing (Ctx+) colonies by treating them first with biotin-conjugated anti-rabbit immunoglobulin G and then with peroxidase-avidin and horseradish peroxidase color development reagent (Bio-Rad Laboratories, Richmond, Calif.). Ctx+ colonies produced blue spots, while those lacking detectable cytotoxin (Ctx-) produced no color. Since no Ctx- strains of P. aeruginosa were readily available (5), members of several other Pseudomonas species and E. coli were used as negative controls. Of approximately 20,000 colonies from two independently derived pools of mutants screened for Ctx- strains, only one cytotoxin mutant was found. This strain, PA114F5, produced no reaction in the immunological screening proce-

Corresponding author.

t Present address: Infectious Disease Section, Department of Veterans Affairs Medical Center, 113 Holland Ave., Albany, NY

12208. 708

NOTES

VOL. 59, 1991 TABLE 1. Bacterial strains

Species

Relevant propertiesa

Reference

Wild type (Ctx+) Wild type (Ctx+), Apr Tcr Kmr, by conjugation: PA158 x HB101(RP4) Ctx- Kmr, Tpr, Smr, Spr

39 This study

and strain

P. aeruginosa PA158 PA158(RP4)

PA114F5 E. coli HB101(pRI3)

HB101(RP4)

NECO100 (pRKTV14b) a

or source

F- hsdS20 recA13 ara-14 proA2 lacY) galK2 rpsL20 (Sm`) xyl-5 mtl-l supE44 Apr Kmr, by transformation F- hsdS20 recAI3 ara-14 proA2 lacYI galK2 rpsL20 (Smr) xyl-5 mtl-l supE44 Apr Tcr Kmr, by transformation Rif thi leu trp recA Kmr Tpr Smr Spr

Abbreviations: Apr, ampicillin resistance;

Tcr,

This study J. Shaw

This study

50

tetracycline resistance;

Kmr, kanamycin resistance; Tpr, trimethoprim resistance; Smr, streptomycin resistance; spr, spectinomycin resistance. b pRKTV14 was constructed by transposition of Tn7 into the Kmr gene of pRK2013, followed by transposition of TnS into a nonessential region of Tn7

(50).

dure. The mutation in PA114F5 appears stable, since there was no detectable loss of the Kmr phenotype when cells were grown without kanamycin. We were surprised that only one Ctx- mutant was found. Failure to find Ctx- mutants at the expected frequency or any mutant totally lacking cytotoxin together with a previous report that all P. aeruginosa strains tested produced cytotoxin (5) suggests that there may be an as-yet-unidentified selection against the Ctx- phenotype.

A

709

Crude bacterial extracts (5), partially purified cytotoxin (27), and purified cytotoxin (5) were prepared as described previously and then characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting (5). Crude cell extracts of strains PA158 and PA114F5 had indistinguishable protein profiles (Fig. 1A), demonstrating that no major proteins are affected by the mutation. Western blot analysis showed that cytotoxin is produced by both strains but that PA114F5 produces much less cytotoxin than PA158 (Fig. 1B). Quantitative comparison of cytotoxin production was made by scanning the Western blots with a densitometer (Fig. 1C). Although a cytotoxin band was always detected in extracts of PA114F5, the amount of cytotoxin per unit of total protein was only 2% of the wild-type level. Because there is a 50-fold difference in cytotoxin production that cannot be detected by SDS-PAGE of the crude extracts, it is clear that cytotoxin is a relatively minor protein. These data are consistent with the interpretation that PA114F5 is a regulatory mutant that produces a small amount of cytotoxin compared with its parent strain but that neither strain produces much cytotoxin relative to other proteins present in the crude extracts. The other bands in Fig. 1B may be due to aggregates and decomposition products of authentic cytotoxin, since they decrease in proportion to cytotoxin even though other proteins are selectively removed in the purification process. However, the possibility that other proteins are cross-reacting with the antibody cannot be excluded. In similar studies in which Tn5 was used to mutagenize Pseudomonas syringae and Pseudomonas perfectomarina, mutants in which small amounts of the normal gene product were still present were also produced (28, 49). Chemical mutagenesis of P. aeruginosa for creation of toxin A-deficient mutants yielded similar results (35). These studies all suggest that isolation of regulatory Pseudomonas mutants,

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92.569 3021

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14.5-

PA158 PA114F5 Cytotoxin

PA I5B

PA 114F5

PA158 PA114F5 FIG. 1. Comparison of protein profiles and cytotoxin levels in extracts of P. aeruginosa PA158 and PA114F5. (A) Silver-stained SDS-PAGE of crude cell extracts, 10 ,ug of protein per lane. (B) Western blots of partially purified cell extracts (10 ,ug per lane) and G-75-purified cytotoxin (100 ng) with affinity-purified, polyclonal anticytotoxin antibody. Arrow, cytotoxin. (C) Scanning densitometry of Western blots shown in panel B. Arrow, cytotoxin. OD, Optical density.

710

NOTES

whether they are chemically or transpositionally induced, is common (28, 35, 49). Most of the P. aeruginosa toxin A mutants described above were also deficient in protease activity (35). This and previous studies, in which simultaneous decrease in production of more than one extracellular product has been observed, suggest that pleiotropic effects may result from mutation in a regulatory element that controls the expression of several genes coding for extracellular products (35, 47). Therefore, pyocyanin and fluorescein (23), RNase and DNase (21), alkaline protease (43), elastase (34), lipase (17), hyaluronidase (42), sheep erythrocyte hemolysin (35), esterase (17), toxin A (35), and exoenzyme S (31) production by strains PA158 and PA114F5 was compared. No differences were detected, suggesting that the mutation in PA114F5 is in a regulatory region that controls the expression of the cytotoxin gene but not the expression of the others. P. aeruginosa PA158 and PA114F5 were biochemically characterized with the API Rapid NFT system (API Analytab Products, Div. Sherwood Medical, Plainview, N.Y.) and were also tested for growth on cetrimide agar (9). Both strains grew with D-glucose, D-mannitol, N-acetyl-D-glucosamine, D-gluconate, caprate (decanoic acid), adipate (hexanedioic acid), malate, or citrate as the sole carbon source; both failed to grow with L-arabinose, D-mannose, maltose, or phenylacetate. Positive reactions were observed for reduction of nitrate to N2, arginine dihydrolase, oxidase, catalase, and growth on cetrimide agar, while reactions for tryptophanase, urease, P-galactosidase, glucose fermentation, esculin hydrolysis, and reduction of nitrate to nitrite were negative. Serotypes were determined by using a P. aeruginosa Bacto antiserum set (Difco Laboratories, Inc., Detroit, Mich.), and pyocin typing was done according to the method of Gillies and Govan (12). Both strains are serotype 6 and pyocin type 3. PA158 and PA114F5 are motile, gram-negative rods and cannot be differentiated on the basis of microscopic examination or colony morphology. They grow well at 37 and 42°C, and at each temperature the generation times of the two strains are comparable. Susceptibilities to ampicillin, piperacillin, cefoxitin, cefotaxime, amikacin, gentamicin, tobramycin, trimethoprim, trimethoprim-sulfamethoxazole, cefazolin, aztreonam, ceftazidine, ceftriaxone, imipenem, norfloxacin, ticarcillin, kanamycin, streptomycin, and spectinomycin, determined by the microdilution method (22), indicated that PA114F5 is significantly more resistant to kanamycin, trimethoprim, streptomycin, and spectinomycin than PA158 is. We expected PA114F5 to be much more resistant to kanamycin than PA158, because evaluation of the suitability of kanamycin resistance for selecting TnS-induced transpositional mutants with strain PA158(RP4) (Table 1) demonstrated that this strain grows well on VB agar with 1,000 ,ug of kanamycin per ml, while growth of PA158 is completely suppressed on the same medium. However, simultaneous acquisition of the other antibiotic resistances specified by pRKTV14 suggests that the mutation in PA114F5 was actually caused by transposition of Tn7::TnS. For further genetic characterization, plasmid DNA was isolated by the method of Godson and Vapnek (14), as described by Sambrook et al. (38). Although plasmid DNA was readily recovered from PA158(RP4), none was recovered from PA114F5 or PA158 itself. A probe for Tn5 was constructed by isolating a BamHI-HindIII fragment from the Kmr gene of pRI3 (pBR322:: Tn5) and used as previously

INFECT. IMMUN.

described (36). Examination of PA158 and PA114F5, as well as several other pseudomonads of various species, other P. aeruginosa strains, plasmidless E. coli, and E. coli containing pRI3 for the presence of the Kmr gene showed that it is present only in strain PA114F5 and E. coli(pRI3). Since PA114F5 has no plasmids but hybridizes strongly to a probe for the Kmr gene of Tn5 while PA158 does not, transposition of TnS to the chromosome of PA114F5 must be responsible for its kanamycin resistance. More detailed analysis, using Southern blots on which restriction digests of PA114F5 DNA were probed for the Kmr gene of TnS, showed that only one copy was present. Cytotoxicity was assessed by using a human granulocyte cell-swelling assay in which granulocytes are exposed to the test material and then examined by phase-contrast microscopy for swelling (13). No swelling was seen when granulocytes were exposed to an extract of strain PA114F5. However, exposure to the same amount of extract from PA158 (as measured by total protein) caused 4+ swelling (on a scale of 0 to 5, where 0 indicates no swelling and 5 indicates maximal swelling). This confirms that the decreased level of cytotoxin produced by strain PA114F5 has functional significance. In vitro and in vivo studies in which the cytotoxicity and virulence of PA158 and PA114F5 are being compared are now under way. Because PA114F5 appears to be a regulatory mutant, it should also be useful in helping us understand how expression of the cytotoxin gene is regulated. To that end, detailed analysis of the chromosomal segment containing the mutation is now in progress. We thank Donald Woods for performing the toxin A and exoenzyme S assays. This research was supported by the Department of Veterans Affairs Medical Research Service and by National Cystic Fibrosis Foundation grant 1065 8-01.

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