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(Investigative Ophthalmology and Visual Science. 2002;43:1437-1444.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Pseudomonas aeruginosa Exotoxin A and Keratitis in Mice

Christopher M. Pillar and Jeffery A. Hobden

From the Department of Immunology and Microbiology, School of Medicine, Wayne State University, Detroit, Michigan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the importance of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in corneal disease.

METHODS. Isogenic mutants deficient in ETA were constructed in P. aeruginosa strains PAO1 and ATCC 19660 by allelic exchange and then evaluated for virulence in a mouse model of bacterial keratitis. The effect of ETA on adherence to scarified corneal epithelium was assessed in an in vitro organ culture model.

RESULTS. Mutants of either P. aeruginosa PAO1 or 19660, deficient in ETA, adhered to wounded corneal tissue and initiated ocular disease similar to that in wild-type strains. However, in contrast to wild-type strains, ETA mutants were quickly cleared from the eye, inflammation diminished, and the cornea healed.

CONCLUSIONS. Although ETA has no effect on the ability of P. aeruginosa to adhere to corneal wounds or to initiate Pseudomonas keratitis, it is crucial for the organism to persist in the eye and ultimately cause disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Gram-negative bacillus Pseudomonas aeruginosa is a significant cause of bacterial keratitis in patients who use extended-wear contact lenses.1 Infection with P. aeruginosa results in a particularly rapid and destructive disease2 that, if untreated, can result in corneal perforation and loss of the eye in 2 to 4 days.3 The pathogenesis of Pseudomonas keratitis consists of the colonization of the cornea, the induction of a number of proinflammatory cytokines, such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1ß,4 5 and the migration of polymorphonuclear leukocytes (PMNs) into the cornea to clear the pathogen. PMN influx is due primarily to induction of chemokines responsible for attracting PMNs, such as macrophage inflammatory protein (MIP)-2.4 The resultant pathologic damage in Pseudomonas keratitis is due to factors associated with the colonizing bacteria, as well as the resultant response of PMN.6

P. aeruginosa produces a variety of extracellular factors that are potentially damaging to ocular tissues. The organism is capable of producing a number of proteases (alkaline protease, staphylolytic protease, elastase, and protease IV), a heat-labile and heat-stabile hemolysin, phospholipase C, and toxins (exoenzyme [Exo]-T, -S, and -U, and exotoxin A [ETA]). A number of these factors have been shown to be damaging to corneal tissue when applied exogenously to the eye7 8 9 10 11 12 or have been linked to ocular virulence through the study of mutants deficient in these factors made by chemical or transposon mutagenesis.7 13 14 15

ETA is a type II secreted16 66-kDa A-B toxin, whose function is identical with that of diptheria toxin produced by Corynebacterium diphtheriae. It is bound by the host through the {alpha}2-macroglobulin receptor,17 and on its internalization, the enzymatic A subunit is released into the cytoplasm.18 19 This subunit transfers adenosine diphosphate (ADP)-ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor (EF)-2 and thus inhibits protein translation.20 21 Production of ETA is tightly regulated and is highly dependent on availability of iron. Transcription of ETA is under the direct control of its transcriptional regulator regA,22 and is also under the indirect control of global regulators vfr23 and lasR.24 ETA is also transcriptionally activated under conditions of low iron availability through an interaction between fur and pvdS, the latter encoding an alternative sigma factor required for regA transcription.25 26

Several studies have suggested that ETA could be a critical factor in Pseudomonas keratitis. Addition of exogenous ETA results in ocular damage consistent with keratitis in mice8 and in corneal ulceration and cell death in rabbits.9 Mutants of the P. aeruginosa strains PA103 and PAO1, which are deficient in ETA, generated by chemical mutagenesis result in less severe disease in a mouse model of ocular infection.15 ETA has been associated with adherence of P. aeruginosa to tracheal epithelial cells in vitro.27 ETA also has been shown to be toxic to murine and human PMNs and to inhibit phagocytosis and killing of P. aeruginosa by PMNs in vitro.28

In this study, the role of ETA as an important virulence factor in P. aeruginosa ocular disease was demonstrated with isogenic mutants of two well-characterized and ocularly virulent strains of P. aeruginosa, PAO1 and ATCC 19660, in a murine model of bacterial keratitis. These two strains differ phenotypically in their expression of type III secreted toxins and were selected to determine whether ETA affects ocular virulence, regardless of these strain variations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacteria and Media
Strains used in this study are listed in Table 1 . Two strains of P. aeruginosa were selected for mutagenesis. P. aeruginosa PAO1 is a wild-type strain widely used for genetic studies, including sequencing of the entire P. aeruginosa genome.29 ATCC 19660 is a strain frequently used to study P. aeruginosa keratitis in murine models of infection.30 31 32 33 These two strains differ phenotypically in that PAO1 is invasive to corneal epithelium, whereas 19660 is cytotoxic.34 35


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  		Table 1. Strains and Plasmids Used in the Study

 
Escherichia coli were grown in LB (1% wt/vol bacto-tryptone [Difco Laboratories, Detroit, MI], 1% wt/vol NaCl [Sigma, St. Louis, MO], 0.5% wt/vol bacto-yeast extract [Difco]), and P. aeruginosa were grown in LB, PTSB (5% wt/vol peptone [Difco], 0.25% wt/vol tryptic soy broth [Difco]), or dialyzed and chelated tryptic soy broth (DTSB) prepared as described by Kozak and Saelinger36 for the production of ETA. ETA-producing cultures were grown under conditions described by Frank and Storey.37 For maintenance of plasmid vectors, E. coli strains were grown in the presence of 100 µg/mL ampicillin (Sigma) or 50 µg/mL chloramphenicol (Sigma). All strains were stored in either PTSB or LB containing 7.5% wt/vol glycerol at -70°C. Cultures of bacteria were grown overnight in PTSB or LB at 37°C and 100 rpm, unless otherwise specified. Colonies of E. coli were maintained on LB plates, whereas colonies of P. aeruginosa were maintained on Pseudomonas isolation agar (PIA; Difco).

Construction of the Allelic Exchange Vector pEXTOXA{Omega}
All molecular cloning techniques used in the construction of the allelic exchange vector were performed essentially as described by Sambrook et al.38 with reagents from Life Technologies (Gaithersburg, MD). Mutagenesis by allelic exchange was conducted essentially as described by Schweizer and Hoang.39 Plasmids used in this study are listed in Table 1 .

Construction of the allelic exchange vector pEXTOXA{Omega} is illustrated in Figure 1 . A 2.4-kb fragment containing toxA was excised from pMS151 with EcoRI and EcoRV, and was blunt-ended by Klenow treatment. The fragment was then cloned into the SmaI-digested calf intestinal alkaline phosphatase (CIAP)–treated vector pEX100T, creating the intermediate vector pEXTOXA. A 4.1-kb {Omega} cassette40 41 encoding chloramphenicol resistance was removed from pHP45-Cm (American Type Culture Collection [ATCC], Manassas, VA) by digestion with EcoRI. This cassette was then cloned into the BglII restriction site within the toxA gene on pEXTOXA by blunt ending both the cassette and the vector with Klenow, treating the vector with CIAP, and ligating with T4 DNA ligase. The resultant vector was named pEXTOXA{Omega} and was used for allelic exchange. Insertion of the chloramphenicol cassette into the toxA gene of pEXTOXA{Omega} resulted in 1.2 and 1.3 kb of P. aeruginosa DNA flanking the insertion. All plasmid constructs were confirmed by restriction pattern analysis with agarose gel electrophoresis.



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  		Figure 1. Construction of allelic exchange vector pEXTOXA{Omega}. A 2.4-kb EcoRI-EcoRV fragment containing toxA was blunt ended with Klenow and ligated into pEX100T, creating intermediate vector pEXTOXA. Vector pEXTOXA{Omega} was formed by removing a 4.1-kb EcoRI fragment containing cat from pHP45-Cm, and ligating it into pEXTOXA within the toxA reading frame. lacZ: ß-galactosidase, sacB: levansucrase, bla: ß-lactamase, toxA: exotoxin A, cat: chloramphenicol acetyltransferase (not drawn to scale).

 
Construction of ETA-Deficient Mutants of PAO1 and 19660
The allelic exchange vector was introduced into 19660 and PAO1 by triparental mating between 19660 or PAO1, E. coli JM109 with pEXTOXA{Omega}, and E. coli HB101 carrying a mobilization plasmid, pRK2013. Cultures of each were grown in 10 mL LB broth for 24 hours at 37°C with shaking. Aliquots (200 µL) from each of the three cultures were mixed with 2 mL fresh LB broth, and the bacteria were vacuum filtered onto a 0.45-µm filter (Pall Life Sciences, Ann Arbor, MI). The filter was incubated on LB agar overnight at 37°C. Bacteria were resuspended in 50 mM phosphate-buffered saline (PBS, pH 7.4) by vortexing the filter, and 100-µL aliquots were plated onto PIA supplemented with 500 µg/mL carbenicillin to select for integration of the allelic exchange vector within the toxA gene on the chromosome. Double recombinants, in which the genomic copy of toxA was replaced with toxA insertionally inactivated by a chloramphenicol cassette, were selected by growing carbenicillin-resistant colonies overnight in LB alone, subculturing overnight into LB with 250 µg/mL chloramphenicol, and finally subculturing overnight into LB with 250 µg/mL chloramphenicol and 5% wt/vol sucrose (showing loss of the sacB marker encoding levansucrase, which confers sensitivity to sucrose). Colonies able to grow in sucrose and chloramphenicol were streaked for isolation and screened for loss of resistance to carbenicillin (showing loss of the bla marker encoding ß-lactamase which confers resistance to carbenicillin). These carbenicillin-sensitive, chloramphenicol- and sucrose-resistant colonies were renamed PAO1 tox (-) and 19660 tox (-) and were subjected to further characterization, as described in the following sections.

Complementation of ETA Mutants
A 2.5-kb HindIII–EcoRI fragment containing toxA and flanking P. aeruginosa sequence was cloned from pMS151 (Steven Lory, Harvard Medical School, Boston, MA) into an HindIII–EcoRI digested P. aeruginosa expression vector, pUCP21 (Herbert P. Schweizer, Colorado State University, Fort Collins, CO). The resultant construct, pUCPTOXA,was electroporated into PAO1 tox (-) and 19660 tox (-) as described by Diver et al.,42 with a commercial electroporation apparatus (Gene Pulser; Bio-Rad, Hercules, CA). The presence of either pUCPTOXA or pUCP21 within PAO1 tox (-) and 19660 tox (-) was confirmed by restriction digestion of recovered plasmid DNA and also by restoration of ETA secretion by Western blot analysis.

Characterization of PAO1 Tox (-) and 19660 Tox (-)
Southern Blot Analysis.
Introduction of the chloramphenicol cassette into toxA resulted in the incorporation of two new BamHI sites at that region on the chromosome allowing for the detection of the cassette by Southern blot analysis of BamHI digested chromosomal DNA (Fig. 1) . PCR primers 5'-CGACCTCTGGAACGAATGC-3' and 5'-AGCAGGCACAACACCTTGC-3' (Life Technologies) resulting in a 580-bp amplification product were designed (MacVector; Oxford Molecular Group PLC, Oxford, UK) to anneal to toxA sequence upstream of the chloramphenicol cassette insertion site. This 580 bp was amplified from pEXTOXA (PCR Supermix; Life Technologies; and Touchdown PCR) as described by Roux.43 The PCR product was desalted with concentrators (Microcon-100; Gelman) and 25 ng was then labeled with [{alpha}-32P] dCTP (Amersham, Arlington Heights, IL) using a random primer DNA labeling system (Life Technologies).

Genomic DNA was isolated from wild-type PAO1 and 19660 and ETA-deficient PAO1 tox (-) and 19660 tox (-), as described by Sambrook et al.38 A total of 5 µg genomic DNA was digested to completion with BamHI (with the exception of 19660, for which only 1 µg DNA was available for digestion). Plasmids pEXTOXA (1 ng) and pEXTOXA (1 ng) were also digested with BamHI as a control. Digested DNA was loaded onto an agarose gel (0.8% wt/vol), and samples were run at 120 V. DNA was denatured by soaking the gel in 0.5 N NaOH, and 1.5 M NaCl at room temperature (RT) for 30 minutes. The gel was then soaked in neutralizing buffer (1 M Tris-HCl [pH 8.0] and 1.5 M NaCl) at RT for 30 minutes. Transfer of DNA to a membrane (Ny+ Immobilon; Millipore, Bedford, MA) was achieved by capillary action with blotting paper (QuickDraw; Sigma) in 20x SSC buffer (Sigma) overnight. Transfer was confirmed by staining the gel with ethidium bromide and checking for absence of DNA.

The blot was rinsed in 5x SSC and baked at 80°C for 2 hours. For prehybridization, the blot was moistened with 5x SSPE,38 sealed in a bag containing hybridization solution (5x Denhardt’s, 0.1% wt/vol SDS, and 100 µg/mL fragmented salmon sperm in 5x SSPE), and incubated at 68°C for 2 hours. Probe (25 ng) was then added, and the blot was incubated at 55°C for 20 hours. The membrane was then washed according to the manufacturer’s instructions and exposed to x-ray film (Bio-Max; Eastman Kodak Co.; Rochester, NY) overnight at -70°C.

Western Blot Analysis.
The presence of ETA in DTSB culture supernatants recovered from cultures grown under conditions to maximize ETA production37 was determined by Western blot analysis. Culture supernatants were concentrated 10-fold (Centricon-30 concentrators; Pall Life Sciences). Equal concentrations of protein, as determined by a protein assay (DC Protein Assay; Bio-Rad), were loaded onto a 12.5% wt/vol SDS-polyacrylamide gel and run at 120 V at RT for 2 hours under denaturing conditions. Pure ETA (100 ng; List Biological Laboratories, Campbell, CA) was used as a control. Protein was transferred to transfer medium (Trans-Blot; Bio-Rad) at 120 V at 4°C for 1 hour. The blot was blocked (Blotto, Santa Cruz Biotechnology, Santa Cruz, CA; 3% bovine serum albumin and 3% skim milk in TBS) at RT for 1 hour and was incubated in primary antibody (goat anti-ETA 1:100; List Biological Laboratories) overnight at 4°C. The blot was then incubated in the presence of a horseradish peroxidase–conjugated secondary antibody (mouse anti-goat IgG 1:10,000; Sigma). Secondary–primary antibody immune complexes were detected with 4-chloro-1-naphthol tablets (Sigma), as described by Sambrook et al.38

Adherence Assay.
The ability of wild-type and mutant strains to adhere to scarified corneal epithelium was determined essentially as described by Gupta et al.44 All animals used in this study were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Female 10- to 12-week-old 129/Sv mice45 were anesthetized with isoflurane (Mallinckrodt Veterinary, Inc., Mundelein, IL) and killed by cervical dislocation. Corneas of each eye were scarified with a sterile 26-gauge needle. Eyes were then enucleated and placed into organ culture, as previously described.46 Eyes (three per strain) were inoculated with 5 µL 107 colony-forming units (CFU) of bacteria on the corneal surface. After incubation for 1 hour at 37°C, eyes were rinsed to remove nonadherent bacteria and prepared for scanning electron microscopy, as previously described.44 Eyes were examined with a scanning electron microscope (JSM-840A; JEOL, Tokyo, Japan), and bacteria adhering to corneal wounds were quantitated as described by Hazlett et al.30 In brief, five representative fields were photographed from each group of three eyes at a magnification of x3000. Negatives from these five photographs were enlarged to x6000 m and three fields (80 mm2 each) were counted on each photograph. Counts are presented as the mean number of bacteria bound per field ± SEM. Statistical comparisons between strains were conducted by an unpaired, two-tailed Student’s t-test (n = 15/strain) at P <= 0.05 significance.

Murine Scarification Model of Bacterial Keratitis.
ETA-deficient mutants were tested in a mouse scarification model of bacterial keratitis, as described by Kwon and Hazlett.47 In brief, female C57BL/6J mice (B6; 6–8 weeks; Jackson Laboratories, Bar Harbor, ME) were anesthetized. The cornea of the left eye was scarified three times with a needle and then topically inoculated with 106 CFU of wild-type or mutant strains in normal saline (0.85%, pH 7.2). Five mice were infected per strain of P. aeruginosa, and the infection experiments were repeated to assure reproducibility of results. Individual mice were identified by marking their tails with nontoxic colored markers. The ocular disease response of the animals was recorded in a masked manner on days 1, 3, 5, and 7 postinfection (pi), according to a scale of 0 to +4: 0, clear or slight corneal opacity, partially covering the pupil; +1, slight opacity fully covering the anterior segment; +2, dense opacity, partially or fully covering the pupil; +3, dense opacity covering the entire anterior segment; and +4, corneal perforation or phthisis bulbi (i.e., shrunken eye).31 Data from separate infection experiments were pooled by strain. Median scores between mutant, wild-type strains, and restored strains were compared by ANOVA at a significance of P <= 0.05.

In a separate experiment, three mice were infected per time point for quantitation of viable bacteria and PMN infiltrate in corneas 6, 12, 24, 36, and 48 hours PI, as described by Hobden et al.48 Mean log10 CFUs and PMNs/eye were calculated in triplicate for each strain and were compared by an unpaired, two-tailed Student’s t-test at a significance of P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of Isogenic ETA-Deficient Mutants of 19660 and PAO1
A suicide vector was constructed carrying toxA interrupted with an {Omega} cassette,40 41 encoding resistance to chloramphenicol. Introduction of this vector into P. aeruginosa PAO1 or 19660 resulted in a double homologous recombination event, in which the disrupted toxA gene was incorporated into the host genome.39

Genomic DNA from PAO1 tox (-) and 19660 tox (-) and their respective isogenic parents was analyzed by Southern blot analysis at the region of the insertion of the chloramphenicol cassette within toxA. A probe was constructed by PCR to hybridize to toxA sequence upstream of the chloramphenicol cassette’s insertion site (Fig. 2A) . In the absence of the chloramphenicol cassette, the probe hybridized to a 1.5-kb fragment of BamHI-digested DNA. Successful disruption of toxA resulted in a hybridization product of 700 bp, because of the presence of a BamHI site within the {Omega} cassette. Figure 2B illustrates that the chloramphenicol cassette was successfully detected within toxA in BamHI-digested genomic DNA isolated from both PAO1 tox (-) and 19660 tox (-), with plasmid DNA from the toxA allelic exchange vector pEXTOXA{Omega} serving as a positive control. Genomic DNA isolated from isogenic parents PAO1 and 19660 digested with BamHI as well as plasmid DNA from pEXTOXA containing a wild-type copy of toxA resulted in a predicted hybridization product of 1.5 kb.



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  		Figure 2. Southern blot for the detection of the chloramphenicol resistance (Cm) cassette within toxA. (A) BamHI cut sites within toxA disrupted by the insertion of the Cm cassette on pEXTOXA{Omega}. ***, probe hybridization site. (B) Blot of BamHI-digested DNA for the presence of the Cm cassette. Lane 1: pEXTOXA{Omega}; lane 2: pEXTOXA; lane 3: PAO1; lane 4: PAO1 tox (-); lane 5: 19660; and lane 6: 19660 tox (-).

 
Supernatants were isolated from cultures of PAO1 tox (-), 19660 tox (-), and their respective wild-type isogenic parent strains grown under conditions optimal for ETA production. The supernatants were then analyzed by Western blot analysis for the detection of ETA. Culture supernatants from wild-type PAO1 and 19660 contained immunoreactive protein, consistent with that observed with pure ETA standard (66 kDa; Fig. 3 ). No such product was detected in the supernatants isolated from PAO1 tox (-) and 19660 tox (-).



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  		Figure 3. Western blot for the detection of ETA in culture supernatants of PAO1 and 19660. Lane 1: molecular weight marker; lane 2: pure ETA (200 ng); lane 3: wild-type; lane 4: ETA mutant; lane 5: ETA mutant with pUCPTOXA; and lane 6: ETA mutant with pUCP21.

 
The ability of pUCPTOXA to restore ETA production in these ETA-deficient mutants was also confirmed by Western blot analysis. Introduction of pUCPTOXA into PAO1 tox (-) and 19660 tox (-) restored secretion of full-length ETA. As expected, culture supernatants from PAO1 tox (-) and 19660 tox (-) carrying vector control pUCP21 were negative for ETA, by Western blot analysis (Fig. 3) .

Ocular Virulence of ETA-Deficient Mutants of P. aeruginosa
The ability of ETA-deficient mutants of P. aeruginosa to adhere to wounded corneal epithelium was assessed using an in vitro organ culture model.44 46 Bacterial counts made from micrographs obtained through scanning electron microscopy46 allowed for the determination of the bacteria’s ability to adhere to tissue. No statistically significant difference (P >= 0.20) in mean adherent organisms per counted field (n = 15) was observed between wild-type and ETA-deficient mutants of either PAO1 (PAO1, 9.5 ± 1.5; PAO1 tox (-), 7.6 ± 0.9) or 19660 (19660, 16.7 ± 2.3; 19660 tox (-), 12.7 ± 2.0).

Determination of the effect of ETA on the pathogenesis of Pseudomonas keratitis was made by examining the infections initiated by ETA-deficient mutants in comparison with those in their isogenic parent strains, using a mouse model of bacterial keratitis.47 Figure 4A illustrates that infection with 19660 tox (-) resulted in a statistically significant decrease in mean clinical score at all time points examined (n = 20; P <= 0.001). In wild-type infected eyes at day 7 PI, the cornea perforated, whereas mutant infected eyes at this time point had healed (Fig. 3C) . Expression of toxA in trans in 19660 tox (-) resulted in complete restoration of ocular disease (Fig. 3A) , whereas the vector control exhibited virulence similar to that with 19660 tox (-) (data not shown). Similar results were obtained in mice infected with PAO1 and PAO1 tox (-) (Figs. 4B 4C) .



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  		Figure 4. Effect of ETA during experimental Pseudomonas keratitis. Mean clinical scores ± SEM in B6 mice infected with (A) 19660 (n = 20), 19660 tox (-) (n = 20), or 19660 tox (-) pUCPTOXA (n = 10) and (B) PAO1 (n = 20), PAO1 tox (-) (n = 20), or PAO1 tox (-) pUCPTOXA (n = 10). Differences in clinical scores between wild-type and ETA mutant infections were statistically significant at P <= 0.05 at all time points examined. (C) Slit lamp examination photographs of mouse eyes at day 7 PI. Infection with wild-type P. aeruginosa resulted in severe ocular disease (left), whereas eyes infected with tox (-) mutants appeared nearly normal (right). Magnification, x50.

 
Host Response against P. aeruginosa ETA-Deficient Mutants
The host response in the cornea against P. aeruginosa, comprised primarily of PMNs,6 49 can be quantitated by measuring the presence of PMN-derived myeloperoxidase (MPO) activity in ocular homogenate.48 As early as 24 hours PI, significantly less (n = 3; P <= 0.05) PMNs were detected in the homogenate of eyes infected with ETA-deficient mutants 19660 tox (-) and PAO1 tox (-) (Fig. 5) . Whereas PMN numbers in ocular homogenate of ETA mutant infected mice remained low, ocular homogenate of wild-type infected mice exhibited increasing numbers of PMNs as the infection progressed.



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  		Figure 5. Quantitation of host inflammatory infiltrate (PMNs) as MPO activity in infected eyes of B6 mice at 6, 12, 24, 36, and 48 hours PI. Mean MPO U/mL ± SEM (n = 3 eyes/strain per time point). ND, not determined; *significant at P <= 0.05.

 
Viability of ETA-Deficient Mutants In Vivo
The ability of bacteria to persist in the cornea during infection was determined by serial dilution plating of ocular homogenate from infected mice. As early as 24 hours PI, there were significantly fewer bacteria recovered from eyes infected with PAO1 tox (-) (Fig. 6A ; n = 3, P <= 006). PAO1 tox (-) was cleared from the eye by 48 hours PI. Significantly fewer viable bacteria were detected in 19660 tox (-)–infected eyes at 36 hours PI (Fig. 6B ; n = 3; P <= 0.0005) and fewer than 10 CFU/mL bacteria were detectable as late as 72 hours PI.



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  		Figure 6. Viable bacteria in eyes of B6 mice infected with (A) 19660 or 19660 tox (-) at 6, 12, 24, 36, 48, and 72 hours PI and (B) PAO1 or PAO1 tox (-) at 6, 12, 24, 36, and 48 hours PI. Mean log10 CFU/eye ± SEM (n = 3 eyes/strain per time point). *Significant at P <= 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ETA has been implicated in the ocular virulence of P. aeruginosa based on previous studies investigating the cytotoxicity of ETA exogenously added to the eye8 9 and the reduced ocular virulence in vivo of chemically generated mutants deficient in ETA.15 Results presented in this study, resulting from the construction of isogenic mutants deficient only in ETA, demonstrate definitively that this protein is a factor in virulent ocular disease.

Because no significant difference in ocular disease was observed in this study early during infection between ETA-deficient mutants and their respective wild-type strains, we hypothesized that ETA itself has no essential role in adherence of P. aeruginosa to scarified corneal epithelium. Results from the examination of ETA-deficient mutants in an in vitro organ culture model of bacterial adherence support this hypothesis.

The significant difference in disease between ETA-deficient mutants and their respective isogenic parents after the infection is established, correlates with the clearance of P. aeruginosa that is incapable of expressing enzymatically active ETA. Our data demonstrate that bacteria are capable of adhering to the cornea and surviving early during the infection, regardless of production of ETA, but that ETA’s absence affects their ability to persist and cause disease.

Although both ETA-deficient strains of P. aeruginosa examined in this study were eventually cleared, the 19660 ETA mutant was able to sustain infection comparable to its wild-type parent strain longer than the PAO1 ETA mutant and was not cleared as efficiently as the PAO1 ETA mutant. These differences may be due to the ability of cytotoxic strains such as 19660 to produce an acute cytotoxin designated ExoU, which invasive strains such as PAO1 do not produce.50 51 ExoU has been linked to an acute cytotoxicity exhibited by P. aeruginosa against lung epithelium.51 It is possible that the activities of ExoU early during infection account for the delayed clearance and elevated disease scores observed during infection with 19660 tox (-) in comparison with PAO1 tox (-).

One possible explanation for the inability of ETA-deficient P. aeruginosa to persist in the eye is that ETA may affect the physiology of the corneal epithelium at or near the initial wound site. In a corneal wound, there is an initial period of cellular reorganization accompanied by a large amount of protein synthesis.52 Successful renewal of the epithelium is dependent on the synthesis of cytoskeletal components (e.g., actin, talin, surface-associated glycoproteins, and glycolipids) and other cell surface molecules, such as CD44.52 It is possible that in the presence of ETA, synthesis of these proteins is blocked. ETA could also inhibit the production of growth factors such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor (TGF)-ß1, which are linked to the regulation of corneal epithelial migration, growth, and adhesion.53 The resultant inhibition of wound healing would promote the maintenance of infection by exposing new binding sites for bacterial colonization beyond the initial wound site.

The clearance of ETA-deficient P. aeruginosa may also be attributed to ETA’s modulating the host inflammatory response. ETA has been reported to have an effect on the superstructure of PMNs54 and is capable of inducing the oxidative burst of monocytes,55 an activity associated with tissue damage in the eye. Miyazaki et al.28 demonstrated that ETA is toxic to PMNs in vivo, and also inhibits the ability of PMNs to phagocytose and kill P. aeruginosa. Furthermore, ETA can have an effect on the host response by influencing the immediate cytokine environment. Resolution of P. aeruginosa keratitis depends on a delicate balance between production of cytokine mediators that either promote or diminish an acute PMN response in the eye.4 5

Discovering the mechanism by which ETA affects persistence of P. aeruginosa in the eye is an important step in understanding the molecular basis for the virulence of P. aeruginosa. Regardless of its mechanism, ETA appears to be a potential therapeutic target in the treatment of Pseudomonas keratitis.


   Acknowledgements
 
The authors thank Laura Alionte and Ronald Barrett for technical assistance throughout this study and Linda Hazlett for advice and guidance.


   Footnotes
 
Supported by National Eye Institute Grant R29-EY11483 (JAH).

Submitted for publication October 11, 2001; revised December 26, 2001; accepted January 14, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Jeffery A. Hobden, Wayne State University School of Medicine, Department of Immunology and Microbiology, 540 E. Canfield Avenue, Detroit, MI 48201; jhobden{at}med.wayne.edu.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cohen, EJ, Laibson, PR, Arentsen, JJ, Clemons, CS. (1987) Corneal ulcers associated with cosmetic extended-wear soft contact lenses Ophthalmology 94,109-114[Medline][Order article via Infotrieve]
  2. Laibson, PR. (1972) Annual review: cornea and sclera Arch Ophthalmol 88,630-648
  3. Ogawa, GSH, Hyndiuk, RA. (1994) Bacterial keratitis and conjunctivitis Smolin, G Thoft, RA eds. The Cornea ,125-167 Little, Brown Boston.
  4. Kernacki, KA, Goebel, DJ, Poosch, MS, Hazlett, LD. (1998) Early cytokine and chemokine gene expression during P. aeruginosa corneal infection in mice Infect Immun 66,376-379[Abstract/Free Full Text]
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