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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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Protein expression of S. pneumoniae pneumolysin and P. aeruginosa exotoxins.A. Western blot of pneumolysin in the ATCC reference strain (Lane 1), and four representative clinical isolates (lanes 2–5). B. Western blot of culture supernatants from Pseudomonas aerugenosa reference strains PAO1, which expresses ExoS and ExoT, and PA103, which expresses ExoU and ExoT. C. ExoS, ExoT and ExoU expression in representative Pseudomonas clinical isolates. Lane 1 is similar to PAO1 in expressing ExoS and ExoT; Lane 2 is similar to PA103 in expressing ExoU and ExoT; Lane 3 is P. otitidis, which does not express Type III exotoxins; Lane-4 Clinical isolate express all three effector molecules; Lane-5 Exo U and Exo T expressing clinical isolate similar to PA103. D. percent and total exotoxin production by 49 clinical isolates.
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pone-0064867-g003: Protein expression of S. pneumoniae pneumolysin and P. aeruginosa exotoxins.A. Western blot of pneumolysin in the ATCC reference strain (Lane 1), and four representative clinical isolates (lanes 2–5). B. Western blot of culture supernatants from Pseudomonas aerugenosa reference strains PAO1, which expresses ExoS and ExoT, and PA103, which expresses ExoU and ExoT. C. ExoS, ExoT and ExoU expression in representative Pseudomonas clinical isolates. Lane 1 is similar to PAO1 in expressing ExoS and ExoT; Lane 2 is similar to PA103 in expressing ExoU and ExoT; Lane 3 is P. otitidis, which does not express Type III exotoxins; Lane-4 Clinical isolate express all three effector molecules; Lane-5 Exo U and Exo T expressing clinical isolate similar to PA103. D. percent and total exotoxin production by 49 clinical isolates.

Mentions: Pneumolysin is a major virulence factor of S. pneumoniae, and pneumolysin expressing strains have been isolated from keratitis patients and shown mediate corneal disease in animal models [15], [16]. To determine if S. pneumoniae clinical isolates from patients at the Aravind Eye Hospital express pneumolysin, we examined bacterial lysates by Western blot analysis. As shown in Figure 3A, pneumolysin was expressed by the ATCC reference S. pneumoniae strain ATCC-49619 in addition to four representative clinical isolates. However, all 27 S. pneumoniae ocular isolates were found to express pneumolysin.


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)

Protein expression of S. pneumoniae pneumolysin and P. aeruginosa exotoxins.A. Western blot of pneumolysin in the ATCC reference strain (Lane 1), and four representative clinical isolates (lanes 2–5). B. Western blot of culture supernatants from Pseudomonas aerugenosa reference strains PAO1, which expresses ExoS and ExoT, and PA103, which expresses ExoU and ExoT. C. ExoS, ExoT and ExoU expression in representative Pseudomonas clinical isolates. Lane 1 is similar to PAO1 in expressing ExoS and ExoT; Lane 2 is similar to PA103 in expressing ExoU and ExoT; Lane 3 is P. otitidis, which does not express Type III exotoxins; Lane-4 Clinical isolate express all three effector molecules; Lane-5 Exo U and Exo T expressing clinical isolate similar to PA103. D. percent and total exotoxin production by 49 clinical isolates.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064867-g003: Protein expression of S. pneumoniae pneumolysin and P. aeruginosa exotoxins.A. Western blot of pneumolysin in the ATCC reference strain (Lane 1), and four representative clinical isolates (lanes 2–5). B. Western blot of culture supernatants from Pseudomonas aerugenosa reference strains PAO1, which expresses ExoS and ExoT, and PA103, which expresses ExoU and ExoT. C. ExoS, ExoT and ExoU expression in representative Pseudomonas clinical isolates. Lane 1 is similar to PAO1 in expressing ExoS and ExoT; Lane 2 is similar to PA103 in expressing ExoU and ExoT; Lane 3 is P. otitidis, which does not express Type III exotoxins; Lane-4 Clinical isolate express all three effector molecules; Lane-5 Exo U and Exo T expressing clinical isolate similar to PA103. D. percent and total exotoxin production by 49 clinical isolates.
Mentions: Pneumolysin is a major virulence factor of S. pneumoniae, and pneumolysin expressing strains have been isolated from keratitis patients and shown mediate corneal disease in animal models [15], [16]. To determine if S. pneumoniae clinical isolates from patients at the Aravind Eye Hospital express pneumolysin, we examined bacterial lysates by Western blot analysis. As shown in Figure 3A, pneumolysin was expressed by the ATCC reference S. pneumoniae strain ATCC-49619 in addition to four representative clinical isolates. However, all 27 S. pneumoniae ocular isolates were found to express pneumolysin.

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