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Mary Marquart, Sidney Taylor; Isolation of Two Proteases from Streptococcus pneumoniae that Cause Corneal Erosions. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5221.
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© ARVO (1962-2015); The Authors (2016-present)
Previous work has shown that pneumolysin-deficient strains of Streptococcus pneumoniae retain virulence in the cornea. This study was undertaken to isolate other virulence factors involved in this disease.
S. pneumoniae K1263ΔPLY, a pneumolysin-deficient strain, was used in this study and grown in Todd Hewitt broth containing yeast extract (THY). Bacterial cells grown overnight were removed by centrifugation, and the resulting extracellular milieu (ECM) was filter-sterilized and concentrated. ECM and heat-treated ECM were each injected into rabbit corneal stromas, and corneal damage assessed at 8, 24, and 48 hours. ECM was tested for cytotoxicity of human corneal epithelial cells (HCECs), hemolysis of red blood cells, and protease activity against casein. This ECM was subjected to anion exchange and gel filtration chromatography, and fractions were tested for protease activity, corneal damage, and induction of neutrophil recruitment. All proteins were detected by SDS-PAGE and silver staining.
Forty µg of ECM from K1263ΔPLY, but not heat-treated ECM, produced protease activity in vitro, corneal erosions in vivo by 8 hours, and corneal opacity by 48 hours. Cytotoxicity and hemolytic activity were not detected in vitro. Unbound material from anion exchange chromatography produced protease activity, corneal erosions, opacity, and neutrophil recruitment equivalent to 9,860 neutrophils/mL. Heat treatment abolished all activity and resulted in recruitment of 6,826 neutrophils/mL. Gel filtration produced two fractions with protease activity and ability to form corneal erosions at concentrations of 0.6 µg and 1.9 µg per cornea, which were abolished by heat treatment. One of these fractions contained one detectable protein of >170 kDa and the other fraction contained two detectable proteins of 34 and 30 kDa. The >170 kDa protein (0.6 µg/cornea) caused neutrophil recruitment (9,100 cells/mL), which was abolished by heat treatment (0 cells). The other proteolytic fraction containing the 34 and 30 kDa proteins (1.9 µg/cornea) did not have the ability to recruit neutrophils (0 cells).
S. pneumoniae produces at least two previously unreported proteases that cause corneal erosions. One of these proteases can also recruit neutrophils to the cornea and cause opacity. Future work will entail identification of these novel corneal virulence factors.
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