Abstract
Purpose: :
The purpose of this study was to examine the in vitro effect of Streptococcus pneumoniae pneumolysin on rabbit corneal epithelial cells and to compare this effect to the pathogenesis of pneumolysin-producing S. pneumoniae in a rabbit keratitis model.
Methods: :
An isogenic pneumolysin deletion mutant bacterial strain (K1263ΔPLY) was created from a clinical keratitis strain (K1263) by replacement of the pneumolysin gene with a trimethoprim-resistance cassette. Rabbit corneal cells were extracted from freshly harvested corneas and were cultured as confluent monolayers. Cell monolayers were exposed to media, recombinant pneumolysin, intracellular extracts of K1263 or K1263ΔPLY, or extracellular material of K1263 or K1263ΔPLY for 3, 6, or 24 hours. After incubation, cellular morphology was observed. The corneas of New Zealand white rabbits were injected with 105 colony-forming units (CFU) of either S. pneumoniae strain K1263 or K1263ΔPLY. Severity of keratitis was determined by slit lamp examination (SLE) at 24 and 48 hours post-infection. Following the final examination, rabbits were sacrificed and their corneas were excised, homogenized, serially diluted and plated on blood agar to determine the log CFU per cornea.
Results: :
K1263 had 94% intracellular and 76% extracellular hemolytic activity relative to a 100% control, whereas extracts from K1263ΔPLY caused no detectable hemolysis. Corneal cells exposed to recombinant pneumolysin or K1263 extracts exhibited cellular damage and death, whereas cells exposed to K1263ΔPLY extracts had reduced pathology. SLE scores of rabbit corneas (n = 6) infected with K1263 (4.612 ± 0.5439) were not significantly different from corneas (n = 11) infected with K1263ΔPLY (3.625 ± 0.3309) at 24 hours post-infection (P = 0.1205). However, at 48 hours post-infection, the SLE scores of corneas infected with K1263 were significantly higher (14.55 ± 1.155) than those infected with K1263ΔPLY (9.760 ± 0.8736; P = 0.005). Log CFU at 48 hours post-infection were not significantly different between the parent (6.09 ± 0.521) and mutant (7.10 ± 0.204) rabbit corneas (P = 0.0512).
Conclusions: :
Recombinant pneumolysin and a pneumolysin-producing strain of S. pneumoniae caused direct cellular damage to rabbit corneal epithelial cells in vitro, whereas a pneumolysin-negative isogenic strain was unable to cause cellular damage. These results support the in vivo findings at 48 hours post-infection and thus provide a useful tool for assessing the toxicity of clinical pneumococcal strains.
Keywords: bacterial disease • keratitis • cornea: epithelium