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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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Gene expression of Toll Like Receptors, inflammasome proteins and cytokines in corneal ulcers from patients with bacterial keratitis.RNA was extracted from corneal ulcers, reverse transcribed and processed for Q-PCR. Data points represent individual patients infected with P. aeruginosa (closed circles) or S. pneumoniae (open circles), and the values presented are the log of relative gene expression (log(RQ)) in relation to uninfected donor corneas calculated using the 2−ΔΔct method described in Methods. A. Pro-inflammatory cytokines IL-1α, IL-1β and IFN-γ; B. Toll Like Receptors and C. Inflammasome proteins. There were no significant differences in gene expression between P. aeruginosa and S. pneumoniae (p>0.05).
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pone-0064867-g002: Gene expression of Toll Like Receptors, inflammasome proteins and cytokines in corneal ulcers from patients with bacterial keratitis.RNA was extracted from corneal ulcers, reverse transcribed and processed for Q-PCR. Data points represent individual patients infected with P. aeruginosa (closed circles) or S. pneumoniae (open circles), and the values presented are the log of relative gene expression (log(RQ)) in relation to uninfected donor corneas calculated using the 2−ΔΔct method described in Methods. A. Pro-inflammatory cytokines IL-1α, IL-1β and IFN-γ; B. Toll Like Receptors and C. Inflammasome proteins. There were no significant differences in gene expression between P. aeruginosa and S. pneumoniae (p>0.05).

Mentions: Toll like receptors (TLR) and IL-1β are expressed in corneal ulcers caused by filamentous fungi [6], and mediate disease pathogenesis and bacterial killing in murine models of Pseudomonas keratitis [10], [11]. To examine expression of innate immune genes in P. aeruginosa and S. pneumoniae corneal ulcers, RNA was extracted from 48 corneal ulcer scrapings within 1 week of trauma, reverse transcribed, and quantitative PCR was then performed. Δct data were calculated relative to β-actin, and the fold change with respect to the mean of 10 non-infected donor corneas was derived using the 2−ΔΔCt method as described previously [6]. Figure 2A shows elevated expression of IL-1α, IL-1β and IFN-γ compared with uninfected corneas, with IL-1β and IFN-γ greater than 10,000-fold increased; however, there were no significant differences between P. aeruginosa and S. pneumoniae infected corneas, indicating that both genera of pathogenic bacteria stimulate production of proinflammatory cytokines. Similarly, expression of Toll-like Receptor 2, 4, 5 and 9 was elevated >1000-fold in P. aeruginosa and S. pneumoniae infected corneas compared with donor corneas (Figure 2B).


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)

Gene expression of Toll Like Receptors, inflammasome proteins and cytokines in corneal ulcers from patients with bacterial keratitis.RNA was extracted from corneal ulcers, reverse transcribed and processed for Q-PCR. Data points represent individual patients infected with P. aeruginosa (closed circles) or S. pneumoniae (open circles), and the values presented are the log of relative gene expression (log(RQ)) in relation to uninfected donor corneas calculated using the 2−ΔΔct method described in Methods. A. Pro-inflammatory cytokines IL-1α, IL-1β and IFN-γ; B. Toll Like Receptors and C. Inflammasome proteins. There were no significant differences in gene expression between P. aeruginosa and S. pneumoniae (p>0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064867-g002: Gene expression of Toll Like Receptors, inflammasome proteins and cytokines in corneal ulcers from patients with bacterial keratitis.RNA was extracted from corneal ulcers, reverse transcribed and processed for Q-PCR. Data points represent individual patients infected with P. aeruginosa (closed circles) or S. pneumoniae (open circles), and the values presented are the log of relative gene expression (log(RQ)) in relation to uninfected donor corneas calculated using the 2−ΔΔct method described in Methods. A. Pro-inflammatory cytokines IL-1α, IL-1β and IFN-γ; B. Toll Like Receptors and C. Inflammasome proteins. There were no significant differences in gene expression between P. aeruginosa and S. pneumoniae (p>0.05).
Mentions: Toll like receptors (TLR) and IL-1β are expressed in corneal ulcers caused by filamentous fungi [6], and mediate disease pathogenesis and bacterial killing in murine models of Pseudomonas keratitis [10], [11]. To examine expression of innate immune genes in P. aeruginosa and S. pneumoniae corneal ulcers, RNA was extracted from 48 corneal ulcer scrapings within 1 week of trauma, reverse transcribed, and quantitative PCR was then performed. Δct data were calculated relative to β-actin, and the fold change with respect to the mean of 10 non-infected donor corneas was derived using the 2−ΔΔCt method as described previously [6]. Figure 2A shows elevated expression of IL-1α, IL-1β and IFN-γ compared with uninfected corneas, with IL-1β and IFN-γ greater than 10,000-fold increased; however, there were no significant differences between P. aeruginosa and S. pneumoniae infected corneas, indicating that both genera of pathogenic bacteria stimulate production of proinflammatory cytokines. Similarly, expression of Toll-like Receptor 2, 4, 5 and 9 was elevated >1000-fold in P. aeruginosa and S. pneumoniae infected corneas compared with donor corneas (Figure 2B).

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