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Host response and bacterial virulence factor expression in Pseudomonas aeruginosa and Streptococcus pneumoniae corneal ulcers.

Karthikeyan RS, Priya JL, Leal SM, Toska J, Rietsch A, Prajna V, Pearlman E, Lalitha P - PLoS ONE (2013)

Bottom Line: We found that neutrophils comprised >90% cells in corneal ulcers, and that there was elevated expression of TLR2, TLR4, TLR5 and TLR9, the NLRP3 and NLRC4 inflammasomes and the ASC adaptor molecule.While P. aeruginosa strains expressing both ExoU and ExoS are usually rare, these strains actually outnumbered strains expressing only ExoU in the current study.Further, as neutrophils are the predominant cell type in these corneal ulcers, they are the likely source of cytokines and of the increased TLR and inflammasome expression.

View Article: PubMed Central - PubMed

Affiliation: Dr. G. Venkatasamy Eye Research Institute, Aravind Eye Hospital, Madurai, Tamil Nadu, India.

ABSTRACT
P. aeruginosa and S. pneumoniae are major bacterial causes of corneal ulcers in industrialized and in developing countries. The current study examined host innate immune responses at the site of infection, and also expression of bacterial virulence factors in clinical isolates from patients in south India. Corneal ulcer material was obtained from 49 patients with confirmed P. aeruginosa and 27 patients with S. pneumoniae, and gene expression of Toll Like Receptors (TLR), cytokines and inflammasome proteins was measured by quantitative PCR. Expression of P. aeruginosa type III secretion exotoxins and S. pneumoniae pneumolysin was detected by western blot analysis. We found that neutrophils comprised >90% cells in corneal ulcers, and that there was elevated expression of TLR2, TLR4, TLR5 and TLR9, the NLRP3 and NLRC4 inflammasomes and the ASC adaptor molecule. IL-1α IL-1β and IFN-γ expression was also elevated; however, there was no significant difference in expression of any of these genes between corneal ulcers from P. aeruginosa and S. pneumoniae infected patients. We also show that 41/49 (84%) of P. aeruginosa clinical isolates expressed ExoS and ExoT, whereas 5/49 (10%) of isolates expressed ExoS, ExoT and ExoU with only 2/49 isolates expressing ExoT and ExoU. In contrast, all 27 S. pneumoniae clinical isolates produced pneumolysin. Taken together, these findings demonstrate that ExoS/T expressing P. aeruginosa and pneumolysin expressing S. pneumoniae predominate in bacterial keratitis. While P. aeruginosa strains expressing both ExoU and ExoS are usually rare, these strains actually outnumbered strains expressing only ExoU in the current study. Further, as neutrophils are the predominant cell type in these corneal ulcers, they are the likely source of cytokines and of the increased TLR and inflammasome expression.

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Cellular composition of corneal ulcers from patients with bacterial keratitis.Representative corneal ulcers of patients caused by P. aeruginosa (A), or by S. pneumoniae (B). C, D. Gram staining of corneal ulcer material showing Gram negative bacilli (C), and Gram positive diplococci and chains (D). Original magnification is x1000. E,F: Wrights Giemsa (Diff-Quik) stain of corneal ulcer material from P. aeruginosa (E), or S. pneumoniae (F) infected tissue Original magnification is x400. G. Percent neutrophils and mononuclear cells were determined by counting cells from ten P. aeruginosa and ten S. pneumoniae patients.
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pone-0064867-g001: Cellular composition of corneal ulcers from patients with bacterial keratitis.Representative corneal ulcers of patients caused by P. aeruginosa (A), or by S. pneumoniae (B). C, D. Gram staining of corneal ulcer material showing Gram negative bacilli (C), and Gram positive diplococci and chains (D). Original magnification is x1000. E,F: Wrights Giemsa (Diff-Quik) stain of corneal ulcer material from P. aeruginosa (E), or S. pneumoniae (F) infected tissue Original magnification is x400. G. Percent neutrophils and mononuclear cells were determined by counting cells from ten P. aeruginosa and ten S. pneumoniae patients.

Mentions: Figures 1A, B are representative corneas of patients with culture proven P. aeruginosa and S. pneumoniae keratitis, showing severe corneal opacification and ulceration, and conjunctival inflammation. Examples of Gram stained corneal ulcer material show Gram negative rods typical of P. aeruginosa (Figure 1C), and Gram positive diplococci and chains (Figure 1D), which are indicative of S. pneumoniae.Figures 1E,F show polymorphonuclear cells that are characteristic of neutrophils, although mononuclear and epithelial cells were also detected. Neutrophils were found to comprise 92% infiltrating cells, with 5% being mononuclear cells (Figure 1G).


Host response and bacterial virulence factor expression in Pseudomonas aeruginosa and Streptococcus pneumoniae corneal ulcers.

Karthikeyan RS, Priya JL, Leal SM, Toska J, Rietsch A, Prajna V, Pearlman E, Lalitha P - PLoS ONE (2013)

Cellular composition of corneal ulcers from patients with bacterial keratitis.Representative corneal ulcers of patients caused by P. aeruginosa (A), or by S. pneumoniae (B). C, D. Gram staining of corneal ulcer material showing Gram negative bacilli (C), and Gram positive diplococci and chains (D). Original magnification is x1000. E,F: Wrights Giemsa (Diff-Quik) stain of corneal ulcer material from P. aeruginosa (E), or S. pneumoniae (F) infected tissue Original magnification is x400. G. Percent neutrophils and mononuclear cells were determined by counting cells from ten P. aeruginosa and ten S. pneumoniae patients.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672173&req=5

pone-0064867-g001: Cellular composition of corneal ulcers from patients with bacterial keratitis.Representative corneal ulcers of patients caused by P. aeruginosa (A), or by S. pneumoniae (B). C, D. Gram staining of corneal ulcer material showing Gram negative bacilli (C), and Gram positive diplococci and chains (D). Original magnification is x1000. E,F: Wrights Giemsa (Diff-Quik) stain of corneal ulcer material from P. aeruginosa (E), or S. pneumoniae (F) infected tissue Original magnification is x400. G. Percent neutrophils and mononuclear cells were determined by counting cells from ten P. aeruginosa and ten S. pneumoniae patients.
Mentions: Figures 1A, B are representative corneas of patients with culture proven P. aeruginosa and S. pneumoniae keratitis, showing severe corneal opacification and ulceration, and conjunctival inflammation. Examples of Gram stained corneal ulcer material show Gram negative rods typical of P. aeruginosa (Figure 1C), and Gram positive diplococci and chains (Figure 1D), which are indicative of S. pneumoniae.Figures 1E,F show polymorphonuclear cells that are characteristic of neutrophils, although mononuclear and epithelial cells were also detected. Neutrophils were found to comprise 92% infiltrating cells, with 5% being mononuclear cells (Figure 1G).

Bottom Line: We found that neutrophils comprised >90% cells in corneal ulcers, and that there was elevated expression of TLR2, TLR4, TLR5 and TLR9, the NLRP3 and NLRC4 inflammasomes and the ASC adaptor molecule.While P. aeruginosa strains expressing both ExoU and ExoS are usually rare, these strains actually outnumbered strains expressing only ExoU in the current study.Further, as neutrophils are the predominant cell type in these corneal ulcers, they are the likely source of cytokines and of the increased TLR and inflammasome expression.

View Article: PubMed Central - PubMed

Affiliation: Dr. G. Venkatasamy Eye Research Institute, Aravind Eye Hospital, Madurai, Tamil Nadu, India.

ABSTRACT
P. aeruginosa and S. pneumoniae are major bacterial causes of corneal ulcers in industrialized and in developing countries. The current study examined host innate immune responses at the site of infection, and also expression of bacterial virulence factors in clinical isolates from patients in south India. Corneal ulcer material was obtained from 49 patients with confirmed P. aeruginosa and 27 patients with S. pneumoniae, and gene expression of Toll Like Receptors (TLR), cytokines and inflammasome proteins was measured by quantitative PCR. Expression of P. aeruginosa type III secretion exotoxins and S. pneumoniae pneumolysin was detected by western blot analysis. We found that neutrophils comprised >90% cells in corneal ulcers, and that there was elevated expression of TLR2, TLR4, TLR5 and TLR9, the NLRP3 and NLRC4 inflammasomes and the ASC adaptor molecule. IL-1α IL-1β and IFN-γ expression was also elevated; however, there was no significant difference in expression of any of these genes between corneal ulcers from P. aeruginosa and S. pneumoniae infected patients. We also show that 41/49 (84%) of P. aeruginosa clinical isolates expressed ExoS and ExoT, whereas 5/49 (10%) of isolates expressed ExoS, ExoT and ExoU with only 2/49 isolates expressing ExoT and ExoU. In contrast, all 27 S. pneumoniae clinical isolates produced pneumolysin. Taken together, these findings demonstrate that ExoS/T expressing P. aeruginosa and pneumolysin expressing S. pneumoniae predominate in bacterial keratitis. While P. aeruginosa strains expressing both ExoU and ExoS are usually rare, these strains actually outnumbered strains expressing only ExoU in the current study. Further, as neutrophils are the predominant cell type in these corneal ulcers, they are the likely source of cytokines and of the increased TLR and inflammasome expression.

Show MeSH
Related in: MedlinePlus