CONSTRUCTION AND CHARACTERIZATION OF PSEUDOMONAS AERUGINOSA ATN MUTANTS

S. Elizabeth George^a, Xin Zhou^b and Paul S. Cohen^b

^aNational Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC, (919)541-5036, fax (919)541-0694, george@herl45.herl.epa.gov; and ^bDepartment of Microbiology, University of Rhode Island, Kinston, RI, (401)792-5920, fax (401)792-7148

SUMMARY

Industrial production and agricultural application of biotechnology agents, such as Pseudomonasspp., increase the probability of human exposure to these microorganisms. Therefore, it is important to identify and mitigate potential health effects associated with deliberate or accidental environmental release of these microbial agents. In order to accomplish this goal, an attenuated mutant of P. aeruginosa AC869 (designated atn), was isolated by selection for resistance to bacteriophage E79. Following subculture, mice were challenged intranasally (i.n.) with approximately 2 x 10⁸ CFU/animal and the atn mutant (strain AC869-11) was characterized further. Strain AC869-11 had a higher LD₅₀ than the wildtype strain AC869 (1.55 x 10⁹ vs. 1.91 x 10⁸ CFU) and when lethal, treated animals lived longer following exposure. Clearance from the lungs, small intestine, cecum, and large intestines occurred more rapidly in animals following i.n. treatment of AC869-11 which was accompanied by a decreased pulmonary inflammatory response compared to the wildtype. Translocation to the mesenteric lymph nodes, liver, and spleen showed a similar response. Strain AC869-11 produced 2-fold and 16-fold less of the pathogenicity factors exoenzyme S and extracellular protease than that observed for strain AC869. In addition, the mutant was distinguished by a decreased colony size and increased sensitivity to several antibiotics. Immunoblotting of AC869-11 demonstrated a reduction in both A band and B band LPS. The decline in many dissimilar cell functions suggests that strain AC869-11 harbors a mutation in a regulatory gene.

Key words: Pseudomonas aeuginosa, intestinal colonization, inflammatory response, intranasal exposure, lipopolysaccharide

INTRODUCTION

Microorganisms used for deliberate environmental release applications generally are regarded as safe. Most of the species of microbes used can be readily isolated from soil, air, or water. However, actual numbers used for environmental application may be considerably higher than those that occur naturally. In fact, many of these isolates, such as Pseudomonas aeruginosa and P. cepacia, are considered opportunistic pathogens (Bodey et al., 1983; Rosenstein and Hall, 1980). Consequently, there is a potential for adverse health effects following exposure to high numbers of these agents under optimum conditions.

MATERIALS AND METHODS

Bacterial Strains and Culture Conditions. P. aeruginosa strain AC869, a genetically manipulated environmental isolate that mineralizes 3,5-dichlorobenzoate, was kindly provided by Dr. A.M. Chakrabarty, University of Illinois College of Medicine, Chicago (Chatterjee and Chakrabarty, 1982). P. aeruginosa strain AK1401 was provided by E.J. McGroarty, Michigan State University, East Lansing (Rivera et al., 1992). The attenuated mutant P. aeruginosa AC869-11 was constructed by selection for bacteriophage E79 resistance (E79 was provided by A.M. Kropinski, Queen's University, Kingston, Ontario, Canada; Jarell and Kropinski, 1977) and attenuated pathogenicity in CD-1 mice. P. aeruginosawas incubated at 37C for indicated times for specific applications described below in Luria broth (LB) or cecal mucus (CM) or lung mucus (LM) (1 mg/mg protein in HEPES-Hanks buffer, pH 7.4). Antibiotic sensitivities were determined in LB and LM with identical results.

Isolation and Visualization of Cellular Constituents. Strain AC869 was grown in 1 ml of LB, CM, or LM for 18 hrs. Cells were washed and run on 12 % polyacrylamide gels containing 0.1% SDS. Electrophoresis was performed at 200V for 30 min. The gels were silver stained by the procedure of Dubray and Bezard (1982) or Western blots were reacted with rabbit antiserum prepared against cecal mucus grown strain AC869. Complexes were visualized using gold conjugated goat anti-rabbit IgG and a SilvEnhance kit (Zymed)

Lipopolysaccharide (LPS) was extracted from LB, CM, or LM-grown cells by boiling. Resulting LPS was visualized by silver stain or Western blot serum analysis (using rabbit anti-cecal AC869) as indicated above. A-band LPS was absorbed by repeated exposure of the serum to cell pellets of P. aeruginosa AK1401 (A-Band LPS+, B-Band LPS-).

Pathogenicity. Strain CD-1 male mice were challenged intranasally (50 µl) with indicated concentrations (in phosphate buffered saline) of P. aeruginosa AC869 or AC869-11. LD₅₀ was calculated using linear regression and extrapolation. Clearance and translocation of strains AC869 and AC869-11 were determined by recovery following treatment with 10⁷ CFU as described previously (George et al., 1993). The number of neutrophils/ml lung lavage fluid from intranasally challenged mice was indicative of the pulmonary inflammatory response (George et al., 1993). Extracelluar protease was assayed for on casein agar plates and exoenzyme S was determined by the method of Frank et al. (1994).

RESULTS AND DISCUSSION

Following selection of the mutant, strain AC869-11, by resistance to bacteriophage E79 and attenuated virulence in mice, animals were administered a range of doses and the LD₅₀ determined. As expected, strain AC869-11 was 10-fold less virulent to mice than the wildtype strain AC869 with LD₅₀'s of 1.55 x 10⁹ and 1.91 x 10⁸ CFU respectively. Interestingly, when treated i.n. with 2 x 10⁸ CFU/animal, a dose approximately equal to the LD₅₀ of the wildtype, mice exposed to the mutant strain AC869-11 survived considerably longer than those dosed with strain AC869.

Mice were treated i.n. with a sublethal dose (10⁷ CFU) of strains AC869 and AC869-11 and clearance of both strains from the lungs, small intestine, cecum, and large intestines was determined. The wildtype strain was detectable in lungs 5 days following i.n. treatment whereas the mutant was cleared after 2 days. The decreased survival in the lungs was accompanied by a decreased pulmonary inflammatory response compared to the wildtype (2 x 10⁶ vs. 5 x 10⁵ neutrophils/ml of lung lavage fluid). A similar response was observed in the intestinal tract where the mutant survived 1 to 2 days and the wildtype for 10 to 14+ days. No translocation of the mutant was observed in the mesenteric lymph nodes or spleen. However, the mutant was detected in the liver after 3 hours (0.5 log CFU/g). The wild type, which was observed in all 3 tissues at 3 hours after treatment was cleared by day 1.

In order to establish a mechanistic reason for reduced LD₅₀, clearance, and inflammatory response, the pathogenicity factors exoenzyme S and extracellular protease, were assayed. Strain AC869-11 produced 2-fold and 16-fold less of the pathogenicity factors exoenzyme S and extracellular protease than that observed for strain AC869. A future study will examine exotoxin A activity.

When strain AC869 was grown on cecal or lung mucus, differences (compared to LB grown cells) were observable by SDS-PAGE. Gross analysis of colony morphology indicated that the mutant was a smaller, drier colony compared to the wildtype. Interestingly, a ladder-like pattern characteristic of O-side chain LPS, was observable in the mucus-grown cells. In addition, LB grown cells were untypeable whereas those grown in mucus were serotype O6. When the mutant was grown on mucus, the characteristic LPS ladder was observed, but the resulting bands were lighter, indicating that a regulatory gene may be involved instead of an O-side chain LPS mutant as might be expected from the initial selection method. In order to get a better understanding, LPS was isolated from the wildtype and mutant strains and bands were compared on SDS-PAGE by probing with rabbit antiserum prepared against cecal-grown strain AC869 (anti-cecal AC869). Both high (presumptive B band) and low (presumptive A band) molecular weight LPS were observed in the mutant but bands were less intense. When the A-band LPS was absorbed from the anti-cecal AC869 antiserum (leaving only B-band LPS available), the resulting probe for B-band LPS hybridized to both wildtype and mutant LPS. Again, the mutant bands were lighter than the wildtype.

Because the mutant is less pathogenic and several pathogenicity traits are affected by the mutation, a regulatory gene may be involved in the attenuated virulence. This is characterized by reduced mortality and a reduction in the production of the pathogenicity factors exoenzyme S and extracellular protease. In addition, the mutant is cleared more rapidly from the mouse following i.n. challenge. A significantly higher inflammatory response is induced by the wildtype but appears less effective in eliminating the wildtype strain. In addition, cellular constituents are affected by mucus growth. The mutant produces a similar pattern of bands,as observed by SDS-PAGE, but synthesizes lower concentrations of these bands. When the LPS is isolated and visualized by SDS-PAGE, the mutant bands are present but less intense than that observed from the wildtype strain AC869.

ACKNOWLEDGEMENTS

The authors would like to thank M. Utley, D. Frank, L.D. Claxton, I. Gilmour for their advice and contributions to this study. Additionally, the authors are grateful to P. Mathis and G. Nelson for their technical assistance.

Although the research in this article has been supported in part by the U.S. Environmental Protection Agency, it has not been subject to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred.

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