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Contribution of ExsA–Regulated Factors to Corneal Infection by Cytotoxic and Invasive Pseudomonas aeruginosa in a Murine Scarification Model

¹From the Morton D. Sarver Laboratory for Cornea and Contact Lens Research, School of Optometry, University of California, Berkeley, California; and the ²Touro University College of Osteopathic Medicine, Vallejo, California.

	Abstract

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PURPOSE. The exoenzyme S regulatory protein ExsA regulates a type III secretion system in Pseudomonas aeruginosa. In vitro, cytotoxic strains use this system to secrete exotoxin (Exo)U and ExoT causing cytotoxicity and inhibiting their phagocytosis by epithelial cells. Invasive P. aeruginosa secrete ExoT and ExoS, but exsA mutation has little impact on their short-term interactions with epithelia. In the present study, the contribution of these ExsA-regulated proteins toward corneal infections in vivo was investigated.

METHODS. After anesthesia, the left cornea of C57BL/6 mice was scratch injured and then inoculated with cytotoxic (PA103) or invasive (PAK) P. aeruginosa or with isogenic mutants in exsA-related genes. Inocula of 10³ to 10⁶ bacteria/5 µL were used, and at least five animals were assigned to each experimental group. Corneal disease was quantified at regular intervals for 14 days in masked fashion with two different scoring systems.

RESULTS. For the cytotoxic strain, mutation of either exoU or exoT alone had little effect on virulence, whereas simultaneous mutation of both exoT and exoU or of exsA resulted in a significantly reduced capacity to cause corneal disease. Complementation of the double exoUexoT mutant with exoU alone restored bacterial colonization levels (>3-log increase) and disease severity to wild-type levels. Complementation with exoT alone increased colonization (3-log increase) and increased virulence to almost the same levels as wild-type or exoU-complemented infections. Virulence of the invasive strain was not reduced by mutation of exsA or of genes encoding the ExsA-regulated secreted proteins.

CONCLUSIONS. ExsA contributed to corneal virulence of only cytotoxic P. aeruginosa, with contributions made by both ExoU and ExoT to bacterial survival and disease severity. This differs from cytotoxic P. aeruginosa virulence in the lung, which is ExoU-dependent.

P. aeruginosa strains can be broadly classified as either cytotoxic or invasive based on their effects on epithelial cells.¹⁰ Invasive and cytotoxic strains differ genotypically in some genes of the Exoenzyme S regulon that are coordinately regulated by the transcriptional activator ExsA.¹¹ ExsA-regulated genes that distinguish cytotoxic and invasive strains encode effector molecules that are delivered into mammalian cells through a contact-dependent (type III) secretion mechanism similar to that found in many other Gram-negative bacteria.^{12 13}

In cytotoxic P. aeruginosa, ExsA regulates acute cytotoxic activity toward various mammalian cell types including epithelial cells, macrophages, and polymorphonuclear neutrophils (PMNs).^{11 14 15} The ExsA-regulated effector molecules that have been identified in cytotoxic strains are (1) exotoxin (Exo)U (encoded by exoU), a 70-kDa protein that is required for full cytotoxic activity in epithelial cells and macrophages,^{14 16} and (2) ExoT (encoded by exoT), a 53-kDa form of exoenzyme S with less ADP-ribosyltransferase activity,¹⁷ but which causes epithelial cell rounding and inhibition of bacterial internalization^{18 19} through the activation of RhoGTPases.^{20 21} In vitro research has shown that exsA mutation of a cytotoxic strain changes the way in which it interacts with cultured corneal epithelial cells. ExsA mutants (which do not secrete ExoU or ExoT) lose their acute cytotoxic activity and acquire the ability to invade cells.^{11 22}

In contrast, invasive P. aeruginosa do not induce acute cytotoxicity, and they invade and multiply within epithelial cells in vitro and in vivo.^{23 24 25} Their lack of acute cytotoxic activity is explained by the absence of the exoU gene.^{11 16} However, their ability to invade mammalian cells while encoding ExoT (which inhibits bacterial invasion in a cytotoxic strain background) is not yet understood. In this regard, ExoS, an ExsA-regulated effector (49 kDa) encoded only by invasive strains also has a potential inhibitive effect on invasion, and it exerts this effect when introduced into Yersinia spp.²⁶ or cytotoxic P. aeruginosa.¹⁹ Like ExoT, ExoS can activate Rho-family GTPases to disrupt the actin cytoskeleton.^{27 28} Some invasive strains have also been found to encode a third ExsA-regulated effector molecule, ExoY, which has adenylate cyclase activity.²⁹ Despite the possession of three ExsA-regulated factors that quickly affect cell function in other settings,^{27 28 29} exsA mutation in an invasive strain has little impact on its ability to interact with corneal epithelial cells in vitro, at least over a short (3-hour) period.¹¹

The relative contributions of ExsA-effector molecules in causing corneal disease in vivo are not known for either invasive or cytotoxic strains, although introduction of exoU into an invasive strain through a plasmid was found to enhance its virulence in a lung model of infection.³⁰ In this study, we investigated the roles of ExoU and ExoT in the virulence of a cytotoxic strain and of ExoS and ExoT in the virulence of an invasive strain. Given that type III protein secretion involves translocation of effector molecules directly from the bacterial cytoplasm to the host cell cytoplasm through a specialized delivery apparatus, we used isogenic bacterial mutants, rather than purified exoproducts, to examine the contribution of these ExsA-regulated factors toward P. aeruginosa infectious keratitis.

	Methods

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Bacterial Strains and Preparation of Inocula
ExsA-regulated effectors of cytotoxic P. aeruginosa were investigated with strain PA103 (serogroup O11) and various isogenic transposon mutants that affect type III secretion of ExoU and/or ExoT. These included an exsA mutant (PA103exsA::), that does not produce any ExsA-regulated proteins,³¹ an exoT mutant (PA103exoT::Tc),¹⁶ an exoU mutant (PA103exoU::Tn5Tc),¹⁶ and an exoUexoT double mutant (PA103exoUexoT::Tc).¹⁸ Some experiments involved the use of the exoUexoT double mutant complemented with either exoU or exoT through the plasmid pUCP18. These were compared with wild-type PA103 or with the exoUexoT mutant containing only the empty pUCP18 vector.

ExsA-regulated effectors of invasive P. aeruginosa which produce ExoS and ExoT, but not ExoU, were investigated with strain PAK (serogroup O1) and the following isogenic mutants: an exsA mutant (PAKexsA::),³¹ an exoS mutant (PAKexoS::),³² an exoT mutant (PAKexoT::Gem),³² and an exoSexoT double mutant (PAKexoS::/exoT::Gem).³²

The properties of all the strains and mutants used are summarized in Table 1 . None of the mutants used is defective in growth or survival, even when incubated in vitro with corneal epithelial or other cell types.^{18 19}

Murine Corneal Infection In Vivo
A murine scarification model of corneal infection was used.⁷ Female C57BL/6 mice (5–7 weeks old) were anesthetized, and three scratches (each 1 mm length) were made on the left cornea of each animal with a sterile needle. Scratched eyes were then topically inoculated with strain PA103 or PAK, or their isogenic mutants at inocula of 10³, 10⁴, 10⁵, or 10⁶ total colony-forming units in 5 µL MEM. With the exception of pilot experiments, at least five animals were assigned to each treatment group. Progression of infections was monitored to detect any differences between mutant and wild-type strains during development and resolution of disease. Experiments were repeated at least once when necessary.

The overall severity of corneal infections was scored in a masked fashion at 1, 2, 4, 7, and 14 days after challenge, according to the following previously described grading system³³ : grade 0, eye macroscopically identical with the uninfected contralateral control eye; grade 1, faint opacity partially covering the pupil; grade 2, dense opacity covering the pupil; grade 3, dense opacity covering the entire anterior segment; and grade 4, perforation of the cornea and/or phthisis bulbi (shrinkage of the eyeball). Infections with wild-type bacteria were generally noted to progress in severity until 2 to 4 days after bacterial challenge, after which the severity of infection, as determined by overall scores, tended to remain unchanged. An eye with an overall grade of 2 or greater was considered infected.

An additional five-point grading system (grade 0, no visible disease, to grade 4, severe disease; Table 2 ) was also used for the assessment of multiple disease characteristics, including area and density of the central opacity, density of the peripheral opacity, and quality of the epithelial surface.³⁴

For enumeration of colonizing bacteria, eyes were enucleated and then homogenized in 1 mL tryptic soy broth. To determine the number of viable bacteria, serial dilutions of homogenates were plated on LB agar plates. For plasmid-complemented strains, homogenates were also plated on LB agar plates supplemented with carbenicillin (400 µg/mL) to examine retention of the plasmid during 2 days in vivo.

All procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of California, Berkeley Animal Care and Use Committee.

Statistical Analysis
Data are presented as an overall grade of infection for each mouse (using the system of Beisel et al.,³³ ) or as a median score with lower and upper quartiles (using the system of Beisel et al.,³³ or the extended grading system of Cowell et al.,³⁴ ). Nonparametric statistics were used to determine the statistical significance of differences between groups. Thus, the Mann-Whitney test was used to compare two groups, and the Kruskal-Wallis test was used for comparisons between three or more groups. The ² test was used where indicated. P < 0.05 was considered significant. Bacterial mutants in exsA- and exsA-regulated genes were found to affect the probability of disease occurrence rather than to prevent disease. Thus, some variability occurred among animals in the same treatment group. Because important conclusions related to the role of ExsA in corneal disease caused by cytotoxic and invasive P. aeruginosa strains had emerged from the data already obtained (356 animals), the use of additional animals to perform more detailed statistical analysis was not clearly justifiable.

	Results

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Role of Type III-Secreted Effectors in Corneal Infection by Cytotoxic P. aeruginosa
With an inoculum of 10⁶ cfu total bacteria, both wild-type PA103 and its isogenic exoU mutant infected all inoculated eyes (Table 3) . In contrast, the exoUexoT double mutant and the exsA mutant each caused corneal infection in only one of five inoculated eyes. The significant difference in the infection rates between the exoU mutant and the exoUexoT double mutant with 10⁶ cfu bacteria (P = 0.0367, Mann-Whitney test) suggests a major role for ExoT in disease development. However, subsequent experiments showed that mutation of exoT alone did not reduce virulence compared with PA103 (Table 3) . The difference in infection rates between bacteria lacking both ExoU and ExoT (exsA mutant and exoUexoT double mutant) and those producing one or both of these effectors (exoU mutant and wild-type PA103) was significant (P = 0.0025, Mann-Whitney test). These data suggested that ExoU and ExoT played redundant roles in corneal disease caused by the cytotoxic P. aeruginosa strain PA103.

View larger version (61K): [in this window] [in a new window]   FIGURE 1. Corneal disease caused by bacteria unable to type III-secrete either ExoU or ExoT appeared different despite similar disease scores. Representative photographs of grade 3 infections at 7 days after challenge with 106 cfu bacteria are shown. Clear differences in pigment accumulation and vessel dilation (arrows) were evident between (A) PA103, (B) the exoU mutant, and (C) the exoT mutant when compared with the (D) exoUexoT and (E) exsA mutants.

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Complementation of the exoUexoT mutant with exoT (A) resulted in infections characterized by a dense centrally located opacity surrounded by relatively mild haze compared with those caused by wild-type PA103 (B), in which the entire cornea became opaque (day-2 results).

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Contribution of exoU and exoT to P. aeruginosa ocular survival at 2 days after inoculation. The number of viable bacteria recovered is expressed as the mean ± SEM.

Role of Type III–Secreted Effectors in Corneal Infection by Invasive P. aeruginosa
To determine the potential effects of ExsA-regulated factors on corneal disease induced by invasive P. aeruginosa, a pilot experiment was performed in which eyes were inoculated with 10³, 10⁴, 10⁵, or 10⁶ cfu of strain PAK or its isogenic exsA mutant, and overall disease scores were assigned 4 days after inoculation. The exsA mutation did not result in reduced corneal disease (Table 6) .

	Discussion

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For cytotoxic P. aeruginosa, mutation of exsA, or of both exoU and exoT in PA103 reduced virulence by a similar amount. This suggested that ExoU and ExoT are the principal ExsA-regulated virulence factors in this corneal disease model. The fact that exsA mutation did not decrease virulence below levels noted with the exoUexoT double mutant suggested that other ExsA-regulated virulence mechanisms, such as the type III secretion apparatus itself, which has cytotoxic effects on mammalian cells in vitro,^{15 35} do not play a significant role. Mutation of either exoU or exoT alone did not reduce virulence, which suggests that either of these exoproteins alone was sufficient to confer virulence, whereas neither was absolutely required. Complementation experiments showed that ExoU was somewhat more damaging than ExoT.

Complementation experiments also showed that either ExoU or ExoT was required for bacterial survival in the eye in vivo, which probably relates to their role in the disease process. The inability of exoUexoT double mutants to colonize corneas in vivo is not due to a generalized defect in growth or survival, because they grow efficiently in vitro, even in the presence of mammalian cells.^{18 19} In vitro, ExoT inhibits P. aeruginosa internalization by corneal epithelial cells¹⁹ and disrupts actin cytoskeleton function through the activation of RhoGTPases.^{20 21} It has also been shown to inhibit wound healing of airway epithelial cells in vitro.³⁶ ExoU is essential for acute cytotoxicity in vitro in various mammalian cell types including corneal cells,^{11 16} and it is absolutely essential for virulence of P. aeruginosa in an animal model of acute pneumonia.¹⁶ That ExoT was able to substitute partially for ExoU in the cornea suggests that there could be differences in pathogenesis between the lung and the eye. It also suggests that some of the contribution of ExoU to P. aeruginosa virulence in the cornea may reflect activity separate from its acute cytotoxic effects. The mechanism by which ExoU induces acute cytotoxicity in vitro or disease in vivo is not yet known. It has been proposed that ExoU, like other ExsA-regulated effectors, may have more than one mechanism of action.³⁷ Thus, ExoU and ExoT may share activities that contribute to corneal disease. Otherwise, the role(s) of ExoU and ExoT in P. aeruginosa corneal virulence differ entirely, despite significant redundancy.

A commonly used method of comparing virulence in animal models is calculation of the ID₅₀ (the inoculum at which disease develops in 50% of challenged animals). The exoUexoT double mutant and exsA mutant of PA103 occasionally caused disease in the cornea, albeit with lower probability than wild-type or single exoU or exoT mutants. Furthermore, inoculation with wild-type bacteria at the highest challenge dose used (10⁶ cfu per 5-µL drop) did not always guarantee infection would result, whereas inoculation with the lowest challenge dose (10³ cfu per 5-µL drop) occasionally caused disease. Thus, it was not possible to calculate an ID₅₀ for wild-type or for any of the mutants. A significant reduction, but not total elimination of capacity to cause disease by exsA mutation, shows that ExsA is not absolutely necessary for corneal disease to develop. Rather, the ExsA system, through either ExoU or ExoT, increases the probability that bacterial inoculation will result in disease.

When infections occurred with the exsA or exoUexoT double mutant, there were visible differences in resultant disease that were not detected by either scoring system. Severe limbal blood vessel engorgement and accumulation of pigment and/or blood was observed within corneas infected with wild type or with either the exoU or exoT mutants, but not in those infected with the double exoUexoT or exsA mutant. These pathologic differences developed later in the infection process, approximately 7 days after inoculation. The mechanism and significance of these clinically observed signs are not clear.

The role of ExsA-regulated factors in corneal virulence by an invasive P. aeruginosa strain was investigated using strain PAK, which produces high levels of ExoS and ExoT relative to other known invasive strains.¹¹ Results showed that neither ExoS, ExoT, nor any other ExsA-regulated factor was necessary for full virulence in this infection model. Infections caused by the exoSexoT double mutant or the exsA mutant of the invasive strain PAK occurred as often and were of a severity similar to that of those caused by wild-type bacteria, excluding the possibility that ExoS, ExoT, or any other ExsA-regulated factor makes significant positive contributions. Similar results have been found in a lung model in which an exsA mutant of the invasive P. aeruginosa strain PAO1 was fully virulent.³⁸

Mutation of either exoS or exoT alone resulted in small but significant increases in disease severity compared with wild-type invasive bacteria which secrete both gene products or a exoSexoT double mutant that secretes neither protein. The mechanism for this disease exacerbation is uncertain, but it suggests that these two coordinately regulated factors, which are secreted as a heterologous aggregate, may interfere with each other’s pathogenic potential in some way. This scenario would provide one explanation for the apparent lack of contribution of these ExsA-regulated effectors toward corneal disease by invasive strains, because only invasive strains possess genes for both factors.

Corneal disease caused by strain PAK was generally mild compared with that caused by cytotoxic strain PA103. Hazlett et al.³⁹ also noted that strain PAK exhibited lower corneal infectivity relative to a cytotoxic strain (ATCC 19660; American Type Culture Collection, Manassas, VA). In that study, PAK was mildly virulent, even when scratched mouse corneas were challenged with 5 x 10⁷ cfu bacteria. In the present study, PAK caused significant corneal disease with only 10⁵ cfu bacteria. Differences in PAK virulence between these studies may relate to one or more of the various differences in methodology, which include mouse type (Swiss-Webster versus C57BL/6), anesthesia, and bacterial culture preparation methods.

In summary, the ExsA-regulated type III secretion system of P. aeruginosa was found to make a significant contribution to corneal disease induced by a cytotoxic, but not invasive, strain in the mouse corneal scarification model. That result was consistent with in vitro studies using corneal and other mammalian cell cultures and with in vivo studies of acute pneumonia with cytotoxic and invasive P. aeruginosa strains. However, they also suggest some differences between the lung and the eye with respect to the relative role(s) of both ExoU and ExoT of cytotoxic strains.

In conclusion, the data suggest distinct pathogenic mechanisms for cytotoxic and invasive P. aeruginosa in the cornea. Each causes approximately 50% of P. aeruginosa corneal infections.⁴⁰ Thus, these results are significant for future research into the pathogenic mechanisms of P. aeruginosa and for the design of therapeutic interventions to reduce the probability of bacteria-induced corneal disease.

	Acknowledgements

 
The authors thank Rajana Van for excellent technical assistance and Dara W. Frank (Medical College of Wisconsin) and Shouguang Jin (University of Florida, Gainesville, FL) for supplying the P. aeruginosa mutants used in this study.

	Footnotes

 
Supported National Eye Institute Grant EY11221, University of California Berkeley Faculty Research Grant, a Hellman Family Faculty Award (SMJF), an American Optometric Foundation Ezell Fellowship (EJL), and a C. J. Martin Postdoctoral Fellowship from the National Health Medical Research Council, Australia (BAC).

Submitted for publication December 18, 2002; revised February 27, 2003; accepted March 30, 2003.

Disclosure: E.J. Lee, None; B.A. Cowell, None; D.J. Evans, None; S.M.J. Fleiszig, None

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: Suzanne M. J. Fleiszig, School of Optometry, University of California, Berkeley, CA 94720-2020; fleiszig{at}socrates.berkeley.edu.

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				R. E. Vance, A. Rietsch, and J. J. Mekalanos Role of the Type III Secreted Exoenzymes S, T, and Y in Systemic Spread of Pseudomonas aeruginosa PAO1 In Vivo Infect. Immun., March 1, 2005; 73(3): 1706 - 1713. [Abstract] [Full Text] [PDF]

				G. P. Priebe, C. R. Dean, T. Zaidi, G. J. Meluleni, F. T. Coleman, Y. S. Coutinho, M. J. Noto, T. A. Urban, G. B. Pier, and J. B. Goldberg The galU Gene of Pseudomonas aeruginosa Is Required for Corneal Infection and Efficient Systemic Spread following Pneumonia but Not for Infection Confined to the Lung Infect. Immun., July 1, 2004; 72(7): 4224 - 4232. [Abstract] [Full Text] [PDF]

				E. J. Lee, D. J. Evans, and S. M. J. Fleiszig Role of Pseudomonas aeruginosa ExsA in Penetration through Corneal Epithelium in a Novel In Vivo Model Invest. Ophthalmol. Vis. Sci., December 1, 2003; 44(12): 5220 - 5227. [Abstract] [Full Text] [PDF]

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