January 2005
Volume 46, Issue 1
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Immunology and Microbiology  |   January 2005
Polyphosphate Kinase 1 and the Ocular Virulence of Pseudomonas aeruginosa
Author Affiliations
  • Quinn M. Parks
    From the Department of Microbiology, Immunology, and Parasitology, LSU Health Sciences Center, New Orleans, Louisiana.
  • Jeffery A. Hobden
    From the Department of Microbiology, Immunology, and Parasitology, LSU Health Sciences Center, New Orleans, Louisiana.
Investigative Ophthalmology & Visual Science January 2005, Vol.46, 248-251. doi:10.1167/iovs.04-0340
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      Quinn M. Parks, Jeffery A. Hobden; Polyphosphate Kinase 1 and the Ocular Virulence of Pseudomonas aeruginosa. Invest. Ophthalmol. Vis. Sci. 2005;46(1):248-251. doi: 10.1167/iovs.04-0340.

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Abstract

purpose. To determine the role of polyphosphate kinase 1 (PPK1) in the ocular virulence of Pseudomonas aeruginosa.

methods. Using a mouse model of infection, P. aeruginosa strains PAO1, PAOM5 (an isogenic mutant of PAO1 deficient in PPK1), and PAOM5+PPK1 (the mutant complemented with PPK1 on plasmid pHEPAK11) were compared for ocular virulence. These strains were also characterized with respect to traits associated with survival and pathogenicity in an ocular environment.

results. The PPK1-deficient strain PAOM5 was significantly less virulent than either wild-type PAO1 or the complemented mutant (P < 0.016). Loss of virulence was not associated with serum sensitivity or diminished adherence to the cornea. However, PAOM5 has an increased susceptibility to oxidative stress and was cleared from corneal tissue significantly better (P < 0.006) than either the wild-type or restored strain. Furthermore, the PPK1-deficient mutant produced significantly less (P < 0.022) pyocyanin.

conclusions. PPK1 is essential for a successful ocular infection by P. aeruginosa. The loss of ocular virulence is probably due to the dysregulation of multiple genes, including those responsible for stress response.

The pathogen Pseudomonas aeruginosa, ubiquitous throughout the environment, is a significant opportunistic microbe in immunosuppressed individuals. Furthermore, P. aeruginosa is capable of producing a devastating corneal infection in otherwise healthy wearers of soft contact lenses. In less than 4 days after infection, P. aeruginosa keratitis can result in irreversible corneal scarring at best and at worst the loss of an eye. 1  
P. aeruginosa corneal ulcers are difficult to treat and require an intensive regimen of topical fortified antibiotics (typically a mixture of an aminoglycoside such as tobramycin and a β-lactam such as cefazolin). Fluoroquinolones such as ciprofloxacin are also commonly used as monotherapeutic agents. Resistance to ciprofloxacin has been documented among clinical ocular isolates of P. aeruginosa, 2 thus adding to a sense of urgency in the search for alternative therapeutic agents. 
Polyphosphate kinase (PPK)-1 polymerizes the terminal phosphate of adenosine triphosphate (ATP) into a chain of inorganic polyphosphate (poly P). In reverse, PPK1 generates ATP from poly P and adenosine diphosphate (ADP). PPK1 can be found in numerous bacterial pathogens including P. aeruginosa. 3 This enzyme has a highly conserved amino acid sequence, and thus far there are no reported homologues that exist in mammals. 4 As such, PPK1 would serve as an ideal target for therapeutic intervention. 
Recently, Ayraud et al. 5 reported an increase in the colonization of gastric epithelium by Helicobacter pylori (the etiologic agent of infectious gastric ulcers) associated with an increase in PPK1 activity. Loss of PPK1 in the enteric pathogens Salmonella and Shigella resulted in phenotypes suggestive of decreased virulence, including defective responses to stress and starvation, loss of viability, and diminished invasiveness to epithelial cells. 6 Likewise, an isogenic mutant of P. aeruginosa deficient in PPK1 (strain PAOM5) exhibits traits typically associated with a loss of virulence, including the inability to form a biofilm 7 and a loss of motility. 8 Indeed, strain P. aeruginosa PAOM5 is less virulent in a mouse burn model of disease than its wild-type parent strain PAO1. 7  
In the present study, the PPK1-deficient mutant PAOM5 was also significantly less virulent as a corneal pathogen, despite its ability to adhere to corneal epithelium and to grow in a minimal medium simulating tear film. Evidence is presented that this loss of virulence is in part due to an inability to persist in infected tissue because of an increased susceptibility to oxidative stress. 
Materials and Methods
Bacterial Strains
Bacterial strains used in this study were generously provided by Arthur Kornberg (Stanford University, Stanford, CA). P. aeruginosa PAO1 is the wild-type parent strain of P. aeruginosa PAOM5, a PPK1-deficient mutant constructed by deletion of the PPK1 open reading frame (ORF; Δppk). 9 P. aeruginosa PAOM5+PPK1 is strain PAOM5 carrying a functional ppk allele on plasmid pHEPAK11. 7 These strains have been characterized with respect to motility, acylhomoserine lactone (AHL) production, biofilm formation, and virulence in a burned mouse model of infection. 7 8 Unless otherwise noted, bacteria were grown with shaking at 37°C for 18 hours in 0.25% tryptic soy broth (Difco; BD Diagnostic Systems, Sparks, MD) and 5.0% peptone (Difco; ∼2 × 1010 CFU/mL). 
Evaluation of Ocular Virulence
Virulence of P. aeruginosa strains PAO1, PAOM5, and PAOM5+PPK1 was determined in a mouse scratch model of corneal disease as described by Kwon and Hazlett. 10 Use of animals in this study was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. In brief, female C57BL/6 mice (6–8 weeks old; National Cancer Institute [NCI], Frederick, MD) were anesthetized, and 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 5 μL normal saline (pH 7.2). 
Ten mice were infected per strain of P. aeruginosa, and the infection experiments were repeated four times to assure reproducibility of results. Marking their tails with nontoxic colored markers identified individual mice. The ocular disease response of the animals was recorded in a masked fashion on days 1, 3, 5, 7, 10, 12, and 14 after infection (PI). Disease severity was graded on 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 shrunken eye. 11 Data from separate infection experiments was pooled by strain (n = 40 mice/strain). Disease scores are reported as the mean ± SEM. 
Bacterial Survival in the Inflamed Cornea
In two separate experiments, three mice were infected per strain (n = 6) for quantitation of viable bacteria in corneas at days 1 and 5 PI, as described by Hobden et al. 12 Mean log10 CFU/cornea ± SEM were calculated for each strain. 
Adherence to Corneal Epithelium
The ability of wild-type P. aeruginosa PAO1 and mutant strain PAOM5 to adhere to wounded corneal epithelium was performed essentially as described by Gupta et al. 13 Female 8-week-old C57BL/6 mice (NCI) 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. 14 Eyes (four per strain) were inoculated with 5 μL of 107 CFU bacteria onto 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. 13 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. 15 In brief, representative fields were photographed from each group of eyes at a magnification of 3000×. Negatives from these photographs were enlarged to 6000× and three fields (80 mm2 each) were counted on each photograph. Counts are presented as the mean number of bacteria bound per field ± SEM. 
Growth of P. aeruginosa Strains in Medium Resembling Tear Fluid
To determine whether a loss of virulence was due to an inability to flourish in an ocular environment, P. aeruginosa strains PAO1, PAOM5, and PAOM5+PPK1 were grown in a defined medium formulated to simulate tear fluid. 16 Bacteria were grown with shaking at 37°C overnight in this defined medium and then subcultured 1:50 into fresh prewarmed medium. These subcultures were then grown with shaking at 37°C for 12 hours. Absorbance readings of cultures at 650 nm were recorded every hour. 
Oxidative Stress Response of P. aeruginosa Strains
The ability to survive oxidative stress was determined with a modification of an assay described by Jorgensen et al. 17 P. aeruginosa strains PAO1, PAOM5, and PAOM5+PPK1 were grown at 37°C with shaking for 18 hours in Luria-Bertani broth (LB; BD Biosciences, Sparks, MD). Bacteria were pelleted by centrifugation at 8000g for 10 minutes at 20°C. Pellets were then resuspended in normal saline (pH 7.0) to an optic density (OD) of 1.9 at 650 nM (approximately 5 × 109 CFU/mL). 
Bacteria were diluted 1:100 into prewarmed (37°C) LB containing either 50, 25, 20, 15, 10, 7.5, or 5 mM H2O2. Cultures were then incubated at 37°C for 25 minutes. Dilutions of each culture were made in normal saline (pH 7.0), and 100 μL aliquots were plated onto Pseudomonas isolation agar (PIA; BD Biosciences). Plates were incubated at 37°C for 48 hours to determine survivability (growth or no growth on PIA). 
Serum Sensitivity of P. aeruginosa Strains
To determine whether serum components such as complement could affect the survivability of PPK1-deficient P. aeruginosa in corneal tissue, a serum sensitivity assay was performed as described by Estrellas et al. 18 In brief, P. aeruginosa strains PAO1, PAOM5, and PAOM5+PPK1 were grown overnight, centrifuged, and resuspended in medium 1640 (Invitrogen-Gibco Corp., Grand Island, NY). Serial 10-fold dilutions of each strain were made in 1-mL aliquots of RPMI. A volume of 100 μL of fresh mouse serum was added to each 1-mL dilution. As a control, 100 μL of RPMI was added to an aliquot of each culture. Samples were then incubated with shaking for 2 hours at 37°C. Aliquots of 50 μL were then plated onto PIA, and the plates were incubated overnight at 37°C. The serum sensitivity of a strain is defined as the number of CFU in serum-treated samples divided by the number of CFU in the control (RPMI only) ×100. Serum-sensitive strains have a percentage of <50. A semisensitive strain would have a percentage between 50 and 80. A serum-resistant strain would have a percentage of 80 or higher. 
Assays for Extracellular Virulence Factors of P. aeruginosa
Because the loss of PPK1 has been reported to affect multiple attributes of P. aeruginosa, several secreted products associated with virulence were assayed in strains PAO1, PAOM5, and PAOM5+PPK1. The water-soluble blue phenazine pigment pyocyanin was extracted from culture supernatants and quantitated spectrophotometrically as described by Essar et al. 19  
The yellow-green fluorescent pigment pyoverdin and other iron siderophores were examined with a chrome azurol S (CAS) assay, as described by Schwyn and Neilands. 20  
Statistics
All analyses (descriptive and analytical) were performed on computer (Statview, ver. 4.51; Abacus Concepts, Inc., Berkeley, CA). Median disease scores between the mutant, wild-type, and restored strains were compared by ANOVA at the P ≤ 0.05 confidence interval. Other statistical comparisons between strains were conducted with a Student’s t-test with a P ≤ 0.05 confidence interval. 
Results
PPK1-Deficiency and Ocular Virulence
The effect of the PPK1-deficient mutant of P. aeruginosa on ocular disease is shown in Figure 1 . Wild-type PAO1 produced an infection that resulted in serious ocular disease within 7 days. By day 14, 20% of the corneas infected with PAO1 had perforated (i.e., disease score of +4). The remaining eyes had moderate to dense corneal opacities covering 75% to 100% of the anterior chamber. In contrast to its wild-type parent strain, the PPK1-deficient mutant PAOM5 produced significantly less ocular disease (P < 0.0001) throughout the course of infection. None of the corneas infected with PAOM5 perforated. By day 14, only slight corneal opacities (+1) were observed. 
Complementation of PAOM5 with PPK1 on plasmid pHEPAK11 (PAOM5+PPK1) restored virulence when compared with disease caused by the mutant without complementation (P < 0.014). Similar to the wild-type parent strain PAO1, 20% of the corneas infected with PAOM5+PPK1 had perforated by day 14. Although the rate of corneal perforation in eyes infected with the restored strain was similar to that of the wild-type strain, the severity of disease was significantly less on days 3, 5, and 7 PI (P < 0.017). There were no significant differences observed in disease scores between these two strains on any other day (P > 0.16).  
Bacterial Survival in the Inflamed Cornea
The numbers of viable bacteria per cornea recovered on days 1 and 5 PI (expressed as mean log10 CFU/cornea ± SEM) are shown in Table 1 . There was no significant difference between any of the three strains with respect to CFU recovered from corneas on days 1 and 5 PI (P > 0.08). There was no significant decrease (P > 0.291) in CFU in corneas infected with either wild-type PAO1 or with the restored mutant PAOM5+PPK1 from days 1 to 5 PI. However, the number of PPK1-deficient PAOM5 CFU recovered from corneal tissue decreased nearly 50% from days 1 to 5 PI (P < 0.006). 
Adherence to Wounded Corneal Epithelium
The PPK1-deficient strain PAOM5 adhered to wounded corneal epithelium (143 ± 40 CFU per field) equally as well as its wild-type parent strain PAO1 (116 ± 26 CFU per field; P = 0.613). 
Growth and Survival in the Ocular Environment
The capacity to thrive in the ocular environment despite innate host defenses can define the success or failure of an infection. To establish that the reduced virulence of the PPK1-deficient mutant PAOM5 was not due to a growth deficiency, growth curves were constructed using a minimal medium formulated to simulate tear film. 16 As shown in Figure 2 , strain PAOM5 grew as well as its wild-type parent strain PAO1 or the complemented mutant PAOM5+PPK1 in this medium containing bactericidal tear proteins. Moreover, all three strains were resistant to the effects of normal mouse serum. 
In addition to coping with antibacterial substances such as complement and lysozyme, P. aeruginosa must also contend with the harsh environment generated by the large number of polymorphonuclear leukocytes (PMN) which infiltrate the tear film, cornea, and anterior chamber hours after infection. PMN release toxic oxygen-derived products such as H2O2, which are harmful to not only P. aeruginosa but to host tissue as well. A 25-minute exposure to a concentration of 15 mM H2O2 was lethal for the PPK1-deficient mutant PAOM5. In contrast, a concentration of 25 mM H2O2 was lethal for strains PAO1 and PAOM5+PPK1. 
Production of Extracellular Virulence Factors
Routine culture of the PPK1-deficient strain PAOM5 on solid or in liquid medium revealed a deficiency in the blue phenazine pigment pyocyanin. When extracted from culture supernatants and quantitated spectrophotometrically, there was significantly less pyocyanin produced (P > 0.017) by PAOM5 (0.60 ± 0.16 mg/mL) when compared to either the wild-type parent strain PAO1 (2.36 ± 0.14 mg/mL) or the complemented mutant PAOM5+PPK1 (4.43 ± 0.56 mg/mL). Although PAOM5+PPK1 produced twice as much pyocyanin as PAO1, this difference was not significant (P = 0.100). As determined with a CAS assay, the production of iron siderophores such as the yellow-green pigment pyoverdine was similar for all three strains. 
Discussion
In this study, the loss of PPK1 (a unique bacterial enzyme with no mammalian homologue) negatively affected the pathogenesis of P. aeruginosa keratitis. The means by which PPK1 affected ocular virulence is not related to the initial step of adherence because the PPK1-deficient mutant PAOM5 adhered equally as well to wounded corneal epithelium as wild-type P. aeruginosa PAO1. Our results also show that loss of PPK1 did not affect growth of P. aeruginosa either in vivo 1 day PI or in vitro in media formulated to mimic tear film. However, loss of PPK1 did affect the degree in which P. aeruginosa was cleared from the cornea. P. aeruginosa PAOM5 was found to be susceptible to oxidative stress. This susceptibility may explain why it was more efficiently eradicated from corneal tissue than its wild-type parent (PAO1). 
In principle, inhibitors of PPK1 may be effective as therapy in the treatment of P. aeruginosa corneal ulcers. A PPK1 inhibitor may make P. aeruginosa more susceptible to killing by PMNs because of the mutant’s increased susceptibility to oxidative stress. Furthermore, an inhibitor may lessen corneal tissue damage from bacterial products since strain PAOM5 was deficient in several secreted virulence factors such as pyocyanin and elastase. 7 To date, no such inhibitor exists but Zhu et al. 21 have crystallized and characterized PPK1 from Escherichia coli in anticipation of discovering such a compound. 
Restoration of PPK1 to a P. aeruginosa mutant deficient in this enzyme restored virulence similar to that with wild-type P. aeruginosa. It is unclear why the complementation of the mutant strain PAOM5 with a wild-type copy of PPK1 did not restore virulence equivalent to the wild-type parent strain PAO1. A similar observation was made by Rashid et al. 7 when these strains were evaluated for virulence in a mouse burn model of infection. The authors of that study suggested that the lack of complete complementation may be due to the PPK1 gene’s being expressed on a high copy number plasmid. Attempts have been made in our laboratory without success to express PPK1 on a low copy number vector, such as a cosmid. 
The pathogenesis of P. aeruginosa corneal disease results from a complex interaction between the microorganism and the ensuing host response to infection. 22 23 Secreted bacterial proteases and toxins can directly damage corneal tissue or indirectly cause tissue damage through stimulation of several potentially deleterious host processes. Without such a secreted virulence factor (or factors), a large number of P. aeruginosa could be present in corneal tissue without any apparent disease. Eventually, however, disease would become evident as the host inflammatory response is triggered by the presence of increasing amounts of LPS. The PPK1-deficient mutant PAOM5 does not produce numerous secreted products, including elastase 7 and pyocyanin. One day after infection with this strain, corneas had significantly less disease compared with corneas infected with PPK1-producing strains, despite similar numbers of CFU present in tissue. What missing secreted virulence factor or factors of P. aeruginosa PAOM5 that allow it to grow in corneal tissue without eliciting damage in the early stages of infection remains to be elucidated. 
The molecular mechanism by which PPK1 regulates virulence gene expression in P. aeruginosa remains unknown. In E. coli, the accumulation of poly P (due to PPK1 activity) causes an increase in the expression of RpoS, a stationary-phase alternate sigma factor. RpoS is required for the expression of >100 genes involved in protection from various environmental pressures including starvation, osmotic and oxidative stress, and heat shock. 24 In the present study, the loss of PPK1 correlated with an increase in sensitivity to oxidative stress. However, Bertani et al. 25 demonstrated that PPK1 apparently has no effect on RpoS expression in P. aeruginosa
In conclusion, P. aeruginosa requires PPK1 activity for full virulence in the eye. Loss of PPK1 affected the ability of the organism to elaborate virulence factors and contend with the host inflammatory response. The pleiotropic deficiencies in a PPK1-deficient mutant of P. aeruginosa suggest that a global mechanism of gene regulation has been affected. 
 
Figure 1.
 
Ocular virulence of wild-type (PAO1), PPK1-deficient (PAOM5), and restored PPK1-deficient (PAOM5+PPK1) strains of Pseudomonas aeruginosa.
Figure 1.
 
Ocular virulence of wild-type (PAO1), PPK1-deficient (PAOM5), and restored PPK1-deficient (PAOM5+PPK1) strains of Pseudomonas aeruginosa.
Table 1.
 
Log10 CFU/Cornea at Days 1 and 5 after Infection
Table 1.
 
Log10 CFU/Cornea at Days 1 and 5 after Infection
Day PAO1 PAOM5 PAOM5+PPK1
1 4.58 ± 0.33* 3.39 ± 0.17, † 4.74 ± 0.29, ‡
5 3.78 ± 0.98 1.36 ± 0.47 3.45 ± 1.10
Figure 2.
 
Growth of wild-type (PAO1), PPK1-deficient (PAOM5), and restored PPK1-deficient (PAOM5+PPK1) strains of Pseudomonas aeruginosa in minimal medium formulated to simulate tear film.
Figure 2.
 
Growth of wild-type (PAO1), PPK1-deficient (PAOM5), and restored PPK1-deficient (PAOM5+PPK1) strains of Pseudomonas aeruginosa in minimal medium formulated to simulate tear film.
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Figure 1.
 
Ocular virulence of wild-type (PAO1), PPK1-deficient (PAOM5), and restored PPK1-deficient (PAOM5+PPK1) strains of Pseudomonas aeruginosa.
Figure 1.
 
Ocular virulence of wild-type (PAO1), PPK1-deficient (PAOM5), and restored PPK1-deficient (PAOM5+PPK1) strains of Pseudomonas aeruginosa.
Figure 2.
 
Growth of wild-type (PAO1), PPK1-deficient (PAOM5), and restored PPK1-deficient (PAOM5+PPK1) strains of Pseudomonas aeruginosa in minimal medium formulated to simulate tear film.
Figure 2.
 
Growth of wild-type (PAO1), PPK1-deficient (PAOM5), and restored PPK1-deficient (PAOM5+PPK1) strains of Pseudomonas aeruginosa in minimal medium formulated to simulate tear film.
Table 1.
 
Log10 CFU/Cornea at Days 1 and 5 after Infection
Table 1.
 
Log10 CFU/Cornea at Days 1 and 5 after Infection
Day PAO1 PAOM5 PAOM5+PPK1
1 4.58 ± 0.33* 3.39 ± 0.17, † 4.74 ± 0.29, ‡
5 3.78 ± 0.98 1.36 ± 0.47 3.45 ± 1.10
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