Contribution of ExsA-regulated factors to corneal infection by cytotoxic and invasive Pseudomonas aeruginosa in a murine scarification model

Ellen Lee, Brigitte A. Cowell, David J. Evans, Suzanne M J Fleiszig

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3892-3898
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number9
DOIs
StatePublished - Sep 1 2003
Externally publishedYes

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Pseudomonas aeruginosa
Virulence
Corneal Diseases
Infection
Mutation
Exotoxins
Proteins
Inbred C57BL Mouse
Phagocytosis
Cornea
Genes
Epithelium
Anesthesia
Epithelial Cells
Bacteria
Lung

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Contribution of ExsA-regulated factors to corneal infection by cytotoxic and invasive Pseudomonas aeruginosa in a murine scarification model. / Lee, Ellen; Cowell, Brigitte A.; Evans, David J.; Fleiszig, Suzanne M J.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 9, 01.09.2003, p. 3892-3898.

Research output: Contribution to journalArticle

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abstract = "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.",
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T1 - Contribution of ExsA-regulated factors to corneal infection by cytotoxic and invasive Pseudomonas aeruginosa in a murine scarification model

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N2 - 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.

AB - 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.

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